Role and Regulation of Starch Phosphorylase and Starch Synthase IV in Starch Biosynthesis in Maize Endosperm Amyloplasts

Home , Starch phosphorylase, Starch synthase

Renuka M. Subasinghe

A Thesis presented to The University of Guelph

In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Molecular and Cellular Biology

Guelph, Ontario, Canada

ABSTRACT

ROLE AND REGULATION OF PLASTIDIAL STARCH PHOSPHORYLASE AND

STARCH SYNTHASE IV IN STARCH BIOSYNTHESIS IN MAIZE

ENDOSPERM AMYLOPLASTS

Renuka M. Subasinghe Advisor: University of Guelph, 2013 Dr. Ian Tetlow

Storage starch is synthesized in sub-cellular organelles called amyloplasts in higher plants. The synthesis of the starch granule is a result of the coordinated activity of several groups of starch biosynthetic enzymes. There are four major groups of these enzymes, ADP-glucose pyrophosphorylase (AGPase), starch synthases (SS), starch branching enzymes (SBE), and starch debranching enzymes (SDE). Starch phosphorylase (SP) exists as both dimeric and tetrameric forms in plastids in developing cereal endosperm and catalyses the reversible transfer of glucosyl units from glucose-1-phosphate to the non- reducing end of α-1-4 linked glucan chains, although the precise role in the pathway remains unclear. The present study was conducted to investigate the role and regulation of SP and SSIV in starch biosynthesis in developing maize endosperm. The results of this study showed that the tetrameric form of SP accounts for the majority of measurable catalytic activity, with the dimeric form being barely active and the monomer catalytically inactive. A catalytically active recombinant maize SP was heterologously expressed and used as an affinity ligand with amyloplast lysates to test protein-protein interactions in vitro.

Results showed that the different multimeric status of SP influenced interactions with other enzymes of starch synthesis. Tetrameric SP interacted with SBEI and

SSIIa, whilst the dimeric form of the enzyme interacted with SBEI, SBEIIb. All of these interactions were enhanced when amyloplasts were pre-treated with ATP, and broken following treatment with alkaline phosphatase (APase), indicating these interactions are regulated by protein phosphorylation. In addition, the catalytic activity of SSIV was reduced following treatment with APase, indicating a role for protein phosphorylation in the regulation of SSIV activity. Protein- protein interaction experiments also suggested a weak interaction between SSIV and SP. Multimeric forms of SP regulated by protein-protein interactions and protein phosphorylation suggested a role for SP in starch biosynthesis in maize endosperm.

Acknowledgements

First and foremost I wish to express my gratitude and appreciation to my advisor Dr. Ian Tetlow for providing me the opportunity to conduct a PhD in his laboratory at the Department of Molecular and Cellular Biology, University of

Guelph and for the guidance, encouragement and expert advice given through the program.

I would especially thank to Dr. Michael Emes, for his excellent guidance and contribution given in his area of expertise. I would like to thank Drs. Robert

Mullen and Peter Pauls for serving as the members of my advisory committee. I gratefully acknowledge all members of the examination committee: Dr. Frederic

Marsolais (External examiner), Dr. Anthony Clarke, Dr. Robert Mullen, Dr. Peter

Pauls and Dr. Janet Wood.

The members of the Tetlow/Emes research group have contributed immensely to my personal and professional time at University of Guelph. I am especially grateful to Dr. Fushan Liu for his valuable contribution and Amina

Mahmouduva for technical support given towards my research. My sincere thanks also go to Usha, Zaheer, Nadya, Wendy, Mark, John, Lily, Ruby and all the present and pass members in the lab for their support and friendship.

I gratefully acknowledge the financial support provided by the BioCar

Initiative Project, Ontario and the University of Guelph Graduate Scholarship program.

I sincerely thank to my loving mother, my husband and two daughters for their understanding, sacrifice and encouragement given in my life.

Dedicated to my Loving Family My Husband Wasantha, My daughters Niki and Himi and my mother Karuna

Table of Contents

Title Page

Abstract

Acknowledgements...... ………………………………………………….....………………..iv

Dedication...... v

Table of Contents………...... ………………………………………………………………………………..vi

List of Figures ………………………………………………………………………………………………….....xiii

List of Tables ………………………………………………………………...... …….………………….xviii

List of Abbreviations…………………………………………………………………………………...... ….xvv

Page

CHAPTER 1...... 1

1. General Introduction...... 2

1.1 Starch Metabolism...... 2

1.1.1. Molecular structure of starch...... 3

1.1.2. Starch Biosynthesis...... 7

1.1.2.1. Starch biosynthetic enzymes...... 8

1.1.2.1.1. ADP-glucose pyrophosphorylase (AGPase EC 2.7.7.27)...... 8

1.1.2.1.2. Starch synthase (SS, EC 2.4.1.21)...... 13

1.1.2.1.2.1. Granule bound starch synthases (GBSS)...... 16

1.1.2.1.2.2. Starch synthase I (SSI)...... 16

1.1.2.1.2.3. Starch synthase II (SSII)...... 18

1.1.2.1.2.4. Starch Synthase III (SSIII…...... …………...... …………..…...... 20

1.1.2.1.2.5. Starch synthase IV (SSIV)………...... …….………………...... 21

1.1.2.1.3. Starch branching enzyme (SBEs)...... 25

1.1.2.1.3.1. Starch branching enzyme I (SBEI)...... 25

1.1.2.1.3.2. Starch branching enzyme II (SBEII)...... 26

1.1.2.1.4. Starch de-branching enzyme (DBE)…………....……………...... 27

1.1.2.1.5. Disproportionating enzyme (D-enzyme)...... 28

1.1.2.1.6. Starch phosphorylase (SP)...... 29

1.1.2.1.6.1. Importance of SP in starch metabolism…………..…...... ……..30

1.1.2.1.6.2. The isoforms of SP in higher plants………………...... 30

1.1.2.1.6.3. Characterization of SP…………………………...…...…...... 32

1.1.2.1.6.4. Biochemical characterization of SP……….……………...…...... 33

1.1.2.1.6.5. SP and starch biosynthesis models...... 40

1.1.2.1.6.6. Evidences of interaction of SP with SSIV….....……...... 41

1.1.2.2. Post transitional modification of starch biosynthesis enzymes...... 42

1.2. Objectives of the study……………………………………………………………...... 43

CHAPTER 2: Biochemical Investigation of the Regulation of Plastidial

Starch Phosphorylase in Maize Endosperm….....……....…………………………45

2.1. Introduction……..…………………………………………...... …………...... 46

2.2. Materials and Methods……...... ………...... …...………………….....…………...52

2.2.1. Materials……………………………………………………………...... 52

2.2.2. Methods..…………………………………………………………………...... ………..52

2.2.2.1. Amyloplast purification from maize endosperms...... …………………..52

vii

2.2.2.2. Preparation of whole cell extracts.……………………………………………………53

2.2.2.3. Localization of SP in the plastid..………...... ……………………………54

2.2.2.4. Preparation of granule bound proteins…………….…...... ……………….54

2.2.2.5. Biochemical Characterization of SP in maize endosperm………….…55

2.2.2.5.1. Phosphorylation and dephosphorylation of

amyloplast lysates...... 55

2.2.2.5.2. Enzyme Assays………………………...... ……………………...56

2.2.2.5.2.1. Starch phosphorylase glucan synthetic activity assay………56

2.2.2.5.2.2. Starch phosphorylase glucan degradative activity assay...56

2.2.2.5.3. Gel Filtration Chromatography (GPC)…...…………….………...... 57

2.2.2.6. Protein analysis………………………………………………………………….58

2.2.2.6.1. Quantification of proteins……………………………………………...... 58

2.2.2.6.2. Sodium dodecyl sulfate polyacrylamide gel electrophoresis…...58

2.2.2.6.3. SP-Native affinity Zymogram…………………………………...... 59

2.2.2.6.4. Coomassie blue staining………………………………………………….60

2.2.2.6.5. Silver staining………………………………………………………...... 60

2.2.2.6.6. Mobility shift detection of phosphorylated proteins

(Phos-TagTM)………………………………………………………………..60

2.2.2.6.7. Immunological techniques……………………………………………….62

2.2.2.6.7.1. Preparation of Peptides and Antisera…………………………...62

2.2.2.6.7.2. Antibody Purification………………………………………………...63

2.2.2.6.7.3. Immunoblot analysis………………………………………………...64

2.2.2.6.7.4. Immunoprecipitation……………………………………….…………...... 64

viii

2.3 Results…………………………………………………………………………………………………...... 66

2.3.1. Subcellular localization of SP in maize endosperm...... 66

2.3.2. The synthetic activity of SP in developing maize endosperm...... 69

2.3.3. Investigating the regulation of SP by protein phosphorylation...... 71

2.3.4. Gel filtration chromatography (GPC) analysis of SP...... 74

2.3.5. The synthetic and phosphorolytic activities of SP with

different glucan substrates...... 82

2.3.6. Immunoprecipitation of SP...... 85

2.4. Discussion……………………………………………………………………………………87

3. CHAPTER 3: Using Recombinant Plastidial SP to Understand

The Regulation of Starch Biosynthesis……....……....……………………………..98

3.1. Introduction.………………..……..…………………………………...... 99

3.2. Materials and Methods………………………………………………………………....103

3.2.1. RNA extraction from maize endosperm and synthesis of cDNA……….103

3.2.2. Quantification of nucleic acid…………………………...... ……..………………103

3.2.3. Agarose gel electrophoresis.…...... ……………...... ……………………104

3.2.4. Designing oligo-nucleotide primers and RT-PCR…………...... ………….104

3.2.5. Ligation of complete SP cDNA sequence to the pET29a expression

vector and transformation to DH5α competent cells……...... …...………107

3.2.6. Expression of plastidial maize SP in Escherichia coli………………….….108

3.2.7. Testing the synthetic and degradative activity of recombinant SP

using glycogen affinity native zymogram……………………………………………109

3.2.8. Gel filtration chromatography analysis of the recombinant SP………..109

3.2.9. Immobilization of recombinant SP on S-Protein Agarose beads and

pulldown assay………………………………………...... ……...... 109

3.2.10. Starch phosphorylase glucan synthetic activity assay………....……....111

3.2.11. Starch phosphorylase glucan degradative activity assay……....…..….111

3.3. Results………………….....……………...…………………………...... 112

3.3.1. Comparison of the protein sequence of plastidial SP

of maize endosperm from the cytosolic form and other species…...... 112

3.3.2. Development of recombinant SP…………...... ………………………….118

3.3.2.1. PCR…………………………………………….....……..…...... ….118

3.3.2.2. Testing the expression level and the synthetic and

degradative activity of recombinant SP on

glycogen affinity zymogram...... …119

3.3.3. Gel Filtration Chromatography analysis of recombinant SP…...... …125

3.3.4. Immobilization of recombinant SP on S-Protein Agarose beads…...... 127

3.3.5. The glucan synthetic and phospholytic activity of recombinant SP...... 132

3.4. Discussion…………………………………………...... ……..…………………………135

4. CHAPTER 4: Biochemical Investigation of the Regulation of

Starch Synthase IV in Maize Endosperm...... 146

4.1. Introduction……………………………………………..………………...... …………...147

4.2. Materials and Methods………………………………………………………………………………..156

4.2.1. Analysis of the localization of SSIV in the plastid……………………………….156

4.2.2. Determination of the protein expression of SSIV

in developing endosperm………………………………………………..……....156

4.2.3. Determination of SSIV catalytic activity by zymogram analysis…...... 157

4.2.4. Substrate-affinity electrophoresis…………………………………..………….157

4.2.5. Gel Filtration Chromatography (GPC)………………………...... …………….158

4.2.6. Co-Immunoprecipitation of SSIV…………………………………...... ………..158

4.2.7. Phosphorylation of SSIV using -32P-ATP………………………………...... 158

4.3. Results………………………………………………….…………………..………………160

4.3.1. Testing the specificity of peptide specific anti-SSIV antibodies…...... 160

4.3.2. Localization of SSIV………………….………...………..…………………...... …160

4.3.3. Determination of the expression of SSIV in developing endosperm....162

4.3.4. Determination of the affinity of the SSIV in amyloplast lysates to

Different α-glucan substrates………………………………………………...... 163

4.3.5. Investigating the regulation of SSIV by phosphorylation using

-32P-ATP……………………………………………………………………………..166

4.3.6. Determination of the activity of ATP or APase treated

SSIV on zymogram………………………………………………………………..168

4.3.7. Gel Filtration Chromatography anlysis of SSIV……...... ……….171

4.3.8. Detection of protein-protein interactions of

SSIV by co-immunoprecipitation………………………………………………173

4.4. Discussion………………………………………………………………………………….178

5. General Discussion...... 185

6. List of References...... 200

7. Appendixes...... 218

xii

List of Figures

CHAPTER 1

Figure 1.1: Structural differences between amylose and amylopectin………………5

Figure 1.2: Schematic representation of starch granule structure illustrating

amorphous and semi-crystalline zones of starch granule (a)……………….……6

Figure 1.3: A summary of the role of major groups enzymes involve

in starch biosynthetic pathway……………………………………………………………………7

Figure 1.4. Domain comparison of starch synthase sequences of five

known SS isoforms in cereal…………………………………………………...... …………...15

CHAPTER 2

Figure 2.1: Schematic diagram illustrating the putative roles of plastidial

(Pho1) and cytosolic (Pho2) SP in starch metabolism in plants...... 48

Figure 2.2: Immunoblots showing the subcellular localization of plastidial SP in

maize endosperm; the amyloplast lysates contain soluble amyloplast

proteins, the granule-bound proteins of the starch granules separated

from amyloplast, the soluble protein fraction and starch granule-bound

proteins of whole cell crude extract of the endosperm and the soluble

protein fraction of the amyloplast membrane protein extracts...... 67

Figure 2.3: Analysis of the localization of proteins imbedded in the granule

surface...... 68

Figure 2.4: Analysis of the localization of proteins imbedded in the granule

surface and loosely bound to the granules...... 69

xiii

Figure 2.5: The activity of Pho1 was observed in developing wild-type maize

amyloplast lysates isolated 12-22 DAA, using non-denaturing

affinity native zymogram containing 0.1% glycogen in the gel...... 70

Figure 2.6: The activity of SP in amyloplast lysates at 22 DAA age in the

synthetic and phosphorolytic direction was tested on glycogen affinity

native zymogram contained 0.1% glycogen in the gel...... 71

Figure 2.7: Determination of the different activity levels of plastidial (Pho1)

and cytosolic (Pho2) isoforms of SP following treatment with ATP and

APase...... 72

Figure 2.8: Mobility shift detection of phosphorylated proteins by

Phosphate affinity SDS-PAGE using Phos-TagTM...... 74

Figure 2.9: The standard curve developed to analyze the molecular weights of

the proteins eluted by gel filtration chromatography...... 76

Figure 2.10A: Gel filtration chromatography analysis of maize whole cell

crude extracts at 15 DAA and 35 DAA...... 77

Figure 2.10B/C/D/E: Gel filtration chromatography analysis of

amyloplast lysates...... 78/79/80/81

Figure 2.11: Native affinity synthetic activity SP zymogram of the amyloplast

lysates separated by GPC...... 82

Figure: 2.12: Immunoprecipitation of SP by peptide specific anti-SP

antibodies (30 mg/mL) with 1 mL amyloplast lysates...... 86

xiv

CHAPTER 3

Figure 3.1: Schematic diagram of the consensus and complementary

strands showing the forward and reverse primers use to isolate

the complete cDNA sequence of the plastidial SP from maize...... 106

Figure 3.2: Novagen pET29a vector used to over express plastidial SP…...... 111

Figure 3.3. The protein sequences of the plastidial SP of maize endosperm...115

Figure 3.4. The predicted phosphorylation sites of the plastidial maize

SP protein sequence were analyzed using NetPhos 2.0 Server.....116/117

Figure 3.5: The PCR product of complete mRNA coding sequence (2805 bp) of

plastidial SP of maize was visualized on a 1% (w/v) agarose gel contained

ethidium bromide……………………………………………………….………...... 119

Figure 3.6. Over expression of recombinant SP in Arctic express E.coli was

analyzed by running the soluble recombinant protein on a 10% SDS gel

followed by Coomassie staining and immunoblot analyses by probing

with anti-SP specific antibodies...... 122

Figure 3.7: The synthetic activity of recombinant SP in glycogen affinity

native zymogram………..……………...... ………….....………123

Figure 3.8: Testing the synthetic and degradative activity of recombinant SP

on glycogen affinity native zymogram…………………………………...... 124

Figure 3.9: Gel filtration chromatography (GPC) analysis of recombinant

SP...... 126

Figure 3.10: Immunoblots probed with anti-SP and anti-S-tag peptide specific

antibodies to confirm the immobilization of the recombinant GPC

fractions by S-Agarose beads...... ………………………………………..129

Figure 3.11: Immunoblots of the immobilized GPC fractions of the recombinant

SP by S-Agarose beads probed with anti-SSIIa, anti-SBEI and anti-SBEIIb

peptide specific antibodies………………………………………...... 130

Figure 3.12: Immunoprecipitation of recombinant tetrameric and dimeric forms

of SP by peptide specific anti-SP antibodies bound to Protein A- Sepharose

beads...... 131

Figure 3.13: Schematic diagram summarizing the protein-protein interactions

between tetrameric and dimeric forms of recombinant SP with starch

biosynthetic enzymes present in the amyloplast lysates...... 132

Figure 3.14: Synthetic and degradative activities of tetrameric and dimeric

forms of recombinant SP in different glucan substrates...... 134

CHAPTER 4

Figure 4.1: Amino acid sequence alignment of SSIV in different plant Species...... 151/152 Figure 4.2: A schematic diagram showing major domains found within

the predicted amino acid sequence of SSIV in wheat endosperm…………153

Figure 4.3: Immunoblots of amyloplst proteins probed with purified SSIV-

Specific antibodies………………………...... …...... 161

Figure 4.4: Immunodetection of SSI, SSII, SSIII and SSIV in stroma

and starch granules of wild-type maize amyloplasts at 22 DAA………....162

Figure 4.5: Immunodetection of SSIV at different stages of development

in maize wild-type amyloplasts……………………………………………………………….163

xvi

Figure 4.6A: Determination of the relative mobility of the SSIV in amyloplast

lysates in native affinity gel electrophoresis containing varying

concentrations of amylopectin, glycogen and maltoheptaose

in the gels...... …164

Figure 4.6B: Plots of the reciprocal of the relative mobility (1/Rm) of maize

SSIV against the concentration of different glucan substrates………………165

Figure 4.7: SSIV from amyloplast lysates was immunoprecipitated following

incubation of amyloplast lysates with -32P-ATP. …………………………….167

Figure 4.8A/B: Zymogram analysis of SS activity in amyloplast lysates of wild-

Type maize endosperm at 22 DAA...... 170

Figure 4.8C: Figure 4.8C: The activity of SS in the amyloplast lysates in the

Absence of SSIV...... 171

Figure 4.9: Gel filtration chromatography analysis of SSIV

in amyloplast lysates…………………………………………………………..….....172

Figure 4.10A: Immunoprecipitation of stromal proteins from wild-type

maize amyloplasts using peptide specific anti-SSIV antibodies

to investigate the protein-protein interactions………………………………..175

Figure 4.10B: Co-Immunoprecipitation of stromal proteins from wild-type

maize amyloplasts using peptide specific anti-SSIV antibodies

to investigate the protein-protein interactions………………………………..176

Figure 4.11: Co-immunoprecipitation of ATP and APase treated stromal

proteins from wild-type maize amyloplasts using peptide specific

anti-SSIV antibodies to investigate the protein-protein interactions

of SSIV with other starch biosynthetic enzymes……………………………..……177

xvii

List of Tables

CHAPTER 1

Table 1.1: The Km and Vmax values of starch phosphorylase in different

plant species………………………………………………………………………...... ………34

CHAPTER 2

Table 2.1: The composition of stacking and resolving gels for

SDS-PAGE………………………………………………...... ………………………………59

Table 2.2: Composition of non- denaturing, 8% acrylamide gels (without SDS)

containing 0.1% (w/v) glycogen prepared as follows…….....….…………………60

Table 2.3: The gel preparations for Phos-TagTM analysis……………………...……..…62

Table 2.4. The synthetic peptides sequences derived from the

N-terminal sequences of starch biosynthetic enzyme isoforms of

maize; there location in full length sequence and the GenBank

accession numbers…………………………………………………………………...... 63

Table 2.5: Synthetic and phosphorolytic activities of SP in different glucan

substrates...... 84

Table 2.6: Km and Vmax values of SP in amyloplast lysates in the

phosphorolytic direction...... 85

CHAPTER 3 Table 3.1: The Km and Vmax values of dimeric and tetrameric forms of recombinant SP in phosphorylitic direction...... …………………...134

xviii

CHAPTER 4

Table 4.1: Comparison of Kd values of maize SSIV with SSI, SSII and

SP estimated by Coummri and Keeling, (2001) in different

glucan substrates…………………………………………………………………...... 166

xix

List of Abbreviations

3-PGA – 3-phosphoglycerate ae – amylose extender

ADP – adenosine diphosphate

AGPase – ADP-glucose pyrophosphorylase

AGP-L – AGPase large subunit

AGP-S – AGPase small subunit

AP - amyloplasts

APase – alkaline phosphatase

ATP – adenosine triphosphate

BCIP/NBT – bromo-4-chloro-3-indonyl phosphate/nitro blue tetrazolium

BSA – bovine serum albumin cDNA – complementary DNA

CE – crude extract

D-enzyme – disproportionating enzyme

DBE – debranching enzyme

DAA – days after anthesis

DMSO - dimethylsulphoxide

DP – degree of polymerization

DTT - dithiothreitol

EC – enzyme commission

E.coli – Escherichia coli

EDTA – ethylenediaminetetraacetic acid

G-1-P – glucose-1-phosphate

G-6-P – glucose-6-phosphate

GPC – gel filtration chromatography

GWD – glucan water dikinase

IPTG – isopropyl-3-D-thiogalactopyranoside

Iso – isoamylase

Kd – dissociation constant kDa – kilodalton

MDs – malto dextrins

MOS – malto-oligosaccharide

MW – molecular weight

NAD – nicotinamide adenine dinucleotide

NADH - nicotinamide adenine dinucleotide (reduced form)

NCBI – National Center for Biotechnology Information

OD – optimal density

PAGE – polyacrylamide gel electrophoresis

PBS – phosphate buffered saline

PCR – polymerase chain reaction

Pho1 – plastidial starch phosphorylase

Pho2 – cytosolic starch phosphorylase

PI – phosphatase inhibitor (cocktail)

Pi – inorganic phosphate

PPi – inorganic pyrophosphate

PWD – phosphoglucan water dikinase

RB – rupturing buffer

xxi

RCF – relative centrifugal force

Rm – Relative migration

SBE – starch branching enzyme

SDS – sodium dodecyl sulfate

Ser - serine

SP – starch phosphorylase

SS – starch synthase

TEMED - tetramethylethylenediamine

Thr - threonine

TTBS - tris buffered saline solution

(v/v) – (volume/volume)

UDP – uridine diphosphate

(w/v) – (weight/volume)

Wx – waxy mutant

xxii

CHAPTER 1

1. General Introduction

1.1. Starch Metabolism

Starch is the major form of carbon reserve polysaccharide, being synthesized in plants in cellular organelles called plastids (Joen et al. 2010;

Tetlow et al. 2006). Transient starch and storage starch are two forms of starch available in plants. The chloroplasts in photosynthetic tissues such as leaves produce transient starch during photosynthesis and store it temporally during the light period. Transient starch is converted into sucrose in the dark and which is translocated within the plant to supply the energy and carbon demand required for growth and development. Storage starch is a long-term carbon store in the plant which is synthesized in non-photosynthetic plastids called amyloplasts, found in tuberous tissues (e.g. in potatoes), or as carbon stores in seeds (Tetlow, 2006, 2011). The location of starch production in the plant is reflective of its metabolic role. Storage starch is extremely important to the plant metabolism of higher plants as a supplier of long-term energy requirement

(Gerard et al. 2001). For instance, storage starch in seeds will be broken down during germination to provide the growing seed with energy until it becomes a photoautotrophic plant.

Starch is also an important polysaccharide for humans and represents up to 80% of daily caloric intake in the human diet. Seed storage reserve carbohydrates are produced in cereal endosperms such as in rice, wheat, maize, barley, and sorghum make up 90% of the starch world market alone (Burrell,

2003). Starch is a cheap, natural and renewable raw material and has numerous industrial applications. Aside from the agri-food sector; starch can be fabricated

2 into pulp and paper, paints, textiles, cosmetics, pharmaceuticals, biodegradable plastics, construction materials, and is also used as a source of renewable energy in the form of ethanol (Shigechi et al. 2004).

1.1.1. Molecular structure of starch

Starch exists as water insoluble glucan polymers which form into a semi- crystalline granular structure in the plastid. Starch granules are composed of two different glucosyl polymers called amylose and amylopectin. The ratio of these polymers in a starch granule is largely genetically controlled, and normally amylopectin makes up about 75% of the starch granule mass and amylose around 25%. Glucosyl units of these polymers are connected by (14) bonds.

Amylose is an unbranched, or less branched polymer which is created by 100–

10,000 glucosyl units whereas amylopectin has much larger polymer units

(degree of polymerization is 105–106 glucose units) with both (14) and distinctive (16) branching glycosidic links (Fig 1.1). The number of glucosyl units in (14) linked linear chains and the relative position of (16) branch linkages are determined by the inherent properties of the starch biosynthetic enzymes. There is approximately one branch point for every 20 glucose residues in amylopectin (Manners, 1989).

Amylopectin exhibits a polymodal glucan chain distribution. This allows the condensing of shorter chained glucans and the subsequent development of efficiently packed parallel left-handed double helices which creates crystalline lamella of the starch granule. The compact helices are approximately 6 to 7.5 nm in length. The regular branch point clusters of amylopectin create

3 amorphous lamella, which are approximately 3nm in length. The compact helices coupled with regular branch point clustering gives rise to the organized semi-crystalline nature of the starch granule (Fig. 1.2) (Hizukuri, 1986; French,

1984). Amylose is found predominantly in a single-helical or random-coil form in the amorphous, noncrystalline regions (Jane et al. 1992). The unique semi- crystaline structure of starch differs from its counterpart, glycogen, in archaea, bacterial and animal systems; glycogen exists as a globular shaped molecule, consisting of water-soluble, homogenously branched glucan polymers (Roach,

2002).

(A)

(B)

Figure 1.1: Structural differences between amylose and amylopectin. The starch granule consists of two forms of glucan polymers; amylose and amylopectin. Amylose is a relatively low branched polymer containing (14) bonds (1A). Amylopectin is a highly branched glucan polymer and has both (14) bonds and (16) bonds (1B). = reducing end

Figure 1.2. Schematic representation of starch granule structure illustrating amorphous and semi-crystalline zones of starch granule (a). Enlargement of semi-crystalline growth rings, illustrating the arrangement of the alternating crystalline and amorphous lamellae (b and c). (Tetlow, 2006).

1.1.2. Starch Biosynthesis

A highly complex and organized coordination of various enzymes is required to synthesize starch in the amyloplast. The major enzymes involved in the biosynthetic process catalyze specific reactions and are present in several isoforms in many plants. There are four major groups of enzymes are involved in starch biosynthesis, adenosine 5’ disphosphate glucose pyrophosphorylase

(AGPase), starch synthase (SS), starch branching enzyme (SBE) and starch debranching enzyme (DBE). These enzymes are found in several isoforms, present in all starch synthesizing organelles (Vrinten and Nakamura, 2000).

Major groups of enzymes involved in amylose and amylopectin biosynthesis process are shown in Fig. 1.3.

Figure 1.3: A summary of the role of major groups enzymes involve in starch biosynthetic pathway.

1.1.2.1. Starch biosynthetic enzymes

1.1.2.1.1. ADP-glucose pyrophosphorylase (AGPase EC 2.7.7.27)

ADP-glucose is the soluble precursor and the glucosyl donor for the different classes of starch synthases, the group of enzymes which are involved in elongation of the α-glucan chains in both transient and storage starch biosynthesis in higher plants (Preiss, 1988). ADP-Glucose is produced from glucose-1-phosphate (G-1-P) and adenosine triphosphate (ATP) by the catalytic activity of AGPase. Therefore, AGPase catalyzes the key metabolic step in the synthesis of starch in higher plants and glycogen in bacteria by providing ADP- glucose, the substrate for all SSs (Preiss, 1988). The reversible reaction of ADP- glucose and inorganic pyrophosphate (PPi) synthesis from ATP and G-1-P by the catalytic activity of AGPase is shown in following reaction (Fu et al. 1998).

Glucose-1-phosphate + ATP ADP-glucose + PPi

AGPase is present in all starch synthesizing tissues in higher plants. In spinach leaves (Morell et al. 1987; Copeland and Preiss, 1981), in Arabidopsis thaliana leaves (Lin et al. 1988) and in potato tubers (Okita et al. 1990;

Sowokinos and Preiss, 1982), AGPase is found as a heterotetrameric in structure, containing two large regulating subunits (AGP-L) and two small (AGP-

S) catalytic subunits. In spinach leaves and in potato tubers the large subunits and the small subunits are respectively 54-55 kDa and 50-51 kDa in size (Okita et al. 1990; Morell et al. 1987; Sowokinos and Preiss, 1982) and in the wheat developing endosperm, 58 and 55 kDa, respectively (Tetlow et al. 2003). The primary sequence of the rice endosperm small subunit has 76% identity to the

8 spinach subunit and the small subunit is structurally conserved in plants (Preiss et al. 1989). Similarly, these subunits are coded by at least two different genes shrunken2 (sh2) and brittle2 (bt2), for the large and small subunits of AGPase, respectively in maize (Bae et al. 1990; Bhave et al. 1990). The N-terminus of the small subunit involves its catalytic properties and the heat stability of

AGPase in potato tuber (Ballicora et al. 1995). In contrast, the AGPase shows homotetrameric structure in bacterial systems like Escherichia coli and

Salmonella typhimurium which have 200 kDa and 48 kDa subunits respectively in size (Preiss, 1988).

Biochemical and genetic evidence indicate that there are two distinct

AGPases are localized in the cytosol and in the plastid (Okita, 1990; Denyer et al. 1996b; Tetlow et al. 2003; Tiessen et al. 2011). In dicots, AGPase is exclusively located in the plastid and represents 98% of the total AGPase activity in the cell (Thorbjørnsen et al. 1996; Tiessen et al. 2011). In contrast, the localization of AGPase is predominantly in the cytosol in cereals; for example in wheat endosperm, 60-70% of the AGPase activity is cytosolic (Geigenberger,

2011; Tetlow et al. 2003) in maize endosperm, it is 95% (Denyer et al. 1996) and in developing barley endosperm it represents 80-90% (Beckles et al. 2001;

Tiessen et al. 2011). However, the large and small subunits sizes are slightly smaller in plastidic AGPase than in cytosolic form in the amyloplast (Beckles et al. 2001; Tetlow et al. 2003).

The presence of AGPase in the cytosol of cereal endosperms implies that the synthesized precursor, ADP-glucose, needs to be transported to the amyloplast for starch synthesis. A specialized nucleotide sugar transporter, the

ADP-glucose/ADP transporter, encoded by brittle1 gene is located at the inner amyloplast envelop to import ADP-glucose during storage starch biosynthesis in cereals (Shannon et al. 1998; Tetlow et al. 2003) and the amino acid sequence of the maize endosperm ADP-glucose transporter, termed Bt1, has been determined (Kirchberger et al. 2007). In wheat endosperm, ADP-glucose transport into amyloplasts was shown to be dependent on the adenylates ADP and AMP as counter-exchange substrates (Bowsher et al. 2007). The authors also found that the rate of ADP exported from the amyloplasts to be equal to the rate of ADP-glucose utilization by starch synthases.

AGPase in both photosynthetic and non-photosynthetic plant sources is allosterically regulated by the metabolites within the carbon assimilation pathway. 3-Phosphoglycerate (3-PGA) is the first intermediate in Calvin cycle of photosynthesis and the AGPase is allosterically regulated positively by the 3-PGA and negatively by inorganic phosphate (Pi) in leaf chloroplasts (Neuhaus and

Stitt, 1990). During the light period in photosynthetic tissues the level of 3-PGA in chloroplast stroma increase and the Pi level decreases as it is used as a substrate in ATP synthesis through photophosphorylation process (Buchanan et al. 2000). In non-photosynthetic tissues such as the amyloplast in cereal endosperm (Tetlow et al. 2003), and in potato tubers (Sowokinos and Preiss,

1982; Tiessen et al. 2003) similar regulation by 3-PGA and Pi was shown. Ratios of the allosteric effectors (3-PGA/Pi) are important in controlling AGPase activity.

For example, the plastidial AGPase activity in wheat endosperm is insensitive to

3-PGA activation compared to potato tubers (Gomez-Casati and Iglesias, 2002;

Tetlow et al, 2003; Ballicora et al, 1995; Hylton and Smith, 1992).

The purified wheat endosperm AGPase activity is also inhibited by adenosine diphosphate (ADP) and fructose-1,6-bisphosphate and the inhibition can be reversed by adding 3-PGA and fructose-6-phosphate (Gomez-Casati and

Iglesias, 2002). The regulation of plastidic form of AGPase in wheat amyloplast in synthetic direction required 15 mM 3-PGA to achieve a 2-fold stimulation in rate, and was only inhibited approximately 40% by a 20 mM high concentrations of Pi (Tetlow et al. 2003). In contrast, AGPases from photosynthetic tissues of wheat were regulated by 3-phosphoglycerate (activator; A0.5=0.01 mM), and orthophosphate (inhibitor; I0.5=0.2 mM) shows higher sensitivity of chloroplast

AGPase to 3-PGA (Gomez-Casati and Iglesias, 2002). Interestingly, the subunits of the cytosolic and plastidic forms not only differ in their sizes but also in their kinetic properties in wheat (Tetlow et al, 2003). The ratio of pyrophosphorolytic to synthetic activity indicates a preference towards the pyrophosphorolysis direction in cytosolic form of AGPase and toward synthesis in plastidial AGPase

(Tetlow et al. 2003). The inhibition of the enzyme activity by Pi on the synthetic direction in whole cell extracts could be restored by 3-PGA, whereas the synthetic reaction in amyloplasts was more sensitive to Pi, and this inhibition was not restored by up to 15 mM 3-PGA (Tetlow et al. 2003). Further, pyridoxal phosphate (pyridoxal-P) was shown as an allosteric activator of spinach leaf

AGPase (Morell et al. 1988). Pyridoxal-P covalently binds to both the 54 kDa and

51 kDa subunits at or near the allosteric activator site(s) of the enzyme. AGPase shows higher affinity to pyridoxal-P than 3-PGA and binding of pyridoxal-P to each protein is inhibited by the presence of either the allosteric activator of the enzyme, 3-PGA or the allosteric inhibitor, Pi (Morell et al. 1988). However, the

11 maximum activation by pyridoxal-P is 6-fold and it is comparatively less, compared with 25-fold by 3-PGA (Morell et al. 1988).

The activity of AGPase is also influenced through post-translational redox modulation in several species, which involves in reversible disulfide-bridge formation between the two small catalytic subunits of the enzyme (Tiessen et al.

2002; Hendriks et al. 2003). The catalytic subunits of the enzyme were detected by their mobility in non-reducing SDS gels as a dimer in oxidized form and as a monomer in reduced form where the overall activity of the enzyme was increased in monomeric and lower in dimeric forms (Kolbe et al. 2005). The activity of recombinant AGPase developed from potato was increased in 4-fold by adding a reducing agent dithiothreitol (DTT) (Sowokinos and Preiss, 1982).

Further, AGPase from potato tubers was activated by a small protein (12 kDa) which facilitates the reduction of other proteins called thioredoxin f and m, leading to an increase in catalytic-subunit monomerization and increased sensitivity to activation by 3PGA (Ballicora et al. 2000). In contrast, AGPase activity was partially inactivated following exposure to oxidized thioredoxin, due to formation of disulfide bonds between the N-termini of the AGPase small subunit (ADP-S) in the potato tubers (Fu et al. 1998). Tiessen et al. 2002 also showed that potato tuber AGPase is subject to redox-dependent posttranslational regulation, involving formation of an intermolecular cysteine

(Cys) bridge between the two small catalytic subunits of the heterotetrameric holoenzyme. Hendriks et al. (2003) further analyzed that the intermolecular Cys bridge between the two smaller catalytic subunits is rapidly converted from a dimer to a monomer when isolated chloroplasts are illuminated or when sucrose

12 is supplied to leaves via the petiole in the dark, and from a monomer to a dimer when pre-illuminated leaves are darkened in pea, potato, and Arabidopsis leaves. This redox activation not only responds to the changes in sugars in chloroplast but also in potato tubers (Tiessen et al. 2002). However, the AGPase is regulated by a light-dependent signal in photosynthetic tissues. Further studies carried out by Tiessen et al. 2003 suggested that, sucrose and glucose lead to redox activation of AGPase via two different signaling pathways involving

SNF1-related protein kinase (SnRK1) and hexokinase, respectively which are implicated in a regulatory network that controls the expression and phosphorylation of cytosolic enzymes in response to sugars in potato tubers

(Geigenberger, 2011).

1.1.2.1.2. Starch synthase (SS, EC 2.4.1.21)

The starch synthases catalyze the transfer of the glucosyl moiety of ADP- glucose to the non-reducing end of an - (14)-linked glucan primer in higher plants. Among the entire starch biosynthesis enzymes, SS has the highest number of isoforms (Fujita et al. 2011). This group of enzymes is divided into two groups; first, the granule–bound starch synthases (GBSS) which are encoded by the Waxy (Wx) gene are involved in amylose biosynthesis

(Nakamura et al. 1993; Sano, 1984; Echt and Schwartz, 1981). The second class of starch synthases consists of four major isoforms SSI, SSII, SSIII, and

SSIV which are involved in amylopectin synthesis. Isoforms of the major classes of SSs are highly conserved in higher plants (Ball and Morell, 2003). A region of approximately 60kDa is highly conserved in C-terminus of all these enzymes in

13 higher plants and green algae, whereas this region is distributed across the protein sequence in prokaryotic glycogen synthases (Tetlow, 2011). The K–X–G–

G–L motif is thought to be responsible for substrate (ADP-glucose) binding in prokaryotic glycogen synthase (GSs) and in higher plant SSs (Furukawa et al.

1990, 1993; Busi et al. 2008), and is also found only in the C-terminus of higher plants and green algal SSs (Nichols et al. 2000) where as the K-X-G-G-L domains are distributed across the GSs protein sequence in prokaryotes

(Fukukawa et al. 1990). The presence of lysine in the K–X–G–G–L domain determines glucan primer preference (Gao et al. 2004). Further, the glutamate and aspartate are found as important residues for catalytic activity and substrate binding in maize SSs (Nichols et al. 2000). SSs show considerable variation within the N-terminus upstream of the catalytic core, and this region can vary greatly in length, from 2.2 kDa in granule-bound starch synthase I

(GBSSI) to approximately 135 kDa in maize SSIII (Gao et al. 1998) (Fig. 1.4).

The phylogenetic and sequence analysis of plants SS (Arabidopsis thaliana, wheat and rice) and algal SS and prokaryotic GS isoforms on the basis of predicted amino acid sequence suggests that SSIs, SSIIs and GBSSIs have distinct evolutionary origins as compared to SSIIIs and SSIVs (Leterrier et al.

2008). Especially, the valine residue within the highly conserved K-X-G-G-L motif appears to have faced strong evolutionary selection in SSIII and SSIVs and it may affect primer/substrate binding of these SSs compared to SSIs, SSIIs and GBSSIs (Leterrier et al. 2008). The other prominent difference in SSIII and

SSIV from other SSs is the highly conserved G-X-G motif near the nucleotide- binding cleft (Leterrier et al. 2008).

Figure 1.4. Domain comparison of starch synthase sequences of five known SS isoforms in cereal. The C-terminal catalytic domains (in black color) includes K- X-G-G-L motif which is a putative ADPG-binding domain. SSs vary in the length of the N- terminal region (shown as hatched bars). The N-terminal arm is believed to provide isoform specificity, possibly through binding to other proteins. SSIII in particular has a unique N-terminal extension thought to be involved in controlling protein–protein interactions. (Sequence lengths are not drawn to scale). (Source: Tetlow, 2011).

1.1.2.1.2.1. Granule bound starch synthases (GBSS)

There are two isoforms of GBSS; GBSSI and GBSSII, both of which are only found in the granule matrix of starch biosynthesizing tissues. GBSSI is responsible for elongating amylose in storage tissues and GBSSII in tissues such as pericarp, leaf, stem, and root (Yandeau-Nelson et al. 2010; Vrinten and

Nakamura, 2000). The waxy mutant results in a lack of amylose production

(Vrinten and Nakamura, 2000; Vrinten et al. 1999). All of the GBSSI protein in maize endosperm is remained as granule associated (Mu-Forster et al. 1996).

However, the Waxy or low amylose starches are still able to form a granule and

15 maintain its semi-crystalline property, suggesting that amylose is not required for insoluble granule synthesis (Denyer et al. 1999).

1.1.2.1.2.2. Starch synthase I (SSI)

SSI is responsible for the synthesis of shorter glucan chains up to ten or less than ten glucosyl units in maize endosperm (Commuri and Keeling, 2001). SSI synthesizes shorter glucan chains with the degree of polymerization (DP) less or equal to 10 (DP≤10) in transient starch synthesis in leaves (Delvalle et al.

2005). The soluble SSI in maize is 76kDa in size (Mu et al. 1994). The degree of association of SSI in the starch granule is significant representing 85% of total

SSI content in maize endosperm (Mu-Forster et al. 1996). Further, the affinity of

SSI for amylopectin (Kd= 0.2 mg/mL) was higher compared to starch (Kd= 0.49 mg/mL), glycogen (Kd= 1.0 mg/mL) and amylose (Kd= 0.6 mg/mL) (Commuri and Keeling, 2001).

The amino acid sequence of SSI in maize shares 75.7% sequence identity to rice SSI (Knight et al. 1998). In japonica rice lacking SSII (Nakamura et al.

2005), SSI accounts 70% of the total SSs activity (Fujita et al. 2006). However, the relative contribution of SS isoforms is different in different species (e.g.

SSIII contributes >70% of total SS activity in potato) (Abel et al. 1996). The accumulation of SSI total transcripts was higher at 5–10 days-post-anthesis

(DPA) than at 15–25 DPA in developing wheat endosperm (Peng et al. 2001).

During the endosperm development, the relative abundance of SSI did not vary in starch granules, whereas, SSI concentration in the endosperm soluble fractions was highest from 10-15 DPA, and below detection levels at 5 DPA. The

16 wheat endosperm SSI further exhibited similar concentration per kernel from

15-25 DPA in endosperm soluble fractions, but at considerably higher concentrations in starch granules as compared to endosperm soluble fractions

(Peng et al. 2001). SSI from japonica rice produces chains with a degree of polymerization (DP) 8-12 from short and DP 6-7 chains emerging from the branch point in the A and B1 chains of the amylopectin (Fujita et al. 2006).

Further, SSI mutant, showed decreased number of DP 8-12 glucan chains and increased number of both DP 6-7 and DP 16-19 chains in endosperm amylopectin in japonica rice (Fujita et al. 2006). However, SSI mutants in transgenic potato plants displayed no visible phenotypic changes in starch structure (Kossman et al. 1999). The overlapping function of SSI and SSIII were revealed by creating double-recessive homozygous mutants from SSI null mutants with SSIII null mutants in japonica rice (Fujita et al. 2011). The seeds from these mutants remained sterile and the heterozygous mutants produced fertile opaque seeds further confirmed that SSI or SSIII is required for starch biosynthesis in rice (Fujita et al. 2011).

1.1.2.1.2.3. Starch synthase II (SSII)

Two SSII isoforms are present (SSIIa and SSIIb) in higher plants. SSIIa predominates in cereal endosperm, while SSIIb is mostly confined to vegetative and photosynthetic tissues (Morell et al. 2003). SSII is also partitioned in both the starch granule bound protein fraction and in the soluble protein fraction in the plastid (Li et al. 1999). SSIIa mRNA level showed a higher accumulation during the period of starch accumulation in developing maize endosperm (Harn

17 et al. 1998). SSIIa plays a role in medium chain length extension and appears to be involved in elongating glucan chains produced by SSI leading to the production of medium length chains of DP=12-25 (Zhang et al. 2004; Morell et al. 2003). The sex6 mutant of barley lacking SSII activity in the endosperm has a shrunken endosperm phenotype and reduced starch content due to substantial decrease in amylopectin content. However, the amylose content was increased up to 71% and 62.5% compared with 25% in the wild-type (Morell et al. 2003).

Moreover, these mutants have altered chain-length distribution, whereas the amount of shorter glucan chains (DP= 6-11) increased from 24.15% (in wild- type) to 38.18% and 38.96% and the medium length glucan chains (DP= 12-

30) decreased from 69.12% (in wild-type) to 54.14% and 53.42% in M292 and

M342 mutants respectively (Morell et al. 2003). Interestingly, the lack of SSII causes a reduction in the levels of the branching enzymes SBEIIa, SBEIIb, and

SSI in the starch granule, but not the amount of these enzymes in the soluble fraction in barley amyloplasts (Morell et al. 2003). This suggests that either SSII mutation prevents binding of these proteins to the starch granules, or they are prevented from forming protein complexes in the amyloplast stroma and get trapped in the granule (Morell et al. 2003). The effects on chain length distribution of ss2 mutants observed in barley are similar to sugary2 (su-2) mutants of maize endosperm lacking SSIIa (Zhang et al. 2004) indicating a common function for SSII in starch granule assembly. The su-2 mutants exhibit a significant increase in DP= 6-11 shorter chains and a decrease in DP=13-20 medium length chains (Zhang et al. 2004). In the Arabidopsis thaliana mutant ss2 (Atss2), the growth rate or the starch quantity were not affected, but

18 increased the amylose/amylopectin ratio, increased total amylose (43% of total amylose), and DP=12-28 medium length glucan chains were significantly decreased as similar as in the endosperms of many cereals (Zhang et al. 2008).

In addition, the total SSs activity was recorded as 212 ± 8.7 nmol product/min/mg proteins in wild-type leaf extract, was increased up to 237 ±

8.7 in Atss2 (Zhang et al. 2008). These results suggested that the loss of SSII activity can be restored by any other conserved SS, specifically SSI, GBSSI, or

SSIII or SSIV in transient starch biosynthesis (Zhang et al. 2008).

1.1.2.1.2.4. Starch Synthase III (SSIII)

The SSIII was found as 139.2 kDa size in potato (Abel et al. 1996) and >200 kDa in maize endosperm (Cao et al. 1999) and is expressed throughout the developmental stages of these plants. The calculated molecular masses of the isoforms found in rice endosperm (OsSSIII-1) and leaves (OsSSIII-2) were 138 kDa and 201 kDa respectively (Dian et al. 2005).

SSIII is coded by the DUI gene in maize endosperm (Cao et al. 1999).

The du1 mutations alter starch structure indicates that DU1 provides a specific function(s) that cannot be compensated for by the remaining soluble SS activity

(Abel et al. 1996). The ss3 mutant showed a significant decrease in total SS activity by 13-29% compared to 100% of SS activity in the wild-type, without any significant effect on the plant phenotype, amylose content or the tuber yield in potato (Abel et al. 1996). The granule morphology was changed in ss3 single mutants producing small granule structures (Abel et al. 1996).

SSIII elongates comparatively longer glucan chains than SSII, producing

DP= 25-40 or greater glucan chains in amylopectin (Tomlinson and Denyer,

2003; Edwards et al. 1999; Abel et al. 1996). The frequency distribution of the linear glucan chain in ss2 and ss3 single mutant lines showed strong enrichments at DP= 6-9, and ss2/ss3 double mutant lines showed strong enrichments at both DP= 7–8 and DP= 12–13 (Edwards et al. 2002). The frequency distribution of the linear glucan chain was determined in transient starch from Arabidopsis thaliana Atss3-1, Atss3-2 mutant lines (Zhang et al.

2005). The frequency of shorter chains of DP= 5-10 and DP= 26-37 are respectively increased but, chains of DP= 14-20 and DP= 43-46 are respectively decreased suggesting that SSIII is involved in producing comparatively longer glucan chains compared with SSI and SSII (Zhang et al. 2005; Edwards et al.

2002). SSIII appears to be a vital enzyme in transient starch biosynthesis; starch granule initiation requires the presence of either SSIII or SSIV in

Arabidopsis leaves (Szydlowski et al. 2009).

1.1.2.1.2.5. Starch synthase IV (SSIV)

SSIV is exclusively present in the stroma of the plastids (Leterrier et al. 2008;

Roldan et al. 2007). The role of SSIV in chain length distribution is not clear, but it may play a selective role in priming starch granule formation (Roldan et al.

2007). SSIII and SSIV in rice have two isoforms in each enzyme. OsSSIII1,

OsSSIII2, and OsSSIV1, OsSSIV2 (Dian et al. 2005). The SSIII2 and SSIV1 genes are mainly expressed in rice endosperm, whereas the other two isoforms were expressed mainly in leaves. The cDNA sequence of wheat SSIV

20 preferentially expressed in leaves is most similar to rice SSIV2, which share a similar exon-intron arrangement (Leterrier et al. 2008) suggesting that the SSIV present in leaves and endosperms may have slight variation in amino acid sequences (e.g. as similarly observed in SBEIIa and SBEIIb).

The SSIV protein in Arabidopsis thaliana is 112.99 kDa in size (Roldan et al. 2007); showing 71%, 58.2%, 56.8% and 58.3% sequence identity to Vigna unguiculata (accession number AJ006752), wheat (accession number

AY044844), rice (SSIVa, accession number AY373257) and rice (SSIVb, accession number AY373258) respectively (Roldan et al. 2007). Two independent mutant alleles of SSIV in Arabidopsis thaliana, Atss4-1 [from

Columbia-0 (Col-0) ecotype] and Atss4-2 [from Wassilewskija (WS) ecotype] showed no decrease in total soluble SS activity but lower growth rates were recorded in the mutant plants grown under a 16-h day/8-h night photo period; approximately as 100 mg/FW (fresh weight) and 50 mg/FW of the plant compared with their respective wild types which conserved 550 mg/FW and 275 mg/FW of growth rates (Roldan et al. 2007). However, the fruit size, number of seeds per silique and germination ratios were not altered in the mutant lines but, the starch contents in the leaves were significantly reduced in both mutants by 35% for the Atss4-1 and 40% for the Atss4-2 line with respect to their wild types at the end of the illuminated period. Although, the total SS activity was not affected, the total activity of both cytosolic and plastidial forms of starch phosphorylase (SP) was increased by 1.4–2-fold in both Atss4-1 and Atss4-2 mutants, which may be due to a metabolic alteration that triggers the induction

21 of SP isoform gene expressions in ss4 mutants (Roldan et al. 2007). The exact reason for the increase of SP in ss4 mutant is not known.

In Arabidopsis thaliana leaves, amylose/amylopectin ratio was not changed in Atss4 mutants (Roldan et al. 2007). The chain length distribution pattern was determined in Atss4 mutants and their respective wild types and indicated that the Atss4 mutation had minor effects on the structure of amylopectin and only a slight reduction in the number of shorter chains of DP=

7–10 were observed. The microscopic analysis of starch granules collected at 4 and 12 h during the light phase showed a greater difference in size between Col-

0 and Atss4-1 starch granules, the surface area was increased by 10 times at the end of the day in Atss4-1 mutant plants. A single starch granule was contained in the mutant, whereas in wild-type showed 4–5 starch granules per chloroplast concluding that the mutation at the AtSS4 locus affects both the number and size of starch granules synthesized in the chloroplast. These observations further suggested that the SSIV may be involved in the priming of the starch granule (Roldan et al. 2007). However, this hypothesis was not yet confirmed in any plant species. The essential mechanism of starch granule initiation is largely unknown.

SSIV in wheat endosperm was found as 103.1 kDa protein which is 87% homologous to the OsSSIVb in rice endosperm [Genbank: AAQ82623] (Leterrier et al. 2008). Like all other SSs, the N-terminus of wheat SSIV is unique; the

SSIV-specific region from amino acids 1–405 contains two coiled-coil domains and a 14-3-3-protein recognition site (Leterrier et al. 2008). The coiled-coiled domains are commonly involved in crucial interactions such as transcriptional

22 control (Mason et al. 2004) and 14-3-3 proteins are commonly linked to binding with various signaling proteins such as kinases and phosphatases (Comparot et al. 2003). The mRNA expression of SSIV was highest in non-endosperm tissues such as in leaf, embryo and roots in wheat and the level of expression in the endosperm was comparatively lower and the expression was independent from the regulation of the circadian clock. Therefore, the transcript accumulation levels did not coincide with the period of high carbon flux to starch in the wheat endosperm (Leterrier et al. 2008).

To elucidate the function of SSIV in the priming process of starch granule formation, different combinations of homologous double SSs mutations in ss4 mutant backgrounds were developed in Arabidopsis thaliana; ss1/ss4, ss2/ss4 and ss3/ss4 (Szydlowski et al. 2009). Decreased levels of starch accumulated in ss1/ss4 and ss2/ss4 double mutants were equal with the sum of the decreases starch levels in their respective single mutant lines. At the end of 12h light period, the starch accumulation in the respective ss3 and ss4 single mutants were recorded as 122% (Zhang et al. 2005) and 62% (Rolden et al. 2007) respectively compared to their wild types. However, the ss3/ss4 double mutant did not accumulate any measurable amounts of starch, despite the dark or light conditions (Szydlowski et al. 2009). Therefore, the presence of either SSIII or

SSIV is a crucial requirement in transient starch biosynthesis (Szydlowski et al.

2009). Further, although the ss3/ss4 double mutants did not affect on other starch metabolism enzymes (such as phosphoglucomutase, AGPase, and starch branching enzymes (SBE), they showed a significantly increased SP activity

(more than 8 fold in wild-type) (Szydlowski et al. 2009), which may be due to

23 an alternative route of starch biosynthesis available using hexose phosphates via a starch phosphorylase (SP)-mediated pathway (Fettke et al. 2010) (see the section 1.1.2.1.6. for the details).

SSIV is a newly identified SS isoform existing in the plastids. However, the exact function of SSIV in storage starch biosynthesis has yet to be identified.

The expression of SSIV during the endosperm development is high at the later stage of the grain filling (Dian et al. 2005). The coordination and interactions of the various enzyme classes are explained through the operation of protein- protein interactions (see the section 1.1.2.2.). Chapter 4 of this thesis present an investigation of the catalytic activity and regulation of SSIV by protein- protein interaction with other starch biosynthetic enzymes in maize amyloplasts.

1.1.2.1.3. Starch branching enzyme (SBEs, EC 2.4.1.18)

The branching structural arrangement of amylopectin is generated by starch branching enzymes (SBEs). These enzymes generate the -(16) linkages through cleavage of internal -(14) glycosidic bonds. The reducing ends are then transferred to the C6 hydroxyls forming a new branch point. In common with the other classes of starch biosynthetic enzymes, SBEs have multiple isoforms (e.g. SBEI, SBEIIa, and SBEIIb), some of which are plant, tissue and/or developmental specific in their expression patterns (Regina et al.

2005; Gao et al. 1997).

1.1.2.1.3.1. Starch branching enzyme I (SBEI)

SBEI and the SBEII’s differ in the length of the glucan chain they transfer in vitro and show different substrate specificities. SBEI exhibits a higher rate of branching with amylose and transfers longer chains compared to SBEII, which has a higher affinity towards amylopectin (Guan and Preiss, 1993: Takeda et al.

1993). The amylopectin chain-length profile of the maize SBEI mutant (sbe1) was not affected compared with wild-type (Blauth et al. 2002). However, SBEI has a lower Km for amylose and tends to produce shorter constituent chains compared to SBEIIa or SBEIIb when reacted with amylose in vitro (Gao et al.

1996). In maize, SBEI is expressed moderately during middle stages of kernel development (12–20 DAA), strongly during the later stages of kernel development (22–43 DAA) and is moderately expressed in vegetative tissues

(Kim et al. 1998). When compared to the total SBE activity in mutants of SBEI,

SBEIIa and SBEIIb in maize, a loss of enzyme activity have been identified for only SBEIIa and SBEIIb (Blauth et al. 2002) showed that the lack of SBEI was compensated by other two SBE isoforms. Alternatively, SBEI does not have a significant role in determining starch quantity or quality in leaves or endosperm

(Blauth et al. 2002). SBEI is highly conserved in plants and has been shown to interact with other starch biosynthetic enzymes (Liu et al. 2009; Tetlow et al.

2004) indicating that SBE plays some function in regulating the starch biosynthetic process.

1.1.2.1.3.2. Starch branching enzyme II (SBEII)

In monocots, two SBEII gene products (SBEIIa and SBEIIb) are closely related

(Rahman et al. 2001). However, in wheat endosperm their expression patterns are considerably different where SBEIIa is expressed at a higher level than

SBEIIb (Regina et al. 2005). SBEII mutations show a more pronounced phenotypic change compared to SBEI. A mutation of the gene encoding SBEIIb in maize produces a high-amylose starch phenotype, known as the amylose extender (ae-) (Banks et al. 1974; Yu et al. 1998). Mutations in SBEIIb in maize

(ae mutant) produce resistant starch genotype which characteristically produces less branched and longer glucan chains in amylopectin (Nishi et al. 2001;

Klucinec and Thompson, 2002).

Phenotypic changes in SBEIIa mutations are dependent on the source of starch. In maize there was a visible change in leaf starch in SBEIIa mutants, however no significant changes occurred in storage starches of maize kernels

(Blauth et al. 2001). The catalytic activity of SBEIIa and SBEIIb is regulated by protein phosphorylation in wheat endosperm (Tetlow et al. 2004) and show a high expression of SBEIIa compared to SBEIIa in developinf wheat endosperm

(Morell et al. 1997; Regina et al. 2005). In contrast, in maize endosperm,

SBEIIb is the predominant form, being expressed at approximately 50 times the level of the SBEIIa form (Gao et al. 1997); it is the most abundant protein in the maize endosperm amylopast stroma (Mu et al. 2001).

1.1.2.1.4. Starch de-branching enzyme (DBE, EC 3.2.1.41 and EC

3.2.1.68)

Starch debranching enzymes play an important role in the development of crystalline amylopectin. There are two types of DBEs. The isoamylase-type (ISO) hydrolyzes -(16) linkages in amylopectin and pullulanase-type (PUL) hydrolyzes -(16) linkages in amylopectin and pullulan, a fungal polymer of malto-triose. There are three isoamylase-type DBE isoforms (ISO1, ISO2, and

ISO3). Rice and maize mutants lacking ISO1 (sugary1) demonstrate an increase in the disordered water-soluble, highly and randomly branched polysaccharide called phytoglycogen (Nakamura, 2002, James et al. 1995). Although the respective substrates of isoamylase and pullulanase type DBEs are known, their specific roles in starch biosynthesis are not clear. However, there are two existing models for their function. The glucan trimming model proposes that

DBEs remove any branches that would inhibit crystallization of the developing granule (Ball et al. 1996: Myers et al. 2000). Another model suggests that DBEs clear away any soluble glucan not attached to the granule (Zeeman et al. 1998).

The theory is based on the concept that SS’s and SBE’s will continue to synthesize glucan polymers if sufficient substrate is present, therefore causing phytoglycogen accumulation. Although the latter model would explain the increase of phytoglycogen in DBE mutants, it is possible these models are not mutually exclusive.

ISO1 and ISO2 form a hetero-oligomeric complex to form a functional enzyme (Hussain et al. 2003). This complex is approximately 400 kDa in size, and is also found with a 300 kDa complex containing ISO1, but not ISO2 in

27 maize. Loss of ISO1 prevents formation of the complexes, indicating that ISO1 is required for the complex assembly (Kubo et al. 2010). ISO3 thought to be involved in starch degradation (Dinges et al. 2003). In Arabidopsis leaves, ISO3 is catalytically active on water-soluble polysaccharides that have been produced by β-amylase and starch phosphorylase (Wattebled et al. 2005).

1.1.2.1.5. Disproportionating enzyme (D-enzyme, E, C. 2.4.1.25)

D-enzyme catalyzes the hydrolysis of -(14) linkages of unbranched malto-oligosacharides and subsequent transfer of the glucan released at the non-reducing end to a non-reducing end of the acceptor molecule to form a new

-(14) linkage. D-enzyme mutation in Arabidopsis show reduced rates of nocturnal starch degradation indicating that D-enzyme plays a part in the pathway of chloroplast starch degradation (Critchley et al. 2001). Some research evidence suggested that the D-enzymes work in conjunction with SP, contributing to starch synthesis via the phosphorolytic SP reaction (Takaha et al.

1998). According to this model, the short-chain MOS liberated in the trimming reaction by DBEs are converted to longer-chain glucans by D-enzyme, which are the substrates for phosphorolysis by SP, liberating G-1-P used to synthesize

ADP-glucose by plastidial AGPase (Takaha et al. 1998). In addition, in

Chlamydomonas reinhardtii, the phosphorolytic SP reaction is stimulated by the presence of D-enzyme (Colleoni et al. 1999).

1.1.2.1.6. Starch phosphorylase (SP, EC 2.4.1.1)

Starch phosphorylase, exists in both tetrameric and dimeric states and catalyses the reversible transfer of glucosyl units from glucose-1-phosphate (G-

1-P) to the non-reducing end of α-1-4 linked glucan chains as shown in the following equation.

G-1-P + (1,4-α-D-Glucose)n (1,4-α-D-Glucose)n+1 + Pi

1.1.2.1.6.1. Importance of SP in starch metabolism

SP has often been regarded as a glucan degradative enzyme (Preiss,

1982; Preiss, 1984). The α-glucan phosphorylase (EC 2.4.1.1) found in animals, fungi, and prokaryotes plays a major role in glucan catabolism (Preiss, 1984) and the amino acid sequence of the enzyme is found to be highly conserved among prokaryotes and eukaryotes (Newgard et al. 1989). Genetic analyses in

Chlamydomonas showed that the mutation of plastidial SP affected starch accumulation (Dauvillée et al. 2006). In addition, the mutation of plastidial α- glucan phosphorylase could not change the total accumulation of starch or the starch structure during the day or its remobilization at night when the phosphorylase gene activity was eliminated by T-DNA insertion in Arabidopsis thaliana leaves, where transient starch is synthesized (Zeeman et al. 2004). In contrast, research evidence demonstrated that the SP has a certain effect on the storage starch biosynthesis, that the development of plastidial SP activity coincides with starch accumulation in developing cereal endosperms; in rice

(Satoh et al. 2008), in wheat (Schupp and Ziegler, 2004; Tickle et al. 2009) and

29 in maize (Yu et al. 2001). Above evidence further suggests that the plastidial forms of SP are involved in starch synthesis rather than the degradation in higher plants.

1.1.2.1.6.2. The isoforms of SP in higher plants

Two major isoforms of SP are present in plants and differ in their intracellular localization, and are designated as plastidic (Pho1) and cytosolic

(Pho2) isoforms (Nakano and Fukui 1986). In developing rice endosperm, plastidial Pho1 accounts for about 96% of the total phosphorylase activity and it is restricted to the stroma (Satoh et al. 2008). The predicted protein sequence alignment of Pho1 and Pho2 isoforms show a significant 50 amino acid extension in the N-terminus of Pho1, which represent the transit peptide (Nakano and

Fukui 1986). In this thesis the term SP is generally used for the plastidial form.

The plastidial form of SP (112 kDa in maize; Mu et al. 2001) is known to be the second most abundant protein in the maize amyloplast stroma next to

SBEIIb (Yu et al. 2001). Peptide sequences of plastidial SP in maize showed higher identities to potato, sweet potato and spinach and the N-terminus sequence was unique in maize amyloplast; it can not be aligned with any other

N-terminus sequences of Pho1 available in the gene bank (Yu et al. 2001).

Excluding the N-terminus difference between Pho1 and Pho2, a unique 78-amino acid insertion in the middle of the Pho1 sequence is a prominent characteristic of the plastidial isoform in higher plants (Yu et al. 2001). In potato, Pho1 and Pho2 showed 81% - 84% amino acid sequence similarity over most part of the sequence with the exception of N-terminal transit peptide and the large L-78

30 insertion located between the N and C terminal domains (Albrecht et al. 1998).

Significant variation is found in the molecular mass of the Pho1 and Pho2 in wheat endosperm as 100 kDa and 90 kDa respectively (Albrecht et al. 1998).

The peptide sequence ILDNADLPASVAELFVK is a common sequence fragment found in the L-78 region in maize and potato (Yu et al. 2001, Albrecht et al.

1998). In addition, the sequence comparison among SP from potato tuber, rabbit muscle, and Escherichia coli revealed the presence of the characteristic

78-residue insertion only in the middle of the polypeptide chain of the potato enzyme (Nakano and Fukui, 1986) (Fig. 3.3 in Chapter 3) suggesting the L-78 region is specific to plants. The proposed function of the L-78 insertion is, thought to be the obstruction of the binding of Pho1 to large, highly branched polysaccharides (Albrecht et al. 1998). This idea was further confirmed by the observation that the L-78 insertion in sweet potato (Ipomea batatas) blocked the starch-binding site in Pho1 molecule showing low affinity towards starch

(Young et al. 2006). Several serine phosphorylation sites were also found in the

L-78 insertion suggested that the regulation of Pho1 is phosphorylation dependent (Young et al. 2006). This research group was able to purify a 338 kDa protein kinase activity from sweet potato roots using liquid chromatography methods and which actively phosphorylates the L-78 insertion (Young et al.

2006). Interestingly, this phosphorylation modification was not found in Pho2 isoform or after L-78 insertion was proteolytically removed from Pho1 (Young et al. 2006).

1.1.2.1.6.3. Characterization of SP

All phosphorylases exist as dimers or tetramers of identical subunits, and have similar kinetic and structural properties, but their regulatory mechanisms may vary depending on the source of the enzyme (Dauvillée et al. 2006;

Weinhäusel et al. 1997; Brisson et al. 1989) or its multimeric state (see later).

The α-glucan phosphorylase found in bacterial forms has a homodimeric molecular structure (Dauvillée et al. 2006; Weinhäusel et al. 1997). Gel filtration chromatography studies revealed that the native enzyme consisted of two identical subunits in maize (Mu et al. 2001) which coincides with findings of

Tanabe et al. (1987) on availability of dimeric form (203 kDa) of α-glycogen phopsphorylase in yeast. The purified form of SP from maize endosperm was thermally labile above 50°C where optimum enzyme activity is at pH 6.0 in the synthetic direction and pH 5.5 in the phosphorolytic or degradative direction at

40°C (Mu et al. 2001).

1.1.2.1.6.4. Biochemical characterization of SP

According to their affinities for glucan substrates, SPs are further classified as low affinity (SP-L) and high affinity (SP-H) isoforms respectively in potato tuber and leaf (Mori et al. 1993). When the L-78 insertion in SP-L was replaced by high affinity SP-H sequence, the SP-L showed less affinity to glycogen compared to SP-H form (Km=10,400 and Km=10 μg/mL) (Mori et al.

1993). The L-78 insertion-replaced chimeric enzyme was five times less active than the SP-L isoform but still showed low affinity to glycogen than in SP-L

(Km= 24 μg/mL). However, when the glycogen was replaced by amylopectin

32 and amylose (DP=30), the affinity increased in SP-L (Km= 82 and Km=76

μg/mL respectively), in SP-H form (Km=3.6 and Km=8.7 μg/mL respectively), and in chimeric form (Km=5.3 and Km=2 μg/ml respectively). Among all the isoforms, the SP-H form has the highest affinity to amylopectin, suggesting that the L-78 region has greater affinity towards low molecular weight substrates

(Mori et al. 1993). In addition, two isoforms named Pho1a and Pho1b were identified in potato (Sonnewald et al. 1995). The homodimeric form of Pho1a isoform was immunochemically detectable only in tuber extracts where both

Pho1a and heterodimeric Pho1b were present in leaf extracts in potato (Albrecht et al. 1998). Wheat has three forms of SP (designated as P1, P2, P3) which are distinguished in non-denaturing separation gels containing glycogen (Schupp,

2004). The activity form P3 is plastidic in where as P1 and P2 are cytosolic and found mainly in younger leaves (Schupp, 2004). However, mature leaves only contain the plastidic form which was also strongly evident in the endosperm of the developing seeds. Cytosolic forms are more prominent in germinating seeds

(Schupp, 2004) suggestive of the involvement of cytosolic SP forms in the utilization of α-glucans resulting from starch degradation.

The plastidial and cytosolic SP show different affinity towards high and low molecular glucan polymers in synthetic direction (Table 1.1). Plastidial SP prefers amylopectin than the glycogen potato tuber (Liddle et al. 1961), spinach leaf (Shimomura et al. 1982), and sweet corn (Lee and Braun, 1973) and maize

(Yu et al. 2001). In maize endosperm, the Km value for amylopectin in the synthetic direction of the SP reaction was 3.4-fold lower and the Kd value was

40-fold lower than of glycogen (Yu et al. 2001). The kinetic analysis indicated

33 that the Km value for amylopectin was eight-fold lower than that of glycogen and the phosphorolytic reaction was favored over the synthetic reaction when malto-oligosaccharides (DP= 4 to 7 units) were used as substrates (Mu et al.

2001).

Table 1.1: The Km and Vmax values of starch phosphorylase in different plant species. SP-L =plastidial form of SP, SP-H= cytosolic form of SP, (s) = synthetic direction, (p) = phosphorolytic direction Vmax Km Reference Plant Substrate (umol/min/mg) (mg/ml) Tissue Purified SP Amylopectin 0.58 (s) 0.13 (s) Yu et al. from maize Glycogen 0.63 (s) 0.45 (s) 2001 amyloplast stroma Purified SP Mu et al. from maize Amylopectin 73 (s), 111 (p) 0.017 (s), 0.028(p) 2001 amyloplast Glycogen 71.6 (s), 118.0(p) 0.25 (s), 0.94(p) stroma Maltoheptaose 78 (s), 199.3 (p) 0.08 (s), 0.1 (p)

Sweet potato Starch 0.077 (s) Young et al. tuber crude G-1-P 0.115 (p) 2006 extract Pi 1.052 (s), 1.498(p) Potato tubers Maltopentaose Mori et al. Recombinant SP- L type 39.6 (s), 16.5 (p) 0.13 (s) 1993 proteins of SP- H type 96.1 (s), 36.8 (p) 1.12 (s) SP-L and SP- H types Glycogen SP- L 8.3 (p) 10400 (p) SP- H 9.4 (p) 9.8 (p)

Amylopectin 7.9 (p) 82 (p) SP- L 8.3 (p) 3.6 (p) SP- H

Amylose DP=30 13.9 (P) 7.6 (P) SP- L 18.2 (P) 8.7 (P) SP- H

ADP-glucose, the major precursor for starch biosynthesis has been known for long time as an inhibitor of activity of SP in the synthetic direction (Matheson and Richardson, 1978). ADP-glucose (at 4 mM) reduced the synthetic activity of plastidial SP and G-1-P (at 10 mM) reduced the activity of cytosolic SP by 18% to 22% respectively in pea seeds (Matheson and Richardson, 1978). Low concentration of G-1-P and high Pi/G-1-P ratio increase the degradation activity by glycogen phosphorylase in vivo (Schupp and Ziegler, 2004; Newgard et al.

1989) suggesting SP degradative activity is increased by inorganic phosphate

(Pi). In addition, in developing barley endosperm, cytosolic Pi concentration was very higher (over 23 folds) than G-1-P where cytosolic form of SP required higher level of Pi (Tiessen et al. 2011). However, according to the findings of

Hwang et al. 2010, incorporation of [14C]-G-1-P into starch was only partially affected by Pi. Even under physiological G-1-P substrate levels (0.2 mM), plastidial SP from rice was still able to carry out the biosynthetic reaction, although at low rates, in the presence of 50-fold excess of Pi in vitro. Hence, under conditions that would favor the degradation of starch, plastidial SP preferentially carries out biosynthesis.

The animal orthologue of SP, glycogen phosphorylase, consists of two identical subunits, each of which have a highly conserved C-terminal region incorporating a pyridoxal phosphate molecule which is essential for activity and a site effecting non-catalytic glucan binding (Newgard et al. 1989). The activities of animal glycogen phosphorylases in releasing glucose for dissimilative metabolism are highly regulated by allosteric effectors and covalent modifications (Johnson, 1992; Newgard et al. 1989). All known α-glucan

35 phosphorylases require pyridoxal 5-phosphate for activity as a cofactor (Yanase et al. 2006). The maize shrunken-4 mutant is found to be lacking SP activity in the endosperm and the mutants had reduced the starch content and the soluble protein content by two-third than in the wild type kernel (Tsai and Nelson,

1969). The activities AGPase and SS, are also reduced in the shrunken-4 mutant while reducing the total amount of pyridoxal-5-phosphate in the endosperm by

8-fold than in the wild type endosperm (Tsai and Nelson, 1969). This reduction was identified as the lack of SP cofactor pyridoxal-5-phosphate in the shrunken-

4 mutant in the maize (Tsai and Nelson, 1969). However, external addition of pyridoxal-5-phosphate to the assay system for SP activity of the mutant did not affect the activity (Yu et al. 2001). Thioreactive agents, such as diethyl pyrocarbonate, phenylglyoxal have also been identified as some of the chemical inhibitors of SP (Mu et al. 2001).

The pho1 mutants developed in rice endosperm have helped to elucidate the in vitro role of SP on the other major starch biosynthetic enzyme isoforms

(Satoh et al. 2008). Induced mutagenesis of SP by N-methyl-nitrosourea treatment led to the creation of a series of mutants with a considerable reduction in starch contents from the seed morphologies varies from white-core, pseudonormal to shrunken in rice particularly at different temperatures (varied from 20oC to 30oC) (Satoh et al. 2008). The white-core phenotypes made approximately 18 and 20 mg of grain weight, in pseudonormal, approximately

18 and 19 mg and in shrunken made 10 and 8 mg of grain weight, where the wild type approximately made 22 mg both at 30oC and 20oC temperatures respectively. Scanning electron microscopy showed that the sizes of the starch

36 granules were decreased (shrunken phenotype had the smallest granules than in the wild type) in the mutant lines and some granules were more spherical than the irregular polyhedron-shaped granules typical of wild-type starch grains.

High-resolution capillary electrophoresis technique was used to measure the chain length distribution of the amylopectin in the endosperm. The mutants created a higher proportion of DP=11 shorter glucan chains with a decrease in the proportion of intermediate chains with a DP= 13-21. Even though, the seed weight was varied within the white-core, pseudonormal and shrunken phenotypes of the mutants, they have demonstrated a similar change in chain length distribution in the amylopectin. In contrast, this study also showed that the Pho1 mutants did not have any effects on the measurable activity levels of the other major starch biosynthetic enzymes such as AGPase, DBE isozymes

(isoamylase and pullulanase), SBE isoforms (SBEI, SBEIIa, and SBEIIb), and SS isoforms (SSI and SSIIIa) (Satoh et al. 2008). Based on these results, the authors suggested that the SP could operate at two distinct phases of starch biosynthesis; one phase consisting of starch initiation and a second phase is in starch elongation (Satoh et al. 2008). The in vitro analysis of chain length elongation properties of recombinant SP and SSIIa from rice were compared on

MOS of DP=4, DP=6, or DP=7 glucan primers. Despite the type of primer used in the reaction, the two enzymes showed different product distributions to each other (Satoh et al. 2008). SP produced a broad distribution of MOS products of increasing size mostly DP= 6-11, SSIIa showed a much narrower distribution

(DP= 6-7) of MOS products. The results clearly indicated that SP can synthesize much longer linear glucans (DP= 16) than SSIIa (DP= 7-9) (Satoh et al. 2008).

In addition, the catalytic activity of SP from rice is significantly higher (75 mmoles G-1-P/mg protein/min) toward MOS than SSIIa is (24 nmoles

ADPglucose/mg protein/min). Therefore, these results support a role for SP in extending small MOS, whereas rice SSIIa is unlikely to be involved in this process. The authors further suggested that these longer linear glucan chains which are produced by SP could presumably be the linear substrates for SBE to form branched glucans in the starch initiation process (Satoh et al. 2008).

Functional interactions between SP and SBE isoforms were observed in vitro, and showed that purified SP from rice endosperm synthesized glucans from G-1-P in the presence of different isoforms of SBE even without any exogenous glucan primer (Nakamura et al. 2012). Glucan production was higher by SP when SBEI was present, compared to SBEIIa or SBEIIb and produced glucan polymers with DP =11, 7 and 6 respectively (Nakamura et al. 2012).

Activities of SP and SBE were depended on the mutual availability SP and SBE and showed mutual capacities for chain elongation and chain branching

(Nakamura et al. 2012).

The isoforms of the major enzymes involved in starch biosynthesis are regulated by protein phosphorylation, and protein-protein interactions (Liu et al.

2009; Hennen-Bierwagen et al. 2008; Tetlow et al. 2008; Tetlow et al. 2004).

Plastidial SP in wheat endosperm is also involved in formation of active protein complexes with the SBEI and SBEIIb particularly in wheat amyloplast stroma in a phosphorylation-dependent manner (Tetlow et al. 2004). Novel complexes of starch synthesis enzymes assembled in the amylose extender (ae-) mutant

(lacking SBEIIb) of maize (Liu et al. 2009). The complex formed by SSI, SSII

38 with SBEIIb in wild-type was replaced by forming SBE1 combined with SP in the ae- mutant (Liu et al. 2009). Genetic analyses further revealed that the loss of

SBEIIb in ae mutant could cause a significant increase in the SBEI, SBEIIa,

SSIII, and SP in the starch granule (Liu et al. 2009; Grimaud et al. 2008).

1.1.2.1.6.5. SP and starch biosynthesis models

Based on recent genetic and biochemical evidence, some researchers suggested that SP may play a role in the initiation of starch biosynthesis (Satoh et al. 2008; Leterrier et al. 2008; Roldan et al. 2007). Tickle et al. (2009) recently suggested a model in which SP plays a role in starch synthesis via two pathways. First, SP degrades the soluble malto-oligosaccharides (MOS) which are made from starch via the action of DBE, into G-1-P in the amyloplast stroma. This G-1-P can then be converted to ADP-glucose by AGPase and to recycled back into starch. The second mechanism suggests that SP can directly act on the surface of the starch granule; where it could phosphorolytically modify the structure of starch to produce G-1-P (Tickle et al. 2009). Recent mutant analysis in Arabidopsis suggests plastidial SP is not required in starch degradation in chloroplasts (Zeeman et al. 2004). The leaves of mature SP mutant plants had small, white lesions on the tips or margins of fully expanded leaves. It was suggested that SP may play a role in creating tolerance to abiotic stresses in leaves by providing an alternate route for starch degradation

(Zeeman et al. 2004).

The existence of a complementary path of forming reserve starch was discussed in potato by analyzing the effect of the G-1-P-dependent intracellular carbon flux (Fettke et al. 2010). The tuber discs of wild-type and various transgenic potato lines expressing an antisense construct directed against the plastidial SP isofoms were incubated with 14C-lablled G-1-P, G-6-P, sucrose, and maltose. Highest amount of starch was measured in G-1-P substrate compared to G-6-P, sucrose, and maltose, indicating that the path of starch biosynthesis is functional that is selectively initiated by the uptake of the anomeric glucose phosphate ester (Fettke et al. 2010). The initiation of this path is separated against external glucose 6-phosphate. Rice SP mutants grown at 300C produced about 6% of the shrunken phenotypes (compared to 100% in wild-type), the starch content was similar in the wild-type and the percentages of shrunken phenotype was increased in SP mutant plants when the temperature was decreased to 250C and 200C by 35-39% and 66% respectively with a severe reduction in starch accumulation. It was suggested that SP may play an important role in starch biosynthesis during fluctuating and/or adverse temperature conditions in rice (Satoh et al. 2008).

1.1.2.1.6.6. Evidence of interaction of SP with SSIV

Research evidence suggested potential interactions between the SP and

SSIV enzymes. In Arabidopsis thaliana leaves, the activity of SP increased in ss4 mutants by 1.4 -2 fold compared to the wild-type without changing starch structure or the amylose/amylopectin ratio and the concentrations of soluble sugars remain unchanged (Roldan et al. 2007). However, granule size was

40 increased in ss4 mutants with a reduction in the granule number to 2-3 granules per amyloplast compared to the 4-5 granules in wild-type (Roldan et al. 2007).

Interestingly, the double mutant of ss4 and sp produced granule size of at least

4 times higher than starch granules originating from the wild-type plants

(Planchot et al. 2008).

1.1.2.2. Post translational modification of starch biosynthesis enzymes

Protein phosphorylation, allosteric and redox modification are the major post translational modifications, which take place in order to control the activity of enzymes. Phosphorylation of major starch biosynthetic enzymes was recently discovered by Tetlow et al. (2004) who investigated the role of protein phosphorylation as a mechanism of regulation of the starch synthesis in developing wheat endosperm. After incubating intact plastids from wheat with -

[32P]-ATP, it was found that three isoforms of SBE’s (SBEI, SBEIIa, and SBEIIb) were phosphorylated on serine residues (Tetlow et al. 2004). The activity of

SBEIIb in amyloplasts and SBEIIa in chloroplasts was stimulated by phosphorylation whereas dephosphorylation using alkaline phosphatase reduced catalytic activity (Tetlow et al. 2004).

There is increasing evidence that starch synthesis does not consist of several isolated and simple reactions as indicated in Figure 1.3. The interaction and coordination of starch biosynthetic enzymes appears to be a general feature of starch biosynthesis in plants. Starch biosynthetic enzymes form heteromeric protein complexes that are probably involved in starch synthesis (Hennen-

Bierwagen et al. 2008, Tetlow et al. 2008, Tetlow et al. 2004). Co-

41 immunoprecipitation experiments revealed that SP, SBEIIb and SBEI form a protein complex of three enzymes when only these enzymes are phosphorylated within the soluble protein fraction in wheat amyloplasts lysates, (Tetlow et al.

2004). Dephosphorylation with alkaline phosphatase disassembled the complex formed (Tetlow et al. 2004), suggesting that the protein-protein interactions are likely to be phosphorylation-dependent. In developing endosperm of barley, the sex6 mutant lacking SSIIa, resulted a reduction in amylopectin synthesis to less than 20% of the wild-type levels and production of high amylose starches

(Morell et al. 2003). A pleiotropic effect of the SSIIa mutation abolished the binding of SSI, SBElla and SBEIIb to the starch granules, while not significantly altering their expression levels in the soluble fraction (Morell et al. 2003). In wheat endosperm, physical interactions between SS’s and SBE’s were detected and two distinct complexes identified (Tetlow et al. 2008). The authors found one complex consisting of SSI, SSII, and SBEIIa, and another complex with SSI,

SSII, SBEIIb. Furthermore, both of these complexes are phosphorylated and in vitro dephosphorylation with alkaline phosphatase resulted in disassociation of the proteins. In maize amyloplasts, a multi-subunit complex containing SSIIa,

SSIII, SBEIIa, and SBEIIb was detected using gel permeation chromatography

(Hennen-Bierwagen et al. 2008). The authors also located another complex consisting of starch synthesizing enzymes SSIIa, SBEIIa, and SBEIIb. In the ae- mutant lacking SBEIIb, a novel protein complex was found in which SBEIIb was replaced by SBE1 and SP (Liu et al. 2009). Analyses further revealed that eliminating SBEIIb in ae- mutant caused significant increases in the abundance of SBEI, SBEIIa, SSIII, and SP in the granule (these proteins are not found in

42 the granule in the granules of wild-type maize), without affecting SSI or SSIIa

(Grimaud et al. 2008). Staining the internal granule-associated proteins using a phospho-protein specific dye revealed phosphorylation of at least three proteins,

GBSS, SBEIIb, and SP (Grimaud et al. 2008). This evidence added weight to the hypothesis that starch synthesizing enzymes exists as hetero complexes in developing cereal endosperm and these proteins eventually become granule- associated via as yet unknown mechanisms.

1.2. Objectives of the study

As the research evidence indicates SP may have the potential to be involved in starch synthesis possibly involving the formation of protein complexes with other enzymes. Therefore, the first aim of this research project was;

 To determine whether the SP is involved in starch biosynthesis in maize

endosperm by interacting with starch biosynthetic enzymes and forming

protein complexes.

 The second objective was to understand the involvement of SP in starch

synthesis in maize and explore possible interactions with SSIV.

 The third objective was to investigate if the SP-involved protein-protein

interactions are regulated by protein phosphorylation.

The results in this thesis discuss the possible interaction of SSIV and SP and the mechanisms of their regulation through phosphorylation in wild type developing maize endosperm using the amyloplast lysates and partially purified

43 recombinant SP. This research aims to provide further insight into our growing understanding of coordinated activity of different enzymes associated in starch synthesis through protein-protein interactions and complex formation in developing maize endosperm. The results in the thesis outline the biochemical characterization of SP and SSIV in developing maize endosperm and explore possible protein-protein interactions of SP and other starch biosynthetic enzymes. The protein complexes in amyloplasts could influence the quality as well as the quantity of starch in developing endosperm through the modulation of the granule structure. Understanding of the basis of these modulations in starch is therefore essential. Starch produced in plastids provides up to 80% of the food calorie requirement of humans with various potential applications in non–food industries. Application of starch in food and non-food industries is depends on different structural and functional properties of starch which can be modified with the knowledge of its genetic manipulations. This research expected to enhance our understanding of the basics of starch biosynthesis to develop models of starch structure assembly.

CHAPTER 2

Biochemical Investigation of the Regulation of Plastidial Starch

Phosphorylase in Maize Endosperm

2.1. Introduction

Starch phosphorylase (SP) is a tetrameric or/and dimeric enzyme which catalyses the addition of glucosyl units from glucose-1-phosphate (G-1-P) to the non-reducing end of α-1-4 linked glucan chains liberating inorganic phosphate

(Pi) in forward reaction and produces G-1-P while degrading glycosyl units in reverse reaction. SP is potentially involved in both starch synthesis and degradation as shown in the following equation;

G-1-P + (1,4-α-D-Glucose)n (1,4-α-D-Glucose)n+1 + Pi

Two isoforms of SP are found in higher plants; designated by their subcellular localization; the plastidial (Pho1) and the cytosolic (Pho2) (Zeeman et al.

2004; Steup et al. 1988/1981; Nakano and Fukui, 1986). The plastidial form

(Pho1) in maize endosperm is designated as SP in this thesis.

2.1.1. Cytosolic form of SP (Pho2)

The extraplastidic (Pho2) starch phosphorylases do not contain L-78 amino acid insertion as in plastidial form (Pho1) and they are much more effective in degrading processes (Zeeman et al. 2004; Steup et al. 1988). Pho2 preferentially degrades branched starch molecules, and can even attack starch

46 granules in vitro (Steup et al. 1988). However, in starch-accumulating tissues like developing seeds and leaves which maintain intact amyloplasts or chloroplasts, cytosolic Pho2 has no direct access to the starch inside the plastid.

Cytosolic SP may be involved in regulating the cytosolic G-1-P level by glucosylating and trimming a heteropolysaccharides found in the cytosol produced mainly from maltose (a product of starch breakdown inside the plastid) which is translocated to the cytosol through MEX1 transporter located in the plastidic membrane (Yang and Steup 1990, Steup et al. 1991, Buchner et al.

1996; Pyke, 2009; Rathore et al. 2009). The production of metabolites such as maltose and glucose, which are exported to cytosol are involved in glycan metabolism by the action of cytosolic phosphorylase (Pho2), disproportionating enzyme, cytosolic transglucosidase, and Pho2 produces G-1-P in the cytosol

(Pyke, 2009; Zeeman et al. 2004). Fig. 2.1 illustrates the putative roles of plastidial (Pho1) and cytosolic (Pho2) SP in starch metabolism in plants.

2.1.2. Plastdial SP (Pho1)

The plastidial isoform of SP, Pho1 is present throughout endosperm development in cereals (Schupp and Ziegler, 2004; Satoh et al. 2008; Tickle et al. 2009). The Pho1 also contributes the highest proportion of the total SP activity in the endosperm, and remains active throughout the endosperm development in rice endosperm (Satoh et al. 2008). Also, the mutation in Pho1 in rice endosperm produces a shrunken phenotype endosperm with reduced starch content and altered starch granule structure in rice (Satoh et al. 2008).

The shrunken 4 mutants lacking plastidial SP activity in maize endosperm

47 produce endosperms with reduced starch contents (Tsai and Nelson, 1969), and the fact that Pho1 does not appear to influence starch degradation in

Arabidopsis thaliana (Zeeman et al. 2004) suggests plastidial SP may play a role in the storage starch biosynthesis, or play a subsidiary role in to the α- amylolytic pathway in starch in starch degradation.

Figure 2.1: Schematic diagram illustrating the putative roles of plastidial (Pho1) and cytosolic (Pho2) SP in starch metabolism in plants. The dashed lines indicate that there may be intermediate steps in the pathways. ADGP=ADP glucose pyrophosphorylase, SS= starch synthases, SBE= starch branching enzymes, DBE= debranching enzymes, DPE1/DPE2= Disproportionating enzymes, GWD= glucan water dikinase, PWD=phospho-glucan water dikinase, Glc-1-P= glucose- 1-phosphate, GT= glucose transporter, MEX1= maltose transporter, TPT= triose phosphate transporter (Modified from Rathore et al. 2009).

The biochemical characteristics of plastidial SP such as the lower affinity towards the high molecular starch and the higher affinity towards the low molecular weight linear malto-oligosaccharides (MOS) in sweet potato tubers

(Young et al. 2006) suggested the possibility that SP acts on elongating the shorter glucan chains, and might be also involved in the process of granule initiation. The 78 amino acid insertion (L-78) in the middle of the sequence in

Pho1 but not in cytosolic Pho2 is a prominent molecular characteristic in all the plant species investigated. This insertion prevents the binding of SP to large, highly branched polysaccharides in sweet potato tubers (Young et al. 2006). In contrast, in cereals, SP showed higher affinities towards to amylopectin than glycogen in synthetic direction and to MOS in phosphorylitic direction (Mori et al.

1993; Mu et al. 2001; Schupp and Ziegler, 2004).

The plastidial form of SP in maize endosperm amyloplasts is 112 kDa in size and known to be the second most abundant enzyme presence next to the

SBEIIb (Yu et al. 2001). In addition to the presence of the L-78 insertion in the middle of the maize SP protein sequence, the N-terminus of maize amyloplast

SP does not align with any other N-terminus sequences of SP available in the gene bank (Yu et al. 2001). Due to the variability in the N- terminus of the enzyme, SP in maize and other plastidial SP forms may have different regulatory mechanism, for example, the N-terminus of the protein generally contain signal recognition peptides, targeting peptides and important in enzyme regulation

(Fig. 2.2).

The first evidence for the post translational regulation of SP described the phosphorylation of SP and its involvement in phosphorylation-dependent

49 protein-protein interactions in wheat amyloplast stroma with SBEI and SBEIIb

(Tetlow et al. 2004). In the maize ae.1 mutant amyloplasts lacking SBEIIb, novel protein complexes are found with SP; these include SSI, SSIIa, SBEI and

SBEIIa (Liu et al. 2009). The ae.2 mutant, contains an inactive form of SBEIIb found to be associated in complex formation with SSI, SSIIa and SBEI both in the stroma and the granule (Liu et al. 2012). Interestingly the SP is not involved in complex formation in ae.2 mutant as seen in ae.1 mutant (Liu et al. 2012).

Indirect evidence implicates interactions between SP and SSIV in mutants of Arabidopsis. The activity of both Pho1 and Pho2 increased in SSIV mutants (Atssiv1 and Atssiv2) by 1.4 -2 fold compared with the wild-type in

Arabidopsis thaliana leaves where transient starch is synthesized (Roldan et al.

2007). However, there was no significant influence on starch structure or the amylose/amylopectin ratio in these mutants and the concentrations of soluble sugars remain unchanged (Roldan et al. 2007). A double mutant produced by the insertion of an heterologous T-DNA within the nucleic sequence of an intron or an exon, lacking both Pho 1 and SSIV activity produced 1-2 granules per plastid (3-4 granules per plastid in wild-type) but increased the granule size by at least four times higher than the starch granules originating from the their single mutants plants in Arabidopsis (Planchot et al. 2008; patent EP1882742).

However, no evidence is currently available to show any direct relationship between SP and SSIV in storage starch synthesizing tissues.

The active Pho1 enzyme exists as an assembly of dimeric or tetrameric subunits in maize and different multimeric forms of SP in maize might be involved in the formation of different protein complexes (Liu et al. 2009; Mu et

50 al. 2001). Previous studies confirmed that SP activity can be modulated by the substrates ratio of G-1-P/Pi (Schupp and Ziegler, 2004; Mu et al. 2001) and

ADP-glucose (Matheson and Richardson, 1978). Comparatively less information is available on SP regulation by protein phosphorylation in storage starch synthesizing tissues. Unlike the SP mutant lines developed in rice (Satoh et al.

2008) and Arabidopsis (Roldan et al. 2007; Planchot et al. 2008); there are no genetically developed mutants lines available in maize. The shrunken-4 mutant has reduced SP activity, but this is probably due to alterations in levels of pridoxal-5-phosphate, the essential cofactor for SP activity in the endosperm

(Tsai and Nelson, 1969).

The objectives of this study were to characterize and investigate the role and regulation of Pho1 in maize wild-type amyloplasts by protein phosphorylation and protein-protein interactions. Moreover, the possible involvement of SP in granule initiation was tested specifically by testing the possibility of interactions between SP and SSIV in the amyloplast.

2.2. Materials and Methods

2.2.1 Materials

2.2.1.1. Plant materials

The wild type maize (C. G. 102) (Zea mays) was used in all experiments.

The cobs were collected at different growth stages (5-35 days after anthesis) from wild type maize plants grown under the normal field conditions. Cobs were kept at +40C cold room until use for amyloplast extractions. The kernels were also collected and frozen at -800C for future use for whole cell (crude) extracts.

2.2.1.2. Chemicals

All chemicals were obtained from Sigma Aldrich unless otherwise stated.

2.2.2. Methods

2.2.2.1. Amyloplast purification from maize endosperms

Endosperms harvested at 22 days after anthesis (DAA) from the wild-type of maize plants were mainly used to purify the amyloplasts in the experiments unless otherwise stated. This stage of endosperm development was found to be the major grain-filling period (Liu et al. 2009). Amyloplasts are purified to remove any contaminating proteins that may be found in maize whole cell lysates. Maize amyloplast extraction was performed as described by Liu et al.

2009.

Approximately, 100g of the endosperms were taken from the developing kernels with a spatula and gently chopped with a razor blade in 40-50 mL of ice- cold amyloplast extraction buffer (50 mM N-(2-hydroxyethyl) piperazine-N’-

52 ethanesulphonic acid (HEPES)/KOH, pH 7.5, containing 0.8 M sorbitol, 1 mM

KCl, 2 mM MgCl2, and 1 mM Na2-EDTA) on a petri dish on ice until firmly chopped in to creamy solution. The resulting whole cell extract was then filtered through four layers of Miracloth (CalBiochem, catalogue no. 475855) wetted in the same buffer. Then the filtrate was then carefully layered onto 15 mL of 3%

(w/v) Histodenz (Nycodenz, Sigma, catalogue no. D2158) in amyloplast extraction buffer followed by centrifugation at 100xg at 40C for 20 min and the supernatant was carefully removed. The pellet with intact amyloplasts was ruptured with 1 mL of ice-cold rupturing buffer containing 100mM N-tris

(hydroxymethyl) methyl glycine (Tricine)/KOH, pH 7.8, 1 mM dithiothreitol

(DTT), 5 mM MgCl2, and a protease inhibitor cocktail (5μl per 1 mL buffer) (see

Appendix 09 for details). Then the mix was transferred into micro-centrifuge tubes and centrifuged at 13,000xg at 40C for 5 min to remove starch. The soluble fractions were frozen in liquid nitrogen and stored at -800C until further use. The amyloplast lysates were ultra-centrifuged at 100,000xg for 15 min before use to remove plastidial membranes.

2.2.2.2. Preparation of whole cell extracts

Whole cell extracts were prepared as described previously by (Tetlow et al. 2003). Approximately, 10 g of endosperm tissue was quickly frozen in liquid nitrogen and immediately ground into a fine powder adding liquid nitrogen on ice using a chilled mortar and pestle. The frozen powder was mixed with ice-cold rupturing buffer (same rupturing buffer used in amyloplast purification) and a protease inhibitor cocktail (5 μL per 1 mL buffer) (see Appendix 09 for details).

The mixture was further mixed and allowed to stand on ice for 5 min followed by centrifugation at 13,000xg for 5 min at 40C. The supernatant was subjected to ultracentrifugation at 100,000x g for 15 min in a Beckman Coulter Optima-Max–

XP ultracentrifuge to remove membranes and particulate material. The supernatant obtained following the ultracentrifugation was used for experiments.

2.2.2.3. Localization of SP in the plastid

To investigate the proportional of SP and other starch biosynthetic proteins in the stroma-granule interface, where the proteins are imbedded on granule surface, the remaining pellet (approximately 1 g of fresh weight) from the isolation of amyloplast lysates (as described in section 2.2.2.1.), was subjected to a series of washings (for up to 10 times) with rupturing buffer (0.3 mL/washing stage) used in amyloplast extraction. The supernatant was collected after centrifugation at 13,000xg for 5 min. and the proteins were separated on the SDS gels and the proteins are visualized by silver staining and identified by immunoblotting.

2.2.2.4. Preparation of granule bound proteins

The granule bound protein was isolated as the method described by

(Tetlow et al. 2004). After rupturing of the amyloplasts and the separation of soluble protein fractions by centrifugation (as described in section 2.2.2.1.), the remaining pellets (approximately 1g) were resuspended in 1 mL of cold aqueous washing buffer [50 mM Tris (hydroxymethyl) aminomethane (TRIS)-acetate, pH

7.5, 1 mM Na2 -EDTA, and 1 mM DTT] and centrifuged at 13,000 rpm for 1 min

54 at 40C. This washing step was repeated 8 times. The pellet was then washed three times with 1 mL acetone each time followed by three washes with 2%

(w/v) SDS (1 mL each time). Starch granule bound proteins were extracted by boiling the washed starch in 2XSDS loading buffer [62.5 mM TRIS-HCl, pH 6.8,

2% (w/v) SDS, 10% (w/v) glycerol, 5% (v/v) β-mercaptoethanol, 0.001% (w/v) bromophenol blue] for 5 min. at 900C. The boiled samples were cooled and centrifuged at 13, 000xg for 5 min and supernatants separated by SDS-PAGE.

2.2.2.5. Biochemical characterization of SP in maize endosperm

2.2.2.5.1. Phosphorylation and dephosphorylation of amyloplast lysates

The amyloplast lysates/crude extracts were incubated with 1 mM ATP to stimulate protein phosphorylation by protein kinases present in the endosperm.

To prevent in vitro dephosphorylation, the lysates were also incubated with phosphatase inhibitor cocktail (10 μl/1ml lysates) in a separate tube as a control. Another treatment involved the incubation of maize amyloplast lysates with alkaline phosphatase conjugated to agarose beads (APase, insoluble form suspension in (NH4)SO4, final conc. 25 units/mL) to promote non-specific dephosphorylation. Untreated amyloplast lysates were used as the control in all phosphorylation experiments. All samples had >1 mM MgCl2. Rupturing buffer was added to balance the total end-volumes of the treatments. Phosphatase inhibitor (PI) was added to inhibit the endogenous alkaline phosphatases in the sample as a control (see appendix 09, section 1 for the details about PI).

2.2.2.5.2. Enzyme assays

2.2.2.5.2.1. Starch phosphorylase glucan synthetic activity assay

The synthetic activity of SP was assayed in vitro by using amylopectin, glycogen and maltoheptaose as the substrates. 80 μL of glucan substrates

(2.5% [w/v]; prepared in 100 mM MES-NaOH [pH 6.0], only amylopectin was gelatinized before adding to the mixture) and 20 μL [U14C]-G-1-P (GE Health care, catalogue No., CF0113; 10 mM stock; 0.1μCi; prepared in 100 mM MES

14 [pH 6.0]) were added to a clean 1.5 mL micro centrifuge tube. [U- C]-G1P was used. The reaction was initiated by adding 100 μL extract in 10 second intervals and terminated after incubated for 30 minutes at 37°C by the addition of 1 mL stop solution (75% [v/v] methanol, 1% [w/v] KCl). Samples were then centrifuged at 10000g for 5 minutes. The supernatant was removed and the remaining pellet was resuspended in 300 μL H 0 before the addition of 1 mL 2 stop solution. Samples were then centrifuged for a further 5 minutes at

10,000xg for 5 min. and the supernatant was removed. The pellet was resuspended in 300 μL H 0 and added to 3.7 mL Ecoscint™ scintillation cocktail 2 and radioactivity was measured in a liquid scintillation analyzer (Bekman

14 Coulter-USA, ls-6500 Multi-purpose scintillation counter). Amount of [U- C]-G-

1-P incorporated into glucan was calculated.

2.2.2.5.2.2. Starch phosphorylase glucan degradative activity assay

SP phosphorolytic activity was determined based on the procedure described by (Tickle et al. 2009). The G-1-P formed in the phosphorolysis

56 direction was converted to glucose-6-phosphate (G-6-P) by phosphoglucomutase and then the G-6-P converted to 6-phopsphogluconate by glucose-6-phosphate dehydrogenase. The amount of NADH was released at this step was analyzed at 340nm; the amount of NADH was equal to the amount of

G-1-P produced in the reaction. In the procedure, for one reaction (1 mL final volume), final concentration of 20 mM HEPES (pH 7.0) was added to a 1 mL plastic cuvette with final concentrations of 5 mM MgCl , 0.25 mM NAD, 0.024 2 mM glucose-1,6-bisphosphate, and 1% [w/v] substrate (glycogen, amylopectin and maltoheptose) (all solutions were prepared in 50 mM HEPES [pH 7.0]), 3.7

-1 μL phosphoglucomutase (0.5 units.μL ), 100 μL of amyloplast lysates (0.95 mg/mL concentration) and 1.6 μL glucose-6-phosphate dehydrogenase (0.32

-1 units.μL ). This reaction was initiated by the addition of 4.5 mM Na HPO as the 2 4 source of Pi.

2.2.2.5.3. Gel filtration chromatography (GPC)

Extracts of soluble proteins from maize amyloplasts and whole cell extracts (500 μL loading volume) were separated through a Superdex 200

10/300GL gel permeation column (equilibrated with two column volumes of the rupturing buffer) on an AKTA- FPLC system (Amersham Pharmacia Biotech model No. 01068808). The column was calibrated using commercial protein standards from 13.7 kDa to 669 kDa (GE Healthcare Gel Filtration Calibration

Kits low molecular and high molecular weight). The column was pre-equilibrated with two column volumes of running buffer containing 10 mM HEPES-NaOH, pH

7.5, 100 mM NaCl, 1 mM DTT, and 0.5 mM PMSF, at a flow rate of 0.25 ml min_1. 0.5 ml fractions were collected.

2.2.2.6. Protein analysis

2.2.2.6.1. Quantification of proteins

Protein content was determined using the Bio-Rad protein assay (Bio-Rad

Laboratories, Canada) according to the manufacturer’s instructions and using bovine serum albumin as the standard.

2.2.2.6.2. Sodium dodecyl sulfate polyacrylamide gel electrophoresis

(SDS-PAGE)

SDS-PAGE was performed using a Mini-Protean III Vertical Electrophoresis

System (Bio-Rad) according to the manufacturer’s instructions. Proteins were separated on SDS-PAGE on 10% acrylamide gels. The compositions of 3% stacking gel and the separation gel was shown in Table 2.1. Prior to electrophoresis, proteins were mixed with SDS sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% [w/v] SDS, 10% [w/v] glycerol, 5% [v/v] b-mercaptoethanol,

0.001% [w/v] bromophenol blue) and boiled for 5 min at 900C. The gel was run using 0.25 M Tris (pH 7.2), 192 mM Glycine, 0.4% SDS running buffer at 120V for 1.5hr.

Table 2.1: The composition of stacking and resolving gels for SDS-PAGE.

Stock solution SDS-PAGE (10 mL) Stacking gel Resolving gel (5% acrylamide) (10% acrylamide) ® ProtoGel Acrylamide:bisacrylamide 1.68 3.4 (30%[w/v]) 0.5M Tris (pH 6.8) 1.28 - 1.5M Tris (pH 8.8) - 2.6 10% (w/v) SDS 0.1 0.1 10% (w/v) ammonium persulfate 0.112 0.1 Distilled water 7 3.8 TEMED 0.008 0.01

2.2.2.6.3. SP-Native affinity zymogram

Zmogram analysis was used to identify the activity of the proteins as described by (Tickle et al. 2009). The extracts were separated by substrate- affinity (glycogen, amylopectin and maltoheptaose) non-denaturing PAGE. The non-denaturing gels were prepared as 8% (w/v) polyacrylamide gels containing glycogen (0.1% w/v) (Table 2.2). The composition of the stacking gel and the resolving gel are shown in Table 2.2. Following electrophoresis, the gels were incubated for 16 hours at 28°C in substrate buffer containing (0.1% [w/v] glycogen, 20 mM G-1-P, made up in 100 mM sodium citrate [pH 6.5]) to test the synthetic activity. Phosphorylitic activity was tested by incubating the gel containing (0.1% [w/v] glycogen, 20 mM Na2HPO4, made up in 100 mM sodium citrate [pH 6.5]) and incubated under same conditions as used in synthetic activity gels. Gels were then rinsed briefly in sodium citrate (100 mM, pH 6.5) before covering the gel for up to 1 minute in Lugol solution (0.2% [v/w] iodine,

2% [v/w] potassium iodide). Gels were subsequently rinsed in distilled water and photographed immediately.

Table 2.2: Composition of non- denaturing, 8% acrylamide gels (without SDS) containing 0.1% (w/v) glycogen prepared as follows.

Stock solution Resolving gel (10 mL) Stacking gel (5 mL)

H2O 4.7 (mL) 3.55 (mL) 30%Acrylamide 2.6 (mL) 0.84 (mL) 1.5M Tris, pH 8.8 2.6 (mL) - 1M 5M Tris, pH 6.8 - 0.64 (mL) 10% APS 0.1 0.056 0.1% glycogen 10 (mg) - TEMED 10 μL 4 μL

2.2.2.6.4. Coomassie blue staining

Polyacrylamide gels were stained in Coomassie Blue stain (42% [v/v] methanol, 18% [v/v] acetic acid, 0.1% [w/v] Coomassie Brilliant Blue R 250) for

1hr and destained overnight in 42% [v/v] methanol/ 18% [v/v] acetic acid.

Then the gel was washed in distilled water.

2.2.2.6.5. Silver staining

Following the electrophoresis, the polyacrylamide gel was kept in 50 mL fixing solution (50% Methanol [v/v], 5% acetic acid [v/v]) for 20min on a shaker and washed the gel in washing buffer (50% Methanol [v/v]) for 10min and with distilled water at least for 1hr. Then, the gel was transferred to sensitizing buffer (0.02% Na2S2O3 [w/v]) for 1min and washed the gel twice in distilled water for 2 min each time. The gel was stained in ice-cold silver nitrate buffer (0.1% AgNO3 [w/v]) for 20 min and washed the gel in distilled water for 2 min each time. Developed the gel in developing solution (2% Na2CO3 [w/v],

0.04% formalin [v/v]) for 5-7 min until the proteins bands were visualized.

Staining was stopped by adding the stop solution (5% acetic acid [v/v]) for 5 min and transferred to distilled water.

2.2.2.6.6. Mobility shift detection of phosphorylated proteins

(Phosphate affinity SDS-PAGE using Acrylamide-pendant Phos-TagTM

The Phos-Tag affinity ligand (10 μmol/L Phos-tagTM AAL) was used to detect phosphorylated proteins using the SDS-PAGE gels. A dinuclear metal complex (Mn2+) acts as a selective phosphate-binding tag molecule and the

Phos-Tag binds to the phosphate group of the phosphorylated protein and retards the movement of the phospho protein in the SDS-PAGE gel. The phosphorylated and nonphosphorylated proteins were detected by immunoblot analysis. The composition of the gel prepared (see in Table 2.3) and the experimental procedure is described as below. The gel was run at 10 mA for 14 hours at room temperature.

Solutions

1. Stock solution of 5.0 mmol/L Phos-tagTM AAL Solution containing 3% (v/v)

MeOH (Phos-tagTM AAL-107 10 mg was mixed with methanol 0.10 mL and distilled water 3.2 mL). This oily product, was stored in dark at 40C until use.

2. 10 mmol/L MnCl2 Solution was prepared by dissolving 0.10 g MnCl2 (H2O)4

(FW: 198) in 50 mL of distilled water.

Table 2.3: The Gel preparations for Phos-TagTM analysis.

Stock solution Resolving Gel (10 mL) Stacking Gel (10 mL)

10% (w/v) acrylamide and (5% (w/v) acrylamide) 50 μmol/L Phos-tag TM AAL)

30% (w/v) Acrylamide Solution 4.0 mL 1.50 mL 1.5 mol/L Tris/HCl Solution, pH 8.8 2. 2.5 mL 2.50 mL (pH 6.8) 5 mmol/L Phos-tag AAL Solution 0.1 mL - 10 mmol/L MnCl2 Solution 0.1 mL - 10% (w/v) SDS Solution 0.1 mL 0.10 mL 10% (w/v) Diammonium Peroxydisulfate 0.1μL 0.10 μL Distilled Water 3.1 mL 5.0 mL TEMED (tetramethylethylenediamine) 10 μL 8.0 μL

2.2.2.6.7. Immunological techniques

2.2.2.6.7.1. Preparation of peptides and antisera

Polyclonal antibodies were raised in rabbits against the synthetic peptides derived from the sequence of maize SP (YSYDELMGSLEGNEGYGRADYFLV) corresponding to residues 917–943 of the full length sequence (GenBank accession no. AAS33176). Synthetic polypeptides were raised to the polyclonal rabbit antisera targeted to maize SSI, SSIIa, SBEI, SBEIIa, SBEIIb, Iso-1, and

Iso-2. The specific peptide sequences used for the various antibodies were as follows (Table 2.4).

Table 2.4. The synthetic peptides sequences derived from the primary amino acid sequences of starch biosynthetic enzyme isoforms of maize; their location in the full length sequence and the GenBank accession numbers.

Enzyme Peptide Location GeveBank Accession Isoform Sequence in Full Length Number Sequence

SSI AEPTGEPASTPPPVPD 72-87 AAB99957 SSIIa GKDAPPERSGDAARLPRARRN 69-89 AAD13341 SSIV ANHRNRASIQRDRASASI 55-72 AAC197339 SBEI KGWKFARQPSDQDTK 809-823 AAC36471 SBEIIa FRGHLDYRYSEYKRLR 142-157 AAB67316 SBEIIb PRGPQRLPSGKFIPGN 641-656 AAC33764 Iso-1 FTKHNSSKTKHPGTYIAC-NH2 269-286 AAA91298 Iso-2 ARSYRYRFRTDDDGVV 37-52 NP001105666 GBSSI QDLSWKGPAKNWENV 442-456 ABW95928

2.2.2.6.7.2. Antibody purification

The peptide affinity columns were used to purify the various crude antisera. The columns were prepared as follows. To make a 1 mL column, the respective synthetic peptide (2 mg) was dissolved in 1 mL of TRIS-HCl, pH 8.5

(50 mM TRIS-HCl, 5 mM EDTA). 2 mL sulpholink resin slurry (Pierce) was washed in 1 mL TRIS-HCl, pH 8.5 for six times. The dissolved peptide was added to 1 mL washed resin in a falcon tube and incubated on a rotor for 15min in room temperature and for additional 30 min without rotating and added to the column and column was washed with 3 mL TRIS-HCl, pH 8.5 and then blocked with 1 mL of 50 mM cysteine in the same washing buffer. 5 mL antisera containing the polyclonal maize antibodies were applied to the column and mix on a rotator for overnight at 4oC with 3 mL of PBS /0.01% Na azide [w/v}. Then

63 the column was washed with 10 ml RIPA [50 mM TRIS-HCl, pH 7.5, 150 mM

NaCl, 1% (w/v) nonyl phenoxylpolyethoxyl ethanol (NP-40), 0.5% (w/v) Na- deoxycholate, 0.1% (w/v) sodium dodecyl sulphate (SDS)]. The column was further washed with 10 ml sarcosyl buffer [NETN (20 mM TRIS-HCl, pH 8.0, 1 M

NaCl, 1 mM Na2-EDTA, and 0.5% (w/v) NP-40)], followed by washing again with

10 ml of 10 mM TRIS-HCl pH 7.8. The antibody bound to the column was eluted with 0.5 mL of 100 mM glycine pH 2.5 to a tube contained 0.5 mL 1M TRIS-HCl pH 7.8 and the protein contents were measures. The column was neutralized by adding 10 mL of 10 mM TRIS-HCl pH 7.8/0.05% [w/v] sodium azide.

2.2.2.6.7.3. Immunoblot analysis

After electrophoresis, the proteins in polyacrylamide gels, were transferred to nitrocellulose membranes (Pall Life Sciences) using a Mini Trans-

® Blot Electrophoretic Transfer Cell (Bio-Rad) according to the manufacturer’s instructions. The transfer buffer contains 10% running buffer, 20% methanol and 70% water. Then the membrane was blocked with 1.5% bovine serum albumen (BSA) in 1XTBA buffer, and incubated overnight in diluted antibodies using the methods described by (Tetlow et al. 2004). The anti-maize antisera were used in immunoblot analyses were diluted in 1.5% BSA in 1XTBA buffer as follows 1:1000 for SSI, SSIIa, SSIIb, SBEI, SBEIIb and 1:500 for SP, SSII and

SSIV. The bound antibodies were detected with alkaline phosphatase-conjugated anti-rabbit IgG using a 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium liquid substrate developing solution (BCIP/NBT).

2.2.2.6.7.4. Immunoprecipitation

Immunoprecipitation was performed with amyloplast lysates, using methods previously described by (Tetlow et al. 2004). The SP, SSIIa and SBEIIb antibodies were added at 30 mg/mL concentration and the SSIV antibodies at 60 mg/mL to 1.0 mg of amyloplast lysates and incubated for 1hr on a rotator at room temperature. Proteins were immunoprecipitated by adding 40 μL of 50%

(w/v) Protein A-Sepharose slurry (60 μL of slurry for SSIV). The Protein A-

Sepharose slurry was made by adding the phosphate buffer saline (137 mM

NaCl, 10 mM Na2HPO4, 2.7 mM KCl, 1.8 mM KH2PO4) to the Protein A-Sepharose beads and incubated for 1hr at room temperature. Protein A-Sepharose/protein complex was centrifuged at 100 g for 2 min at 40C in a refrigerated micro centrifuge, and the supernatant was collected and denatured with the sample running buffer containing SDS to use as an indicator of the immunoprecipitation efficiency. The remaining pellet, Protein A-Sepharose/protein complex was washed eight times each with 1 mL phosphate buffered saline (PBS), followed by three similar washes with 10 mM HEPES/KOH, pH 7.5 buffer (at 100 g, 1 min centrifugation). The immunoprecipitation pellet was boiled in 2X SDS loading buffer for 8 min. Co-immunoprecipitation was tested by probing with specific anti-peptide antibodies of major starch biosynthetic enzymes.

2.3. Results

2.3.1. Subcellular localization of SP in maize endosperm

To determine the subcellular localization of SP, the amyloplast lysates, the granule-bound proteins of the starch granules separated from amyloplast, the whole cell crude extracts of the endosperm, and the amyloplast membrane protein were extracted from 22 DAA wild-type maize plants. Immunoblot analysis using peptide specific anti-Pho1 antibodies showed that there is no SP in granules and in amyloplast membranes (Fig. 2.2). The SP is mainly found in the amyloplast lysates. The proportional existence of the SP in the interface of the soluble fraction and the granule as the granule surface imbedded protein was tested by collecting the extracts as the supernatants after repeatedly washing the granules nine times with rupturing buffer. Fig. 2.3A showed the protein profile of the extracts collected after each washing (silver stained SDS-

PAGE gel). The proteins which were separated on SDS gels were identified by probing the immunoblots with anti-peptide specific antibodies of starch biosynthetic proteins SP, SSI, SSIIa, SSIII, SSIV, SBEI, SBEIIa and SBEIIb (Fig.

2.3B). Most of the SP was found in wash 1 and 2 and slightly in was 3, 4 and 5.

There was no band detectable from wash 6-9, and the protein profile of SP was similar with SSI and SBEIIb (wash 1 to 3) and SBEI (wash 1 to 4). SSIII and

SSIV were found in only the first wash whereas SSII was found clearly from wash 1 to 7 (Fig. 2.3B). To determine the granule bound SP, 0.05 mg (wet weight) of starch was taken out after every centrifugation stage during granule washing and it was boiled with 200 μL of 2XSDS. Immunoblots were probed with

66 anti-SP and anti-SSIIa specific antibodies (Fig. 2.4). SP was not found in the granules as a granule-bound protein while SSIIa, which was found in the granule and could not be removed by the washing treatment (Fig. 2.4).

(A) (B)

Figure 2.2: Immunoblots showing the subcellular localization of plastidial SP in maize endosperm; the amyloplast lysates contain soluble amyloplast proteins, the granule-bound proteins of the starch granules separated from amyloplast, the soluble protein fraction and starch granule-bound proteins of whole cell crude extract of the endosperm and the soluble protein fraction of the amyloplast membrane protein extracts (A). Leaf crude extracts were probed with anti-SP antibodies are shown in (B). All samples were extracted from 22 DAA wild-type maize plants. The blots were developed in two different experiments and both were probed with pastidial peptide specific anti-SP antibodies after equal amounts (25 µg) of proteins were run on SDS-PAGE. Arrows indicate the location of SP.

(A)

(B)

Figure 2.3: Analysis of the localization of proteins imbedded in the granule surface. Approximately 1 g of fresh weight of starch granules from the amyloplast was subjected to a series of washings with the 0.3 mL of 100mM rupturing buffer for 9 times. The supernatant was collected at each time and separated on the SDS gels and the proteins are visualized by silver staining (A) and identified by probing immunoblots with anti-peptide specific antibodies of starch biosynthetic proteins as indicated (B). The numbers indicate the number of washings. L=protein marker. Target protein is indicated by the arrow in each immunoblot.

Figure 2.4: Analysis of the localization of proteins imbedded in the granule surface and loosely bound to the granules. Approximately 1g of fresh weight of starch from the amyloplast lysates was subjected to a series of washings with the 0.3 mL of 100mM rupturing buffer for 9 times. The supernatant (soluble fraction) and 0.05g of the pellet was denatured in 2XSDS (200 μL) at each washing (granule association) was collected at each time and separated on the SDS gels and the proteins are visualized by silver staining and identified by probing the immunoblots with anti-SP and anti-SSIIa peptide specific antibodies of starch biosynthetic proteins as indicated in the blots. The numbers indicate the number of washings. L=protein marker.

2.3.2. The synthetic activity of SP in developing maize endosperm

The synthetic activity of plastidial SP in developing maize endosperm was determined by native affinity zymogram containing 0.1% glycogen in the gel.

The amyloplast lysates were extracted from the endosperm at 12, 15, 19, 22 days after anthesis (DAA). Activity bands were observed for all the developmental stages tested (Fig. 2.4A). The immunoblot was probed with peptide specific anti-SP antibodies confirmed the activity bands are due to plastidial SP (Fig 2.4B). The equal volumes of amyloplast lysates (30 μL/well)

69 were loaded on the gel. The activities of SP shown on the gel did not vary over the various developmental stages tested. Synthetic activity of SP (22 DAA) was slightly reduced when SSIIa was removed from amyloplast lysates, but not the

SSIV (Appendix 01).

The SP in amyloplast lysates at 22 DAA age showed both synthetic and phosphorolytic activities when both activities were tested in a glycogen affinity native zymogram containing 0.1% glycogen in the gel (Fig. 2.6). In synthetic and degradative directions, the gels were incubated at 1, 2, 5, 10, and 20 mM

G-1-P and sodium phosphate dibasic (Na2HPO4) respectively. When the activity bands were visualized by Lugol’s solution, the dark synthetic activity bands were shown in all concentrations of G-1-P tested and the activity band was clear at all concentrations of Na2HPO4. Both synthetic and degradative activities were increased with increasing substrate concentrations (Fig. 2.6).

(A)A B(B) Days After Anthesis Days After Anthesis 12 15 19 22 12 15 19 22

Figure 2.5: The activity of Pho1 was observed in developing wild-type maize amyloplast lysates isolated 12-22 days after anthesis (DAA), using non- denaturing affinity native zymogram containing 0.1% glycogen in the gel (A). Immunoblot of the zymogram gel was probed by peptide specific anti-SP antibodies to detect the SP protein in maize amyloplast (B). Pho1 is localized in the amyloplast stroma, and showed consistent activity in all the developmental stages of amyloplast measured.

G-1-P Na2HPO4 20 10 5 2 1 20 10 5 2 1 mM mM mM mM mM mM mM mM mM mM

SP Synthetic Activity SP Phosphorylitic Activity SP Synthetic Activity SP Phosphorolytic Activity

Figure 2.6: The activity of SP in amyloplast lysates at 22 DAA age in the synthetic and phosphorolytic direction was tested on glycogen affinity native zymogram contained 0.1% glycogen in the gel. Following electrophoresis, the gel was incubated overnight at 280C with the incubation buffer containing 0.1% glycogen and 1, 2, 5, 10, and 20 mM glucose-1-phosphate (G-1-P) or sodium phosphate (Na2HPO4) in synthetic and phosphorolytic directions respectively. The activity bands were visualized by Lugol’s solution. Arrows indicate the bands corresponding plastidial SP.

3.2.3. Investigating the regulation of SP by protein phosphorylation

The activities of the phosphorylated and dephosphorylated isoforms of SP were analyzed on 0.1% glycogen affinity SP-native zymogram using amyloplast lysates, endosperm crude extracts and leaf crude extracts collected at 22 DAA.

The soluble form of plastidial (Pho1) isoforms from maize endosperm amyloplasts (Fig. 2.7A1), both plastidial (Pho1) and cytosolic (Pho2) isoforms of

SP in the whole cell extract of endosperm (Fig 2.7B1) and the isoforms in transient starch biosynthetic maize leaves (Fig 2.7C1) did not show any detectable qualitative differences in the activities in both phosphorylated

(treated with 1 mM ATP) and dephosphorylated (treated with 25 units of APase) extracts when compared with the untreated controls (Fig. 2.7). Immunoblot analyses of the zymograms are respectively shown in A2, B2 and C2 which are probed with peptide specific anti-Pho1.

The mobility shift detection of proteins on phosphate affinity SDS-PAGE using ligand bound Acrylamide-pendant Phos-TagTM showed no retardation in the mobility of ATP-treated and untreated SP from amyloplast lysates (Fig. 2.8).

APase APase

Control ATP APase only Control ATP APase only

A1 A2

Pho1

B1 B2 Pho2

Pho1

C1 C2 Pho1/Pho2

Figure 2.7: Determination of the different activity levels of plastidial (Pho1) and cytosolic (Pho2) isoforms of SP following treatment with ATP and APase. The amyloplast lysates, seed crude extract and leaf crude extracts collected at 22 DAA were treated with either 1mM ATP or with alkaline phosphatase (APase) (25unit/ml) and incubated for 1hr at room temperature. The activity was compared with the untreated controls on native affinity zymograms (0.1% glycogen) in the synthetic reaction. The activities of amyloplast lysates, soluble protein fractions of kernel crude extract and leaf crude extract (90 μg of proteins were loaded in a well) on zymograms are shown in A1, B1 and C1 respectively, with their respective immunoblots A2, B2 and C2 which are probed with peptide specific anti-Pho1 antibodies. APase was used as a negative control.

Figure 2.8: Mobility shift detection of phosphorylated proteins by Phosphate affinity SDS-PAGE using Phos-TagTM Amyloplast lysates (22 DAA) treated with either 1 mM ATP, APase (25unit/ml), or ATP+ PI (phosphatase inhibitor). 30 μg of proteins were loaded in each well. The gel was immunoblot following electrophoresis and probed with peptide- specific anti-SP antibodies and the mobility of the bands was compared with the untreated amyloplast lysates.

2.3.4. Gel filtration chromatography (GPC) analysis of SP

Maize amyloplasts lysates (at 22 DAA) treated with ATP or APase (500

μg/mL of proteins in each) were eluted through a Superdex 200 10/300GL gel permeation column to determine whether ATP or APase treatment influenced the multimeric state of SP. Fractions collected were run on the SDS-PAGE and the elution pattern of the major starch biosynthetic enzymes were analyzed on the immunoblots using peptide specific anti-SP, SSI, SSII, SSIV, SBEI and SBEIIB antibodies (Fig. 2.10B,C,D,E). The elution patterns of SP at early (15 DAA) and

74 late developmental stages (35 DAA) in whole cell crude extracts of the maize endosperm are shown in Fig. 2.10A. The gel permeation column was connected to an AKTA Explorer FPLC was calibrated using commercial protein standards from 13.7 kDa to 440 kDa and the calibration curve developed to estimate the molecular weights of the proteins eluted by GPC is shown in Fig. 2.9.

Both in early and later stages of endosperm development, SP eluted in fractions (fraction 21-23) where the molecular weight corresponds to the tetrameric form of SP (448 kDa). Dimeric forms were not visualized. Amyloplast lysates at 22 DAA, the elution profile of SP was equal in untreated control

(fractions from 7-12) where as the ATP treated and APase treated fractions were respectively from 8-13 and 6-12 (Fig 2.10B). The estimated molecular weights of the eluted SP fractions showed the existence of monomeric (112 kDa), dimeric (112 kDa X 2) and tetrameric forms (112 kDa X 4) of SP. The elution profile of SSI, SSIV, SBEI and SBEIIb were identical regardless of ATP or APase treatments. In contrast, ATP-treated SSII eluted comparatively in low molecular fractions (6-10) compared to APase treated fraction profile (fraction 4-8) (Fig

2.10C). Reprecentative graph of the elution from GPC is shown in Appendix 10.

GPC-fractionated amyloplast lysates (22 DAA) were run on native affinity zymograms. The results indicated that ATP-treated SP eluted in fraction number

25-26 showed SP activity, where as untreated, or APase treated fractions showed SP synthetic activity in fraction number 23-24. Approximate molecular weights of these fractions were investigated as; fraction 23-24 are tetrameric and 25-26 fractions were dimeric forms of SP (Fig. 2.11).

8 7

6 5 4 LogMW 3 2 1 0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 Fraction Number

Figure 2.9: The standard curve developed to analyze the molecular weights of the proteins eluted by GPC. Superdex 200 10/300GL gel permeation column was calibrated using commercial protein standards from 13.7 kDa to 440 kDa. The graph shows the relationship between natural log values of the molecular weight of the commercial proteins versus fraction numbers.

(A)

Figure 2.10A: Gell filtration chromatography (GPC) analysis of maize whole cell crude extracts at 15 DAA and 35 DAA. 0.45 mg of proteins were separated by GPC through a Superdex 200 10/300GL gel permeation column. Elution of SP was detected by immunoblot are shown. The size of the proteins in each fraction was determined by calibrating the column using commercial protein standards from 13.7 kDa to 669 kDa and the sizes of the standards are also indicated. L= protein marker, AP=amyloplast lysates, C=crude extracts before loading onto the column.

(B)

Figure 2.10B: Gel filtration chromatography (GPC) analysis of amyloplast lysates. Maize amyloplast lysates at 22 DAA were treated with 1mM ATP or alkaline phosphatase (APase) (25unit/mL) to the extracts and incubated for 1hr in room temperature. 0.49 mg of protein was separated through a Superdex 200 10/300GL gel permeation column. In total, 45 (500μL each) fractions were collected from each running for the analysis in total; only the fractions where the protein was detected by immunoblot analysis are shown. The SP bands were shown by the arrows. The size of the proteins in each fraction was determined by calibrating the column using commercial protein standards from 13.7 kDa to 669 kDa and the sizes of the standards are also indicated. L= protein marker, AP=amyloplast lysates, C=crude extracts before loading onto the column. Arrows indicate the locations of the corresponding proteins.

(C)

Figure 2.10C: Gel filtration chromatography (GPC) separation of amyloplast stromal proteins. Immunoblots probed with anti-SSI (74 kDa), and anti-SSIIa (85 kDa) antibodies of untreated, ATP- or APase-treated maize amyloplast lysates separated by GPC through a Superdex 200 10/300GL gel permeation column. The protein bands were shown by the arrows. The size of the proteins in each fraction was determined by calibrating the column by commercial protein standards from 13.7 kDa to 669 kDa and the sizes of the standards are also indicated. L= protein marker, AP=amyloplast lysates before loading onto the column.

(D)

Figure 2.10D: Gel filtration chromatography (GPC) separation of amyloplast stromal proteins. Immunoblots probed with anti-SSIV (104 kDa), and anti-SBEI (80 kDa) antibodies of untreated, ATP- or APase-treated maize amyloplast lysates separated by GPC through a Superdex 200 10/300GL gel permeation column. The protein bands were shown by the arrows. The size of the proteins in each fraction was determined by calibrating the column by commercial protein standards from 13.7 kDa to 669 kDa and the sizes of the standards are also indicated. L= protein marker, AP=amyloplast lysates before loading onto the column. Arrows indicate the location of the corresponding proteins.

(E)

Figure 2.10E: Gel filtration chromatography (GPC) analysis of amyloplast lysates. Immunoblots probed with anti-SBEIIb (85 kDa) antibodies of untreated, ATP- or APase-treated maize amyloplast lysates separated by GPC through a Superdex 200 10/300GL gel permeation column. The protein bands were shown by the arrows. The size of the proteins in each fraction was determined by calibrating the column by commercial protein standards from 13.7 kDa to 440 kDa and the sizes of the standards are also indicated. L= protein marker, AP=amyloplast lysates before loading onto the column. Arrows indicate the locations of the protein.

Fraction Numbers Fraction Numbers Fraction Numbers 20 21 22 23 24 25 26 27 28 29 20 21 22 23 24 25 26 27 28 29 20 21 22 23 24 25 26 27 28 29

Untreated Control ATP Treated APase Treated

Figure 2.11: Native affinity synthetic activity SP zymogram of the amyloplast lysates separated by GPC. Untreated, ATP- or APase-treated GPC fractions (10 μg of proteins) were run on native gels containing 0.1% glycogen. Arrows indicate the synthetic activity bands.

2.3.5. The synthetic and phosphorolytic activities of SP with different glucan substrates

The glucan synthetic activity of ATP- or APase-treated SP was quantitatively measured using maltoheptaose, glycogen and amylopectin as glucan primers at 25 mg/mL concentration. Amyloplast lysates were used as the

14 SP source and [U _C]-G-1-P as the glucan donor (Table 2.5). The means of enzyme activities were statistically compared using the One-Way ANOVA (at

P<0.05 level, F=82.74, P=0.00028). (See Appendix 08 for the statistical analysis of ANOVA by Statistix 9 statistical analysis program). The results indicated that synthetic activities of SP were not significantly different in three different glucans in SP present in untreated amyloplast lysates at 25 mg/mL of substrate concentration. The synthetic activity was significantly higher with amylopectin (143.315.2 nmol/mg/min) compared to maltoheptaose 82

(60.0045.6 nmol/mg/min) when amyloplast lysates were treated with ATP.

There was no significant difference in the synthetic activity between maltoheptaose and glycogen within untreated or ATP-treated amyloplasts. SP activities were decreased in all substrates in APase-treated amyloplast lysates compared to both untreated and ATP-treated samples. In addition, synthetic activity was significantly decreased when treated with APase with amylopectin and glycogen compared to ATP-treated SP. The synthetic activity was not significantly decreased in ATP or APase-treated SP when maltoheptaose was used as the glucan primer (Table 2.5).

Phosphatase inhibitor cocktail (PI) was added to inhibit the activity of endogenous phosphatase present in the amyloplast (see Appendix 09 for the details about PI). However, ATP+PI treated SP recorded lower activity compared to ATP treated SP in all three substrates. The APase used was bound to agarose beads (insoluble APase) and it was removed after amyloplast lysates were treated for 1 hour to prevent under estimation of the enzyme activity due to continuous dephosphorylation of substrates in the assays. In general, plastidial

SP had greater activities in synthetic direction over phosphorolytic direction despite ATP or APase treatments or in high or low molecular glucan polymers at

25 mg/mL concentration. SP phosphorolytic activity was not significantly altered within untreated, ATP-treated or APase-treated samples when maltoheptaose was used as the glucan primer (Table 2.5). Phosphorolytic activities of untreated and ATP-treated SP were significantly different from APase-treated SP with amylopectin (Table 2.5) (see Appendix 08 for the statistical analysis on One-way

ANOVA, F= 35.57, P= 0.0004).

The enzyme followed typical saturation kinetics toward amylopectin and maltoheptaose when activity was measured in the phosphorolytic direction. The kinetic data were analyzed using the Michaelis–Menten equation. The Km and

Vmax values of SP in the phosphorolytic direction were analyzed at a range of

(5-25 mg/mL) maltoheptaose and amylopectin concentrations using

Lineweaver–Burk plots (Table 2.6). SP had a higher Km value with maltoheptaose compared to amylopectin in untreated or ATP-treated or APase treated samples. Km values increased with both maltoheptaose and amylopectin with ATP treatment, and decreased with APase treatment compared with the untreated sample values. The Vmax was increased in both substrates following

ATP treatment compared to the untreated samples by 1.3 and 1.2 times in amylopectin and maltoheptaose respectively (Table 2.6).

Table 2.5: Synthetic and phosphorolytic activities of SP in different glucan substrates. Both activities were calculated as nmol/mg/min. Significantly different means (at P<0.05) from the One-way ANOVA followed by LSD are indicated by identical symbols for synthetic and phosphorolytic activities.

Pre-treatment Substrate Untreated ATP ATP+PI PI APase

Synthetic Activity

69.06.0 60.05.6* 60.25.0 59.15.6 27.23.8 Maltoheptaose

99.45.3# 143.35.2*‡ 90.45.0 75.87.4 32.84.5‡# Amylopectin 100.52.6 114.41.0† 73.31.2 74.20.8 37.42.0† Glycogen Phosphorolytic Activity

Maltoheptaose 24.61.5 27.01.4 - - 16.63.4

Amylopectin 46.91.3* 58.84.0# - - 33.41.8*#

Table 2.6: Km and Vmax values of SP in amyloplast lysates in the phosphorolytic direction. The phosphorolytic activity of SP was assayed by spectrophotometry and amount of NADH released was analyzed at 340nm as the amount of G-1-P produced in the reaction. Amylopectin and maltoheptaose concentrations at 5-

25 mg/mL were considered in the calculations. Km and Vmax values were calculated using Lineweaver–Burk plots.

Glucan Substrate Treatment Km (mg/mL) Vmax (nmol/mg/min) Untreated 1.8±0.02 48.3±0.2 Amylopectin ATP 3.1±0.01 65.4±0.06

1.3±0.05 33.7±0.2 APase Untreated 3.3±0.02 27.9±0.01 Maltoheptaose ATP 6.7±0.001 33.9±0.003

2.3±0.001 17.6±0.02 APase

2.3.6. Immunoprecipitation of SP

The immunoprecipitation of SP from the amyloplast lysates was attempted using peptide specific anti-SP antibodies. Native SP protein was not immunoprecipitated by protein A-Sepharose beads (Fig. 2.12), therefore co- immunoprecipitation was not possible. SP was not immunoprecipitated by anti-

SP antibodies bound to Protein A-sepharose beads after removing the SSIIa present in amyloplast lysates indicated that SSIIa is not cover up antibodies binding epitopes of SP (Appendix 02). Recombinant maize SP with a S-tag was produced by over expressing the protein in E.coli. The biochemical and proteomic characterization, and protein-protein interaction studies using the recombinant SP is discussed in Chapter 3.

Figure: 2.12: Immunoprecipitation of SP by peptide specific anti-SP antibodies (30 mg/mL) with 1 mL amyloplast lysates. 40 μL of 50% (w/v) Protein A- Sepharose beads slurry made in phosphate buffer saline (PBS) was used to pull down the Protein A-Sepharose-antibody-proteins complex. The immunoprecipitated pellet was boiled in 2X SDS loading buffer and separated on SDS-PAGE followed by immunoblot analysis. Pre-immune serum was used as a control to show the specificity of the purified antibodies. Figure illustrates the immunoblot probed with SP-specific antibodies. The arrows denote the SP band. L= protein marker.

2.4. Discussion

2.4.1. Subcellular localization of SP in maize endosperm

The overall objective of the study was to elucidate the role and regulation of plastidial starch phosphorylase (SP), and to investigate the possible post translational modifications of SP in wild-type maize endosperm. The subcellular localization of SP was tested at 22 DAA which corresponds with the maximal period of starch synthesis in maize endosperm (9-24 DAA) (Yu et al. 2001; Tsai and Nelson, 1968) and the time when all the major starch biosynthetic enzymes are expressed and active in amyloplasts (Liu et al. 2009; Hennen-Bierwagen et al. 2008). The peptide specific anti-SP antibodies recognized the plastidial SP only in the storage starch synthesizing amyloplasts and not in the transient starch synthesizing chloroplasts (Fig. 2.2B). The plastidial form of SP in chloroplasts may be structurally different from the SP in the amyloplasts within the same species. Degradation of the proteins in crude extracts may be a possible reason for SP not being detected effectively by antibodies. Mutant analysis suggested that the plastidial SP present in Arabidopsis thaliana leaves is not involved in transient starch biosynthesis or degradation (Zeeman et al.

2004). The SP mutants of Arabidopsis showed no change in the activity of other enzymes of starch metabolism or show any significant change in the total accumulation of starch or the starch structure during the day or its remobilization at night (Zeeman et al. 2004). Also leaves contain the cytosolic form of SP (Pho2) abundantly compared to the plastidial SP (Satoh et al. 2008) which was not detected in leaf crude extracts with the antibodies.

The results presented here confirmed the previous findings that the Pho1 is exclusively found in the amyloplast stroma (Satoh et al. 2008; Grimaud et al.

2008; Yu et al, 2001) in the maize amyloplast (Fig. 2.2). The existence of the SP and other SSs and SBE in the interface of the soluble fraction and the granule, as the granule surface imbedded protein suggests their involvement in granule synthesis. The soluble protein fractions collected after the repeated washings of the granules with the amyloplast rupturing buffer and tested on immunoblots indicated that some of the major starch biosynthetic enzymes are present at the granule surface (Fig. 2.3). SP was present up to the fifth wash indicating the tight association with the surface of the starch granule. Similarly, SSI and

SBEIIb (wash 1 to 3) and SBEI (wash 1 to 4) were also associated with the granule periphery. In contrast, SSII was found clearly from wash 1 to 7 (Fig.

2.2B) which is comparatively abundant in the granule surface. By contrast, SSIV and the SSIII were found only in the first extract of the amyloplast and may be regulated as purely soluble. SSIV and SSIII are either not present in the granule surface or present at the extremely low levels in the granule surface. In the wild-type maize amyloplast stroma, it has been demonstrated that the protein present in the assembly of heteromeric protein complexes (SSI, SSII and

SBEIIb) are also entrapped in the starch granule (Liu et al. 2009). However, the

SP is regulated by protein complex formation with SBEI and SBEIIb in wheat amyloplasts (Tetlow et al. 2004) but the components of this protein complex do not appear to become entrapped in the starch granule. SP was only found as a granule-associated protein in the ae- background when it was found to be associated with SSI and SSIIa (Liu et al. 2009; Grimaud et al. 2008).

We were unable to conduct standard immuno-precipitation experiments using the anti-maize SP antibodies since they did not appear to recognize the native protein, and could only detect the protein after SDS-PAGE (Fig 2.12). The reason for this is unclear, but the epitope (SVASDRDVQGPVS located at 73-85 amino acids in N-terminal) present on the SP monomer may well be hidden when the native SP adopts is natural multimeric (dimeric and tetrameric) configuration.

As Tickle et al. 2009 proposed, SP may contribute to starch synthesis by operating in two ways in the cereal endosperm. First, it has been suggested that

SP may degrade soluble malto-oligosaccharides in the stroma, produced via the action of DBE, to G-1-P and then to ADP-glucose by ADP-glucose pyrophosphorylase to produce starch. Second, SP may directly act at the surface of the starch granule, where it functions to phosphorolytically modify the structure of starch to provide suitable substrates for other starch biosynthetic enzymes, ultimately producing G-1-P which can be recycled back to produce starch. Both of the models suggested the effect of SP on starch synthesis by providing G-1-P via the degradative process to produce ADP-glucose by AGPase enzyme. Data in this thesis support a role of SP operating at the granule surface as SP localization experiments clearly show SP associated with starch granule

(Fig. 2.3; 2.4). Analyses of metabolites in the amyloplast also indicate high Pi/G-

1-P levels which could suggest that the phosphorolytic SP reaction is favored in vitro (Fettke et al. 2010; Schupp and Ziegler, 2004). In contrast, previous studies suggested that SP exists in the storage starch biosynthetic tissues, and operates in the synthetic reaction in monocots where it is available throughout

89 the endosperm development (Schupp and Ziegler, 2004; Satoh et al. 2008; Yu et al. 2001). Recently, Hwang et al, (2010) showed that the SP reaction in rice endosperm amyloplasts is predominantly synthetic even in the presence of high

Pi levels.

2.4.2. The synthetic activity of plastidial SP in developing maize endosperm

The synthetic activity of SP in the amyloplast lysates was tested by SP- native zymogram analysis using glycogen as a substrate. SP was active throughout the endosperm development at stages measured (12, 15, 19 and 22

DAA) (Fig. 2.5). The activity of SP is being found as early as 9 DAA in the maize endosperm and remains active throughout the endosperm development (Yu et al. 2001). In the 22 DAA amyloplast lysates, SP showed activity in both synthetic and degradative directions when tested on native affinity zymograms, which were respectively incubated with G-1-P and Pi (Fig. 2.6). Accumulation of

Pho1 was detected throughout the endosperm development in maize was similarly observed in wheat endosperm during 8-31 DAA and Pho1 was undetectable until 8 DAA and reached to the maximum level at 18 DAA and remained constant (Tickle et al. 2009). The presence of Pho1 in cereal endosperm correlates with the presence of other starch biosynthetic enzymes;

SBEI, SBEII, AGPase and SSs (Liu et al., 2009; Tetlow et al. 2003; Morell et al.

1997; Ainsworth et al. 1995) suggesting that Pho1 may be involved in starch biosynthesis or be involved in functional interaction with other starch biosynthetic enzymes.

2.4.3. Investigating the regulation of SP by protein phosphorylation

SP in cereal endosperms has been found to be regulated by protein phosphorylation (Liu et al. 2009; Pollack, 2009; Tetlow et al. 2004). However, the activities of the ATP-treated and APase-treated isoforms of SP on 0.1% glycogen zymograms showed no detectable differences in the activities between treatments (Fig. 2.7). The glycogen affinity SP-native zymogram may not be sensitive enough to detect subtle alterations in catalytic activity associated with phosphorylation.

The mobility shift detection of proteins on phosphate affinity SDS-PAGE using Acrylamide-pendant Phos-TagTM (10 μΜ/mL) (Fig. 2.8) showed no difference in mobility in ATP-treated or untreated SP suggesting SP is not phosphorylated. However, the Pi present in the amyloplast lysates may have affected the activity of SP.

2.4.4. Gel filtration chromatography analysis of SP

Phosphorylases exist as homodimers or homotetramers and have similar kinetic and structural properties, but their regulatory mechanisms may vary depending on the source of the enzyme in higher plants (Brisson et al. 1989) in bacterial forms (Dauvillée et al. 2006) and yeast (Tanabe et al. 1987). Gel filtration chromatography studies revealed that the native enzyme consisted of two identical subunits in maize (Mu et al. 2001). In the present study, the SP was found in multimeric, tetrameric, and dimeric forms in both early (15 DAA) and late (35 DAA) developmental stages (Fig. 2.10 A) in endosperm crude extracts and at 22 DAA in maize wild-type amyloplast lysates (Fig. 2.10B),

91 which has been observed previously (Liu et al. 2009). Seed crude extracts of 15

DAA and 35 DAA had showed similar elution profiles for SP from 21-23 fractions and amyloplast lysates at 22 DAA had wider elution profiles (from fraction 21 to

26) that may be due to less dimeric form of SP in crude extracts. In addition, monomeric, dimeric and tetrameric forms of SP separated by GPC were tested for the synthetic activity by native SP zymogram (Fig. 2.11). Synthetic activity of ATP-treated SP showed an apparent molecular weight approximately similar to the dimeric form (fraction 25-26), untreated and APase-treated SP showed activity in (fraction 22-23) the fractions corresponding to a molecular weight equal to a tetrameric form, suggested that the dimeric forms were more active compared to the tetrameric forms, when the amyloplast lysates were treated with ATP (Fig 2.11).

Phosphorylation may effect the multimeric status of SP. However, no detectable difference in the elution profiles of phosphorylated and dephosphorylated amyloplast lysates was observed (Fig 2.10B). The SP involved in heteromeric protein complex formation with SSI and SSIIa recorded in the ae1 mutant showed the same elution profile as in wild-type (Liu et al. 2009) suggesting that the observed elution profile of SP may be made up of a variety of different SP-containing protein complexes as well as SP monomers.

Immunoblot analysis of the GPC fractions illustrated that SSIV (104 kDa) and SBEI (80 kDa), SBEIIb (85 kDa) showed no difference in their elution profiles following ATP or APase treatment. However, SBEI and ATP-treated

SBEIIb eluted in two different molecular groups; high apparent mass (greater than the expected size of monomer) low apparent mass consistent with the

92 expected monomeric mass. The results of SBEIIb elution can be explained by the phosphorylation dependent SBEIIb complex formation previously observed in maize and wheat amyloplasts (Liu et al. 2009; Tetlow et al. 2008). In contrast, there is no evidence for the existence of homo dimeric or multimeric forms of SBEI in wild-type maize amyloplasts. The higher molecular mass fractions of SBEI are therefore probably due to the formation of heteromeric protein complexes containing SBEI. We observed no alteration of SSIV elution following ATP or APase treatment (Fig.2.10D). In ATP treated lysates, SSI eluted comparatively higher apparent molecular mass fractions (6-13) than in the untreated and dephosphorylated treatments (Fig 2.10B), as previously observed in Liu et al. (2009). In wild-type maize amyloplast stroma, SSI, SSIIa and

SBEIIb form a phosphorylation-dependent heteromeric protein complex (Liu et al. 2009). By contrast, SSIIa eluted in higher molecular fractions when the enzyme was dephosphorylated (Fig 2.8B, Table 2.1) suggesting that the dephosphorylated SSIIa may form protein–protein interactions or complex formation in wild-type maize amyloplasts. This suggestion is further supported by Liu et al. (2009) that the [γ-32P]ATP treated ae1 mutant and wild-type amyloplast lysates immunoprecipitated with anti-maize SSIIa antibodies showed that SBEIIb in wild-type and SBEI and SP in ae1 mutant were phosphorylated but no evidence for phosphorylation of SSII in the complex.

The effect of phosphorylation on the monomeric, dimeric and tetrameric forms of SP and their involvement of protein-protein interactions are discussed in Chapter 3 using a catalytically active recombinant maize SP containing an S- protein affinity tag.

2.4.5. The synthetic and phosphorylitic activity of SP in different glucan substrates

Glucan synthetic activity was significantly less with maltoheptaose cpmpared with amylopectin and glycogen in untreated, ATP or APase-treated SP

(Table 2.3) and the synthetic activity was significantly higher following ATP treatment with amylopectin and glycogen compared to maltoheptaose indicating that the activity of plastidial SP was greater with high molecular mass, branched glucans. This was similarly observed in recombinant plastidial SP in rice; the ratio between the activities of synthetic and dedradative reaction rate

(equilibrium constant) was higher in amylopectin (45), compared to maltopentaose (G5), maltohexaose (G6), maltoheptaose (G7), and amylose, respectively as 22, 19, 15, and 17 (Hwang et al. 2010). Synthetic activity of SP was inhibited by Pi produced in the reaction [inhibition constant (Ki) = 0.69 mM] when amylopectin was used as the primer substrate, but this inhibition is less

(Ki = 14.2 mM) when short α-glucan chains are used as primers and also extends them to synthesize longer MOSs (DP= 4–19) (Hwang et al. 2010). This observation suggested that under physiological conditions of high Pi/G-1-P, Pho1 extends the chain length of short MOSs which can then be used as subsequent primer by starch synthase activities (Hwang et al. 2010).

Phosphatase inhibitor cocktail (PI) was added to inhibit the activity of endogenous protein phosphatases. But its addition did not increase the synthetic activity compared with ATP-treated samples with glycogen and amylopectin, suggesting that some compound in PI cocktail mixture may have inhibited the activity of SP.

The activity of SP in ATP and APase-treated amyloplast lysates in phosphorolytic direction was greater in amylopectin in untreated, ATP or APase- treated SP compared to maltoheptaose (Table 2.5). In contrast, Km was greater in maltoheptaose over amylopectin in ATP-treated SP (Table 2.6). Similarly, the kinetics analysis of purified SP from maize endosperm recorded that the phosphorylitic reaction was favored over the synthetic reaction when malto- oligosaccharides were used as the substrate (Mu et al. 2001). The Vmax and Km values recorded in this study were approximately 7 and 5.8 times lower than with purified SP respectively (Mu et al. 2001). Unlike in the purified form of SP, the activity of SP present in amyloplast lysates may be regulated by other enzymes [e.g. SBEs (Nakamura et al. 2012)], and other metabolites. For instance, G-1-P and Pi present in amyloplast lysates and high Pi/G-1-P ratios are considered as the controlling mechanism of SP activity (Tiessen et al. 2011; Mu et al. 2001; Fettke et al. 2009; Schupp and Ziegler, 2004). However, according to the findings of Hwang et al. (2010), incorporation of [U14_C]-G-1-P into starch was only partially affected by the concentration of Pi in rice. Even under physiological G-1-P substrate levels (0.2 mM) and 50-fold excess of Pi in vitro than the normal physiological level, the Pho1 from of rice was able to carry out the biosynthetic reaction (Hwang et al. 2010). ADP-glucose, the major precursor for starch biosynthesis, inhibits the activity of SP in the synthetic direction

(Dauvillée et al. 2006; Matheson and Richardson, 1978) and may reduce the activity of plastidial SP in amyloplast lysates. The effect of ADP-glucose on plastidial SP in maize was not tested in this study.

The preference of SP for different α-glucans has been studied in many plant species (Young et al. 2006; Dauvillée et al. 2006; Yu et al. 2001; Mori et al. 1993; Shimomura et al. 1982; Liddle et al. 1961). In contrast to maize SP, in sweet potato tubers plastidial SP showed a low binding affinity toward starch and a high affinity toward low molecular weight linear malto-oligosaccharides

(MOS) (Young et al. 2006). In contrast, the cytosolic isoform, has a high affinity towards highly branched polyglucan amylopectin (Young et al. 2006). The synthetic activity of SP with amylopectin (Km =0.13 mg/mL) is higher when compared to the highly branched glycogen (Km=0.45 mg/mL) in maize (Yu et al. 2001), in potato tubers (Liddle et al. 1961) and in spinach leaves

(Shimomura et al. 1982). In sweet potato tubers, the L-78 amino acid peptide insertion located in the middle of the plastidial form of SP, appears to block the binding site of SP to high molecular weight α-glucans (Young et al. 2006). We found no evidence for the proteolytic cleavage of the L-78 peptide in maize endosperm amyloplasts.

In this chapter, experiments were carried out to investigate the regulatory properties of SP in maize amyloplasts. Plastidial SP is present only in the amyloplast stroma and is not found as a granule associated protein which is in agreement with previous studies (Grimaud et al. 2008). SP remains active throughout the endosperm development and it is present in homodimeric or tetrameric configurations throughout the developmental stages analyzed in this study. This study suggested that the tetrameric and dimeric forms have different catalytic activities and may be involved in starch biosynthesis by differential regulation. The SP elution profile by GPC was not altered by ATP or APase

96 treatments suggesting phosphorylation may not alter the multimeric status of

SP. The synthetic and phosphorylitic activity assays showed that SP was active in both directions. However, SP showed greater activities with amylopectin compared to glycogen and maltoheptaose in both synthetic and phosphorylitic directions. ATP treated SP showed higher activities in both directions with amylopectin, indicating that ATP may be involved in regulating SP by phosphorylation. Protein-protein interactions with the plastidial enzyme could not be detected by co-immunoprecipitation, since the native SP was unable to be immunoprecipitated by Protein-A sepharose beads. The development of a S- tagged recombinant SP was used in future experiments to analyze protein- protein interactions involving SP; these experiments are described in Chapter 3.

CHAPTER 3

Using recombinant plastidial SP to understand the regulation of starch biosynthesis

3.1. Introduction

Glucan-phosphorylases are widely distributed enzymes in bacteria, plant and animal tissues (Dauvillée et al. 2006; Weinhäusel et al. 1996; Newgard et al. 1989; Tanabe et al. 1987; Preiss, 1984). SP catalyzes both synthesis and degradation of the α-glucan polymers. The structure and the function of these enzymes are best understood for glycogen phosphorylases, the SP counterpart of animals and bacteria (Dauvillée et al. 2006; Weinhäusel et al. 1996; Newgard et al. 1989). Glycogen phosphorylase (GP) plays an important role by initiating the degradation of glycogen in glycogen metabolism (Dauvillée et al. 2006;

Roach, 2002; Fischer et al. 1971). Predominantly, the physiological function of

SP was considered phosphorolytic rather than to synthesize glucan polymers, is based on the observations in glycogen phosphorylase in animal system and that

SP has a low affinity for G-1-P (Schupp and Ziegler 2004); Preiss and Sivak

1996).

SP has been shown to be regulated by protein phosphorylation in plants

(Pollack, 2009; Grimaud et al. 2008; Tetlow et al. 2004). GP in animal systems is found to be coordinated with the activity of glycogen synthase, GP is required to be phosphorylated in order to activate the glycogen synthases (Carabaza et al. 1992; Johnson, 1992; Madsen 1991) to regulate glycogen synthesis and breakdown. Structural changes of GP occur at the interface of the subunits as a result of conformational transition at the amino terminus by protein

99 phosphorylation; residues surrounding the phosphorylation site (serine-14) that participate in intrasubunit interactions in the dephopsphorylated state are observed to adapt alternate side-chain conformations in the phosphorylated state, enabaling them to form intersubunit interactions to form homodimeric structure of GP (Sprang et al. 1988).

SP present in storage starch synthesizing tissues in plants is suggested to be involved in starch synthesis since SP is active throughout endosperm development in cereals (Tickle et al. 2009; Satoh et al. 2008; Schupp and

Ziegler, 2004; Mu et al. 2001). Also, the shrunken 4 mutants which lack SP activity in maize endosperm resulted in reduced starch contents (Tsai and

Nelson, 1969), and SP mutants in rice produced shrunken endosperm phenotypes with low starch contents (Satoh et al. 2008). Further, SP does not appear to influence the starch degradation in Arabidopsis thaliana (Zeeman et al. 2004) suggesting SP plays a more dominant role in the storage starch biosynthesis. In addition, the SP-L isoform (plastidial form of SP which has lower affinity towards the high molecular starch) in potato tubers and the chimeric form of SP-L enzyme which was developed by replacing the 18% residue sequence of the SP-L isoform including a part of 78-residue insertion, were over expressed in E.coli and the affinities of purified forms of recombinant proteins were compared by Mori et al. (1993). The purified chimeric phosphorylase was five times less active in synthetic direction than the parental type SP-L isoform.

However, the affinity of the chimeric phosphorylase for glycogen (Km= 23.8 mg/mL) and amylopectin (Km=5.3 mg/mL) was much higher than that of the type SP-L isoform (Km=10400, Km=82 mg/mL mg/mL respectively in glycogen

100 and amylopectin) and only slightly lower than that of the cytosolic SP-H, the high affinity isoform. These results provide evidence for the role of the unique

78-residue insertion present in plant plastidial SP sequences, which lowers the affinity of the enzyme for large, branched substrates (Mori et al. 1993).

A possible function of SP in starch biosynthesis is that SP acts on malto oligosaccharide (MOS) which are liberated by the activity of debranching enzymes (DBE) to produce linear maltodextrin (MD) of a length sufficient for a subsequent branching reaction by starch branching enzymes (SBE) (Nakamura et al. 2012; Ball and Morell 2003). In addition, functional interactions between

SP and SBE isoforms were observed in rice endosperm strongly suggesting that

SP and SBE have mutual capacities for chain elongation and chain branching

(Nakamura et al. 2012). Purified SP from rice endosperm synthesized glucans from G-1-P in the presence of SBE, without any exogenous glucan primer and glucan production was higher with SBEI compared to SBEIIa or SBEIIb

(Nakamura et al. 2012). Physical interaction between SP, SBEI and SBEIIb was also recorded in wheat amyloplasts and this protein complex was assembled in a phosphorylation dependent manner (Tetlow et al. 2004). Based on the observations in ss4 and ss4/sp mutants in Arabidopsis leaves, which produce reduced numbers of starch granules with increased granule surface (Roland et al. 2008; Planchot et al. 2008), it has been suggested that SP may be involved in granule initiation in starch biosynthesis process via functional or physical interactions between SP and SSIV (Roland et al. 2008; Planchot et al. 2008).

Investigating possible interactions of SP with other starch biosynthetic enzyme

101 isoforms is therefore important to elucidate the role and regulation of SP in storage starch biosynthesis in maize amyloplasts.

All phosphorylases exist as dimers or tetramers of identical subunits

(Dauvillée et al. 2006; Mu et al. 2001; Brisson et al. 1989; Sprang et al. 1988;

Tanabe et al. 1987). In Chlamydomonas reinhardtii, identical subunits of dimeric form have similar kinetic and structural properties, but their regulatory mechanisms may vary (Dauvillée et al. 2006). In maize amyloplasts, SP is present as dimeric and tetrameric assembles (Mu et al. 2001; Liu et al. 2009).

However, catalytic and regulatory mechanisms of these individual configurations are not well characterized in higher plants.

Previous work showed that available SP antibodies are not capable of effectively immunoprecipitating native SP in protein-protein interaction experiments. We therefore decided to provide a recombinant maize SP for such studies. In this chapter we discuss the production of catalytically active, S- tagged SP recombinant proteins from wild-type maize endosperm and the biochemical characterization of the recombinant SP and the investigations of the possible interactions of SP with other starch biosynthetic enzymes. GPC analysis showed that the S-tagged recombinant SP is present in tetrameric and dimeric forms which were also observed in the amyloplast lysates, and these fractions were found as valuable tools in understanding their diverse regulatory mechanisms. The synthetic and degradative activities of these different recombinant SP configurations in different glucan polymers and their regulation by protein-protein interactions are discussed.

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3.2. Materials and Methods

3.2.1. RNA extraction from maize endosperm and synthesis of cDNA

The RNA was extracted from maize endosperm at 22 DAA by using the QIAGEN,

RNeasy Plant Mini Kit (Catalog No. 74104). Approximately, 100 mg of the frozen maize endosperm was used in a sample. First strand cDNA was synthesized from

RNA by using Fermentas RevertAidTM H Minus Strand cDNA Synthesis kit

(Catalog No. K1631) following manufacturer’s recommendations with some modifications. The mixture of 5 μL RNA (100 μg/mL), 1 μL Oligo DT primer (0.5

0 μg/ μL), 6 μL RNase free H2O was mixed and incubated at 70 C for 5 min. and chilled on ice. Then, 4 μL 5X reaction buffer, 1 μL RiboLock ribonuclease inhibitor, 2 μL 10 mM dNTPs were added to the mix and incubated 370C for 5 min. 1 μL RevertAidTM H Minus M-MuLV-RT reverse transcriptase was added and incubated further at 420C for 1hr. After stopping the reaction by heating at 700C for 10 min, the complementary RNA was removed by RNase H (0.5 Μl/ 29 μL reaction) and further incubated 370C for 20 min. The cDNA was stored in -200C.

3.2.2. Quantification of nucleic acid

The amount of RNA and DNA were measured in a NanoDrop 2000 (Thermo

Fisher Scientific) spectrophotometer at the wavelength of 260 nm, the optical density (OD) of 1 corresponds to a concentration of 50 μg/mL for double- stranded DNA and 38 μg/mL for the RNA.

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3.2.3. Agarose gel electrophoresis

Agarose gel electrophoresis was carried out using standard procedures commonly use. Agarose was added to TAE buffer (0.04M TRIS-acetate, 1 mM

EDTA; pH 8.0) to make the final concentration of 0.8-1% (w/v) and heated in a microwave until completely dissolved. The resulting solution was allowed to cool for approximately 5 minutes before the addition of ethidium bromide to a final

concentration of 0.2 μg/mL and pouring into an appropriately sized horizontal electrophoresis unit. Upon setting, the gel was overlaid with TAE buffer. Samples were subsequently mixed with 0.16 volumes loading buffer (30% glycerol [v/v],

0.25% bromophenol blue [w/v]) and loaded onto the gel. Electrophoresis was carried out at 80V for 1-1.5 hours. Nucleic acids immobilized in agarose gels were visualized on a gel documentation system.

3.2.4. Designing oligo-nucleotide primers and RT-PCR

The complete mRNA sequence (3053 bp) of plastidial maize SP (GenBank:

EU857640.2) was taken from the National Center for Biotechnology Information data base (NCBI). The transit peptide (TP) sequence was detected as 70 amino acids by using ChloroP 1.1 sequence analytical server after analyzing the correct protein frame in the GeneRunner program. The coding sequence including a part of TP sequence was isolated using the forward primer (SP-F1) 5’

GCGGAGGTGGGGTTCTCCT 3’ and the reverse primer (SP-R1) 5’

GCGAAAGAACCTGATATCCAC 3’. The PCR product was purified from the agarose gel by using QIAquick Gel Extraction Kit (QIAGEN, Cat. No. 28704). 50-100

-1 ng.mL was used as the template in next PCR to obtain the complete mRNA

104 sequence of the plastidial SP. The next PCR primers were specifically designed for the CloneEZ® PCR Cloning Kit (GenScript, Cat. No. L00339) with a 15 bp overhang sequence from the vector system pET29a on both forward (SP-F2) and reverse (SP-R2) primers as the forward (SP-F2)

5’GGTTCCATGGCTGATTCAGCGCGCAGCG 3’ and the reverse (SP-R2)

5’GAATTCGGATCCGATCTAGGGAAGGATGGC 3’ (15 bp overhangs are underlined). All forward and reverse primers were used as 30 pmol/ μL final concentration in a 50 μL of the PCR reaction contained final concentration of 50-

-1 100 ng.mL of the DNA template with 10 μL DMSO, 4 μL of 25 mM MgSO4, 10 μL of 2 mM dNTPs, and 2 μL of KOD Hot Start DNA Polymerase (Novagen, 200 U,

Cat. No. 71086-3). The same PCR program was run with both sets of primers as

3 cycles of Loop 1; 980C for 15 seconds, 420C for 30 seconds and 680C for

3.5min. followed by 35 cycles of Loop 2; 980C for 15 seconds, 600C for 30 seconds and 680C for 3.5min and the reaction was further extended at 680C for

10 min. The PCR product was purified from the gel as described before to use in the ligation. The consensus and complementary cDNA sequences and the primers designed are shown in Fig 3.1.

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Forward Primers

15bp overhang from -209bp -221bp pET29a vector SP-F2 SP-F1 GGTTCCATGGCTGATTCAGCGCGCAGCG 127bp- GCGGAGGTGGGGTTCTCCT -145bp

Consensus DNA sequence 121bp- 5’GTGGCGGCGGAGGTGGGGTTCTCCTCGCTGGCGGTTCCGGAGGTGGGGTCGCGCCAGGTTGGGGTCGAGGGCGATTGCAGCGGCGGGTGTCAGCGCGCAGCGTGGCGA 5’ -230bp Complementary DNA sequence 3’CACCGCCGCC TCCACCCCAAGAGGAGCGACCGCCAAGGCCTCCACCCCAGCGCGGTCCAACCCCAGCTCCCGCTAACGTCGCCGCCCACAGTCGCGCGTCGCACCGCT 3’

Reverse Primers

Consensus DNA sequence 2291bp 5’ GCCATCCTTCCCTAG ACCAGGTGGATATCAGGTTCTTT CGCC TATATTT C 3’- 3039bp Complementary DNA sequence 3’ CGGTAGGAAGGGATCTGGTCCACCTATAGTCCAAGAAAGCGGATATAAAG – 5’

CACCTATAGTCCAAGAAAGCG SP-R1 -3010bp 3030bp- GAATTCGGATCCGATCTAGGGAAGGATGGC SP-R2 15bp overhang from -3005bp pET29a vector -2291bp

Figure 3.1: Schematic diagram of the consensus and complementary strands showing the forward and reverse primers use to isolate the complete cDNA sequence of the plastidial SP from maize. The coding sequence including a part of TP sequence was isolated using the forward primer (SP-F1) 5’ GCGGAGGTGGGGTTCTCCT 3’ and the reverse primer (SP-R1) 3’CACCTATAGTCCAAGAAAGCG 5’. The PCR product was purified from the agarose gel and used in next PCR with forward (SP-F2) 5’GGTTCCATGGCTGATTCAGCGCGCAGCG 3’ and reverse (SP-R2) 3’GAATTCGGATCCGATCTAGGGAAGGATGGC 5’ primers with a 15 bp overhang sequence from the vector system pET29a on both primers.

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3.2.5. Ligation of complete SP cDNA sequence to the pET29a expression vector and transformation to DH5α competent cells

The complete coding sequence of SP in the PCR product was confirmed by gene sequence analysis (Appendix 01) The pET29a expression vector encoded a

15 amino acid S-tag (LysGluThrAlaAlaAlaLysPheGluArgGlnHisMetAspSer) at the

N-terminus with a thrombin digestion site (LeuValProArgGlySer), and a T7 promoter (TAATACGACTCACTAT) (Fig. 3.2). 20 μL of ligation mixture was prepared by adding 8 μL of purified PCR (300 ng/ μL), 8 μL of linearized vector

(100-200 ng/μl), 2 μL 10X CloneEZ® buffer, 2 μL CloneEZ® ligation enzyme in the CloneEZ® PCR Cloning Kit (GenScript, Cat. No. L00339) and incubated in room temperature for 40 min. and transferred to ice for 5 min. Then, 8 μL of ligated mix was added to 50 μL of DH5α competent cells and the mix was kept on ice for 30 min. The transformation was done by a heat shock at 420C for 90 seconds with a quick transfer to ice for 5 min. and 600 μL of SOC bacterial growth media (super optimal broth with catabolic repressor, 20 mM glucose) was added to the transformed mix and incubated at 370C on a rotor for 1hr.

Then the cells were plated on 10 mL solid LB media contained 10 μL of 50 mM kanamycin after remove the excess media by centrifugation and incubated overnight at 370C. A single colony was grown in 6 mL of LB media contained 6

μL of 50 mM kanamycin overnight at 370C and the plasmid DNA was extracted by using QIAprep Spin Miniprep Kit. Successful insertion of the SP sequence was identified after restricted enzyme digested plasmid DNA was run on an agarose gel. Then, 2 μL of 100-150 ng/mL of the plasmid DNA with the correct size of the insert was used for transformation into the Arctic Express expression cells

107 described above. The transformed cells were grown on a plate contained 10 mL of solid LB media, 10 μL of 50 mM of kanamycin and 10 μL of 100 mM of gentamycin and incubated overnight at 370C.

3.2.6. Expression of plastidial maize SP in Escherichia coli

An individual colony of the Arctic express E.coli with the insert was grown in 6 μL of liquid LB broth with 6 μL of 50 mM kanamycin and 6 μL of 100 mM of gentamycin and incubated overnight at 370C on a shaker. Then the cultures were further grown in LB liquid media without the selection antibiotics and the expression of the recombinant protein was induced by adding the final concentration of 1 mM IPTG (isopropyl β-D-1-thiogalactopyranoside) when the density of the culture was at 0.5-0.6 at OD600. The cultures were further kept in 100C, and shaken at 250 rpm for 16 hrs. The E. coli cells were collected by centrifugation (at 13,000xg at for 20 min.) lysed using ‘BugBuster’ Protein

Extraction Reagent’ (Novagen catalogue no. 70584) and the soluble fraction containing recombinant SP was collected. The expression level of the protein was tested by running on SDS-PAGE gel followed by Coomassie staining.

(Appendix 05 shows the alignment comparison of the predicted amino acid sequence of SP with the amino acid sequence of the recombinant SP produced in the study).

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3.2.7. Testing the synthetic and degradative activity of recombinant SP using glycogen affinity native zymogram

The glycogen affinity zymogarm analyses were carried out by using soluble recombinant protein of SP. The zymogram gel preparations, electrophoresis and, incubation were carried out as described in chapter 2

(2.2.2.5.3) to test the synthetic and degradative activity.

3.2.8. Gel filtration chromatography analysis of the recombinant SP

The soluble extract of recombinant SP (1.5 mg of total protein) was eluted through a Superdex 200 10/300GL gel permeation column pre-equilibrated with two column volumes of rupturing buffer using an AKTA- FPLC system

(Amershamp Pharmacia Biotech model No. 01068808). In total, 30 (500 μL each) fractions were collected. The column was calibrated using commercial protein standards from 13.7 kDa to 669 kDa (GE Healthcare Gel Filtration

Calibration Kits low molecular and high molecular weight) and the fractions contained different multimeric forms of SP were identified using immunoblotting.

3.2.9. Immobilization of recombinant SP on S-Protein Agarose beads and pulldown assay

The S-tagged GPC fractions of tetrameric, dimeric and monomeric forms of SP were each immobilized to S-protein agarose beads (Novagen, catalogue no. 69704) as described by Liu et al. (2009) with some modifications. 67.5 μg of different recombinant SP GPC fractions were, incubated in room temperature on a rotator with 0.5 mg/mL of amyloplast lysates pretreated earlier with 1mM ATP,

109 or alkaline phosphatase (APase, the insoluble form of suspension in (NH4)SO4 in agarose beads, final conc. 25 units/1ml) or untreated amyloplast lysates. The

APase in beads were removed after incubation by centrifugation. 250 μL of 50%

(v/v/) S-protein agarose beads slurry prepared in buffer (20 mM Tris-HCl pH

7.5, 150 mM NaCl, 0.1% (v/v) Triton X-100, 0.1% (w/v) Na azide) was added and further incubated for 1 hour. The controls were prepared by incubating the

ATP, APase and untreated amyloplast lysates with S-agarose beads without the recombinant GPC fractions. The mixture was transferred onto a 10 mL Bio-Rad

Polyprep chromatography column (Bio-Rad, catalogue no. 731-1550) and washed with 300 mL washing buffer [20 mM TRIS-HCl pH 7.5, 150 mM NaCl,

0.1% (v/v) Triton X-100)] to remove non-specifically bound proteins from the beads. The controls were prepared by incubating the amyloplast lysates with the same amount of S-agarose beads without the recombinant GPC fractions. The washed pellets of S-agarose protein bead complex, was then transferred back into a micro-centrifuge tube and centrifuged at 40C for 5 min at 500xg micro centrifuge. Following the removal of the supernatant, the pellet was boiled in

100 μL of 20mM Tris-HCl pH 7.5 and 5X SDS-loading buffer (0.31M Tri-HCl pH

6.75, 25% (v/v) 2-mercaptoethanol, 10% (w/v) SDS, 50% (v/v) glycerol,

0.005% (w/v) Bromophenol Blue) for 6 min at 950C. The proteins in the samples were analyzed by SDS-PAGE and immunoblotting with primary antibodies of anti

SSI, -SSII, SSIII, SSIV, SBEI, SBEIIa, SBEIIb, SP and S-tag specific antibodies.

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Figure. 3.2: Novagen pET29a vector used to overexpress plastidial SP. The expression vector contained a 15 amino acid S-tag on the N-terminus with a thrombin digestion site, and a T7 promoter.

3.2.10. Starch phosphorylase glucan synthetic activity assay

The synthetic activity of the SP recombinant protein in amylopectin, glycogen and maltoheptaose substrates was analyzed in vitro by using the tetrameric and dimeric forms of the enzyme obtained from the GPC analysis by using the procedure described earlier in Chapter 2, section 2.2.2.4.2.1. Total protein content in a reaction was 15.15 μg.

3.2.11. Starch phosphorylase glucan degradative activity assay

SP phospholytic activity was determined as previously described in Chapter 2, section 2.2.2.4.2.2. by using dimeric and tetrameric forms of recombinant SP.

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3. 3. Results

3.3.1. Comparison of the protein sequence of plastidial SP of maize endosperm from the cytosolic form and other species

The protein sequences of the plastidial SP of maize endosperm (SP1,

Gene Bank: ACF94692.1), Ipomoea batatas tubers (GenBank: AAA63271.1),

Oryza sativa endosperm (Japonica type, GenBank: BAG49328.1), Triticum aestivum endosperm (GenBank: ACC59201.1), Solanum tuberosum tuber

(GenBank: CAA52036.1) and the cytosolic form of maize (SP2, Gene Bank:

ACF94691.1) were aligned by using CLUSTAL 2.1 multiple sequence alignment program (Fig. 3.3). The transit peptide sequence (TP) of maize SP was analyzed and predicted to be 70 amino acids in size using the ChloroP1.1 sequence analytical server, and is indicated in green (Fig. 3.3). The L-80 insertion of plastidial form of maize is located at 510-590 amino acids (highlighted in red).

The epitope sequence of the synthetic peptide used to develop anti SP specific antibodies (YSYDELMGSLEGNEGYGRADYFLV) is located at 916-940 amino acids in the C-terminus. In addition, the serine, threonine and tyrosine residues of predicted phosphorylation sites of plastidial SP were analyzed using NetPhos 2.0

Server. The results indicated that 28 serine residues are present in the protein sequence except the TP, and 25% of the total serine residues are located in the

L-80 insertion. Also 28.5% of the total threonine residues are present in the L-

80 insertion but none of the tyrosine residues are located in the insert (Fig. 3.4).

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CLUSTAL 2.1 multiple sequence alignment

sweetpotato ------MSRLSG---ITPRARDDRSQFQNPR--LEIAVPDRTAGLQRTK 38 potato ------MATANGAHLFNHYSSNSRFIHFTSRNTSSKLFLTKTSHFRRPK 43 SP1 LISPHASHRHSTARAAMATTTSPPLQLASASRPHAS--ASGGGGGGGVLLAGGSGGGVAP 58 rice ------MATASAPLQLATASRPLPVGVGCGGGGGGGLHVGGARGGGAAP 43 wheat ------MATASPP--LATAFRPLAA---AGGAGGGGAHAVGAAG-RVAP 37 SP2 ------

sweetpotato ------RTLLVKCVLDETKQTIQHVVTEKN-----EGTLLDAASIASSIKYHAEFSPA 85 potato ------RCFHVNNTLSEK---IHHPITEQGGESDLSSFAPDAASITSSIKYHAEFTPV 92 SP1 GWGRGRLQRRVSARSVASDRD--VQGPVSPAE-GLPSVLNSIGSSAIASNIKHHAEFAPL 115 rice ------ARRRLAVRSVASDRG--VQGSVSPEE-EISSVLNSIDSSTIASNIKHHAEFTPV 94 wheat R----RGRRGFVVRSVASDRE--VRGPASTEE-ELSAVLTSIDSSAIASNIQHHADFTPL 90 SP2 ------MPEIKCGAAEK---VKPAASPEA------EKPADIAGNISYHAQYSPH 39 . : :: : .: *:..*.:**:::* sweetpotato FSPERFELPKAYFATAQSVRDALIVNWNATYDYYEKLNMKQAYYLSMEFLQGRALLNAIG 145 potato FSPERFELPKAFFATAQSVRDSLLINWNATYDIYEKLNMKQAYYLSMEFLQGRALLNAIG 152 SP1 FSPDHFSPLKAYHATAKSVLDALLINWNATYDYYNKMNVKQAYYLSMEFLQGRALTNAIG 175 rice FSPEHFSPLKAYHATAKSVLDTLIMNWNATYDYYDRTNVKQAYYLSMEFLQGRALTNAVG 154 wheat FSPEHSSPLKAYHATAKSVFDSLIINWNATYDYYNKVNAKQAYYLSMEFLQGRALTNAIG 150 SP2 FSPFAFGPEQAFYATAESVRDHLIQRWNETYLHFHKTDPKQTYYLSMEYLQGRALTNAVG 99 *** :*:.***:** * *: .** ** :.: : **:******:****** **:* sweetpotato NLELTGEYAEALNKLGHNLENVASKEPDAALGNGGLGRLASCFLDSLATLNYPAWGYGLR 205 potato NLELTGDFAEALKNLGHNLENVASQEPDAALGNGGLGRLASCFLDSLATLNYPAWGYGLR 212 SP1 NLEITGEYAEALKQLGQNLEDVASQEPDAALGNGGLGRLASCFLDSLATLNYPAWGYGLR 235 rice NLELTGQYAEALQQLGHSLEDVATQEPDAALGNGGLGRLASCFLDSLATLNYPAWGYGLR 214 wheat NLELTGQYAEALKQLGQNLEDVASQEPDPALGNGGLGRLASCFLDSMATLNYPAWGYGLR 210 SP2 NLGITGAYAEAVKKFGYELEALAGQEKDAALGNGGLGRLASCFLDSMATLNLPAWGYGLR 159 ** :** :***::::* .** :* :* *.*****************:**** ******** sweetpotato YKYGLFKQRITKDGQEEVAEDWLELGNPWEIIRMDVSYPVKFFGKVITGSDGKKHWIGGE 265 potato YKYGLFKQRITKDGQEEVAEDWLEIGSPWEVVRNDVSYPIKFYGKVSTGSDGKRYWIGGE 272 SP1 YEYGLFKQIITKDGQEEIAENWLEMGYPWEVVRNDVSYPVKFYGKVVEGTDGRKHWIGGE 295 rice YKHGLFKQIITKDGQEEVAENWLEMGNPWEIVRTDVSYPVKFYGKVVEGTDGRMHWIGGE 274 wheat YRYGLFKQIIAKDGQEEVAENWLEMGNPWEIVRNDVSYPVKFYGKVVEGTDGRKHWIGGE 270 SP2 YRYGLFKQHIAKEGQEEVAEDWLDKFSPWEIPRHDVVFPVRFFGHVEILPDGSRKLVGGE 219 *.:***** *:*:****:**:**: ***: * ** :*::*:*:* .** :*** sweetpotato DILAVAYDVPIPGYKTRTTISLRLWSTKVPSEDFDLYSFNAGEHTKACEAQANAEKICYI 325 potato DIKAVAYDVPIPGYKTRTTISLRLWSTQVPSADFDLSAFNAGEHTKACEAQANAEKICYI 332 SP1 NIKAVAHDVPIPGYKTRTTNNLRLWSTTVPAQDFDLAAFNSGDHTKAYEAHLNAKKICHI 355 rice NIKVVAHDIPIPGYKTKTTNNLRLWSTTVPSQDFDLEAFNAGDHASAYEAHLNAEKICHV 334 wheat NIKAVAHDVPIPGYKTKTTNNLRLWSTTVPSQNFDLGAFNAGDHAKANEAHLNAEKICHV 330 SP2 VLKALAYDVPIPGYKTKNAISLRLWEAKATAEDFNLFQFNDGQYESAAQLHARAQQICAV 279 : .:*:*:*******:.: .****.: ..: :*:* ** *:: .* : : .*::** : sweetpotato LYPGDESIEGKILRLKQQYTLCSASLQDIIARFERRSGEYVK--WEEFPEKVAVQMNDTH 383 potato LYPGDESEEGKILRLKQQYTLCSASLQDIISRFERRSGDRIK--WEEFPEKVAVQMNDTH 390 SP1 LYPGDESLEGKVLRLKQQYTLCSASLQDIIARFESRAGESLN--WEDFPSKVAVQMNDTH 413 rice LYPGDESPEGKVLRLKQQYTLCSASLQDIIARFERRAGDSLS--WEDFPSKVAVQMNDTH 392 wheat LYPGDESSEGKILRLKQQYTLCSASLQDIISRFESRAGDSLN--WEDFPSKVAVQMNDTH 388 SP2 LYPGDATEEGKLLRLKQQFFLCSASLQDMIARFKERKSDRVSGKWSEFPTKVAVQLNDTH 339 ***** : ***:******: ********:*:**: * .: :. *.:** *****:**** sweetpotato PTLCIPELIRILIDLKGLSWKEAWNITQRTVAYTNHTVLPEALEKWSYELMEKLLPRHIE 443 potato PTLCIPELMRILIDLKGLNWNEAWNITQRTVAYTNHTVLPEALEKWSYELMQKLLPRHVE 450 SP1 PTLCIPELMRILMDVKGLSWSEAWSITERTVAYTNHTVLPEALEKWSLDIMQKLLPRHVE 473 rice PTLCIPELMRILIDVKGLSWNEAWSITERTVAYTNHTVLPEALEKWSLDIMQKLLPRHVE 452 wheat PTLCIPELMRILMDIKGLSWNEAWSITERTVAYTNHTVLPEALEKWSLDIMQKLLPRHVE 448 SP2 PTLAIPELMRLLMDEEGLGWDEAWDITYRTISYTNHTVLPEALEKWSQIVMRKLLPRHME 399 ***.****:*:*:* :**.*.***.** **::*************** :*.******:*

113 sweetpotato IIEMIDEQLINEIVSEYGTSDLDMLEKKLNDMRILENFDIPSSIANLFTKPKETSIVDPS 503 potato IIEAIDEELVHEIVLKYGSMDLNKLEEKLTTMRILENFDLPSSVAELFIKP-EISVDDDT 509 SP1 IIETIDEELINNIVSKYGTTDTELLKKKLKEMRILDNVDLPASISQLFVKPKDKKESPAK 533 rice IIEKIDGELMNIIISKYGTEDTSLLKKKIKEMRILDNIDLPDSIAKLFVKPKEKKESPAK 512 wheat IIETIDEKLMNNIVSKYGTADISLLKQKLKDMRILDNVDLPASVAKLFIKPKEKTG---- 504 SP2 IIEEIDKRFKELVISKH-----KEMEGKIDSMKVLD------430 *** ** .: . :: :: . :: *: *::*: sweetpotato EEVEVSGKVVTESVEVSDKVVTESEKDE------LEEKDTELEKDED------P 545 potato ETVEVH-----DKVEASDKVVTNDEDDTGKKTSVKIEAAAEKDIDKKTPVS------P 556 SP1 SKQKLLVKSLETIVDVEEKTELEEEAEVLSEIEEEKLESEEVEAEEESSED---ELDPFV 590 rice LKEKLLVKSLEPSVVVEEKTVSKVEINEDSEEVEVDSE-EVVEAENEDSED---ELDPFV 568 wheat ---KLLVQSLESIAEGDEKTESQEEENILSETAEKKGGSDSEEAPDAEKEDPVYELDPFA 561 SP2 ------

sweetpotato VPAPIPPKMVRMANLCVVGGHAVNGVAEIHSDIVKEDVFNDFYQLWPEKFQNKTNGVTPR 605 potato EPAVIPPKKVRMANLCVVGGHAVNGVAEIHSEIVKEEVFNDFYELWPEKFQNKTNGVTPR 616 SP1 KSDPKLPRVVRMANLCVVGGHSVNGVAEIHSEIVKQDVFNSFYEMWPTKFQNKTNGVTPR 650 rice KSDPKLPRVVRMANLCVVGGHSVNGVAAIHSEIVKEDVFNSFYEMWPAKFQNKTNGVTPR 628 wheat KYDPQLPRVVRMANLCVVGGHSVNGVAEIHSEIVKQDVFNSFYEMWPTKFQNKTNGVTPR 621 SP2 NSNPQKP-VVRMANLCVVSSHTVNGVAELHSNILKQELFADYVSIWPTKFQNKTNGITPR 489 * *********..*:***** :**:*:*:::* .: .:** ********:*** sweetpotato RWIRFCNPALSNIITKWIGTEDWVLNTEKLAELRKFADNEDLQIEWRAAKRSNKVKVASF 665 potato RWIRFCNPPLSAIITKWTGTEDWVLKTEKLAELQKFADNEDLQNEWREAKRSNKIKVVSF 676 SP1 RWIRFCNPALSALISKWIGSDDWVLNTDKLAELKKFADNEDLHSEWRAAKKANKMKVVSL 710 rice RWIRFCNPELSAIISKWIGSDDWVLNTDKLAELKKFADDEDLQSEWRAAKKANKVKVVSL 688 wheat RWIRFCNPELSAIISKWIGSDDWILNTDKLAGLKKFADDEDLQSEWRTAKRNNKMKVVSL 681 SP2 RWLRFCNPELSEIVTKWLKSDQWTSNLDLLTGLRKFADDEKLHAEWAAAKLSCKKRLAKH 549 **:***** ** :::** :::* : : *: *:****:*.*: ** ** * ::.. sweetpotato LKERTGYSVSPNAMFDIQVKRIHEYKRQLLNILGIVYRYKQMKEMSAREREAKFVPRVCI 725 potato LKEKTGYSVVPDAMFDIQVKRIHEYKRQLLNIFGIVYRYKKMKEMTAAERKTNFVPRVCI 736 SP1 IREKTGYIVSPDAMFDVQVKRIHEYKRQLLNILGIVYRYKKMKEMSTEERAKSFVPRVCI 770 rice IREKTGYIVSPDAMFDVQVKRIHEYKRQLLNILGIVYRYKKMKEMSAKDRINSFVPRVCI 748 wheat IRDKTGYVVSPDAMFDVQVKRIHEYKRQLLNILGIVYRYKKMKEMSAKDRIKSFVPRVCI 741 SP2 VLDVTGVTIDPTSLFDIQIKRIHEYKRQLLNILGAVYRYKKLKGMSAEEK-QKVTPRTVM 608 : : ** : * ::**:*:*************:* *****::* *:: :: ...**. : sweetpotato FGGKAFATYVQAKRIAKFITDVGATINHDPEIGDLLKVIFVPDYNVSAAELLIPASGLSQ 785 potato FGGKAFATYVQAKRIVKFITDVGATINHDPEIGDLLKVVFVPDYNVSVAELLIPASDLSE 796 SP1 FGGKAFATYIQAKRIVKFITDVAATVNHDSDIGDLLKVVFVPDYNVSVAEALIPASELSQ 830 rice FGGKAFATYVQAKRIVKFITDVAATVNHDPEIGDLLKVVFIPDYNVSVAEALIPASELSQ 808 wheat FGGKAFATYVQAKRIVKFITDVAATVNYDPDVGDLLKVVFVPDYNVSVAEKLIPASELSQ 801 SP2 IGGKAFATYTNAKRIVKLVNDVGAVVNNDPEVNKYLKVVFIPNYNVSVAEVLIPGSELSQ 668 :******** :****.*::.**.*.:* *.::.. ***:*:*:****.** ***.* **: sweetpotato HISTAGMEASGQSNMKFAMNGCILIGTLDGANVEIRQEVGEENFFLFGAEAHEIAGLRKE 845 potato HISTAGMEASGTSNMKFAMNGCIQIGTLDGANVEIREEVGEENFFLFGAQAHEIAGLRKE 856 SP1 HISTAGMEASGTSNMKFAMNGCILIGTLDGANVEIREEVGEENFFLFGAEAHEIAGLRKE 890 rice HISTAGMEASGTSNMKFAMNGCILIGTLDGANVEIREEVGEENFFLFGAEAHEIAGLRKE 868 wheat HISTAGMEASGTSNMKFAMNGCILIGTLDGANVEIREEVGEENFFLFGAHAPEIAGLRQE 861 SP2 HISTAGMEASGTSNMKFSLNGCVIIGTLDGANVEIREEVGEDNFFLFGAKADEVAGLRKD 728 *********** *****::***: ************:****:*******.* *:****:: sweetpotato RAEGKFVPDERFEEVKEFIKRGVFGSNTYDELLGSLEGNEGFGRGDYFLVGKDFPSYIEC 905 potato RADGKFVPDERFEEVKEFVRSGAFGSYNYDDLIGSLEGNEGFGRADYFLVGKDFPSYIEC 916 SP1 RAEGKFVPDPRFEEVKEFVRSGVFGTYSYDELMGSLEGNEGYGRADYFLVGKDFPSYIEC 950 rice RAQGKFVPDPRFEEVKRFVRSGVFGTYNYDDLMGSLEGNEGYGRADYFLVGKDFPSYIEC 928 wheat RAEGKFVPDPRFEEVKEYVRSGVFGTSNYDELMGSLEGNEGYGRADYFLVGKDFPSYIEC 921 SP2 RENGLFKPDPRFEEAKQFIRSGAFGSYDYEPLLDSLEGNSGFGRGDYFLVGYDFPSYIDA 788 * :* * ** ****.*.::: *.**: *: *:.*****.*:**.****** ******:. sweetpotato QEKVDEAYRDQKIWTRMSILNTAGSYKFSSDRTIHEYAKDIWNIQPVVFP 955 potato QEKVDEAYRDQKRWTTMSILNTAGSYKFSSDRTIHEYAKDIWNIEAVEIA 966 SP1 QEKVDEAYRDQKLWTRMSILNTAGSSKFSSDRTIHEYAKDIWDISPAILP 1000 rice QEKVDKAYRDQKLWTRMSILNTASSSKFNSDRTIHEYAKDIWDIKPVILP 978 wheat QQKVDEAYRDQKLWTRMSILNTAGSPKFSSDRTIHEYAKDIWDISPVIMP 971 SP2 QDRVDAAYKDKKKWTKMSILNTAGSGKFSSDRTIAQYAKEIWDIKASPVV 838 *::** **:*:* ** *******.* **.***** :***:**:*.. .

114

Figure 3.3: The protein sequences of the plastidial SP of maize endosperm (SP1, Gene Bank: ACF94692.1), the cytosolic form of maize (SP2, Gene Bank: ACF94691.1), Ipomoea batatas tubers (GenBank: AAA63271.1), Oryza sativa endosperm (Japonica type, GenBank: BAG49328.1), Triticum aestivum endosperm (GenBank: ACC59201.1), and Solanum tuberosum tuber (GenBank: CAA52036.1) were aligned by using CLUSTAL 2.1 multiple sequence alignment program. The Transit peptide sequence (TP) of maize SP (70 amino acids) is indicated in green. The L-80 insertion of plastidial form of maize is located at 510-590 amino acids (highlighted in red). The epitope sequence for the synthetic peptide used to develop anti SP specific antibodies (YSYDELMGSLEGNEGYGRADYFLV) is located at 916-940 amino acids.

115

NetPhos 2.0 Server - prediction results

Serine predictions

Name Pos Context Score Pred ______v______Sequence 800 VNHDSDIGD 0.526 *S* Sequence 3 --LISPHAS 0.014 . Sequence 817 DYNVSVAEA 0.179 . Sequence 7 SPHASHRHS 0.927 *S* Sequence 826 LIPASELSQ 0.075 . Sequence 11 SHRHSTARA 0.996 *S* Sequence 829 ASELSQHIS 0.164 . Sequence 22 ATTTSPPLQ 0.159 . Sequence 833 SQHISTAGM 0.013 . Sequence 29 LQLASASRP 0.126 . Sequence 840 GMEASGTSN 0.020 . Sequence 31 LASASRPHA 0.020 . Sequence 843 ASGTSNMKF 0.053 . Sequence 36 RPHASASGG 0.363 . Sequence 911 EFVRSGVFG 0.433 . Sequence 38 HASASGGGG 0.637 *S* Sequence 918 FGTYSYDEL 0.124 . Sequence 52 LAGGSGGGV 0.006 . Sequence 925 ELMGSLEGN 0.913 *S* Sequence 70 QRRVSARSV 0.995 *S* Sequence 946 KDFPSYIEC 0.610 *S* Sequence 73 VSARSVASD 0.987 *S* Sequence 968 WTRMSILNT 0.561 *S* Sequence 76 RSVASDRDV 0.989 *S* Sequence 975 NTAGSSKFS 0.933 *S* Sequence 85 QGPVSPAEG 0.993 *S* Sequence 976 TAGSSKFSS 0.468 . Sequence 92 EGLPSVLNS 0.160 . Sequence 979 SSKFSSDRT 0.988 *S* Sequence 96 SVLNSIGSS 0.495 . Sequence 980 SKFSSDRTI 0.808 *S* Sequence 99 NSIGSSAIA 0.006 . Sequence 995 IWDISPAIL 0.037 . Sequence 100 SIGSSAIAS 0.023 . Sequence 104 SAIASNIKH 0.058 . Sequence 117 APLFSPDHF 0.144 . Sequence 122 PDHFSPLKA 0.242 . Sequence 133 ATAKSVLDA 0.898 *S* Sequence 161 AYYLSMEFL 0.018 . Sequence 199 EDVASQEPD 0.852 *S* Sequence 216 GRLASCFLD 0.007 . Sequence 221 CFLDSLATL 0.003 . Sequence 272 RNDVSYPVK 0.018 . Sequence 321 LRLWSTTVP 0.075 . Sequence 336 AAFNSGDHT 0.018 . Sequence 362 PGDESLEGK 0.826 *S* Sequence 378 YTLCSASLQ 0.009 . Sequence 380 LCSASLQDI 0.882 *S* Sequence 390 ARFESRAGE 0.713 *S* Sequence 395 RAGESLNWE 0.546 *S* Sequence 403 EDFPSKVAV 0.004 . Sequence 432 VKGLSWSEA 0.992 *S* Sequence 434 GLSWSEAWS 0.040 . Sequence 438 SEAWSITER 0.375 . Sequence 460 LEKWSLDIM 0.004 . Sequence 488 NNIVSKYGT 0.777 *S* Sequence 516 DLPASISQL 0.296 . Sequence 518 PASISQLFV 0.004 . Sequence 530 DKKESPAKS 0.994 *S* Sequence 534 SPAKSKQKL 0.584 *S* Sequence 542 LLVKSLETI 0.725 *S* Sequence 563 AEVLSEIEE 0.985 *S* Sequence 572 EKLESEEVE 0.973 *S* Sequence 581 AEEESSEDE 0.996 *S* Sequence 582 EEESSEDEL 0.993 *S* Sequence 592 PFVKSDPKL 0.138 . Sequence 612 VGGHSVNGV 0.038 . Sequence 621 AEIHSEIVK 0.012 . Sequence 631 DVFNSFYEM 0.041 . Sequence 661 NPALSALIS 0.019 . Sequence 665 SALISKWIG 0.004 . Sequence 670 KWIGSDDWV 0.009 . Sequence 694 EDLHSEWRA 0.465 . Sequence 709 MKVVSLIRE 0.953 *S* Sequence 720 GYIVSPDAM 0.052 . Sequence 756 MKEMSTEER 0.996 *S* Sequence 763 ERAKSFVPR 0.944 *S*

116

______^______^______

Threonine predictions Tyrosine predictions

Name Pos Context Score Pred Name Pos Context Score Pred ______v______v______Sequence 12 HRHSTARAA 0.985 *T* Sequence 127 PLKAYHATA 0.057 . Sequence 19 AAMATTTSP 0.074 . Sequence 146 WNATYDYYN 0.411 . Sequence 20 AMATTTSPP 0.697 *T* Sequence 148 ATYDYYNKM 0.262 . Sequence 21 MATTTSPPL 0.660 *T* Sequence 149 TYDYYNKMN 0.559 *Y* Sequence 130 AYHATAKSV 0.546 *T* Sequence 158 VKQAYYLSM 0.035 . Sequence 145 NWNATYDYY 0.020 . Sequence 159 KQAYYLSME 0.385 . Sequence 171 GRALTNAIG 0.117 . Sequence 183 ITGEYAEAL 0.332 . Sequence 180 NLEITGEYA 0.032 . Sequence 227 ATLNYPAWG 0.043 . Sequence 224 DSLATLNYP 0.021 . Sequence 232 PAWGYGLRY 0.042 . Sequence 246 KQIITKDGQ 0.099 . Sequence 236 YGLRYEYGL 0.176 . Sequence 285 VVEGTDGRK 0.186 . Sequence 238 LRYEYGLFK 0.010 . Sequence 311 PGYKTRTTN 0.063 . Sequence 262 LEMGYPWEV 0.023 . Sequence 313 YKTRTTNNL 0.107 . Sequence 273 NDVSYPVKF 0.124 . Sequence 314 KTRTTNNLR 0.341 . Sequence 278 PVKFYGKVV 0.016 . Sequence 322 RLWSTTVPA 0.615 *T* Sequence 309 PIPGYKTRT 0.269 . Sequence 323 LWSTTVPAQ 0.024 . Sequence 343 HTKAYEAHL 0.125 . Sequence 340 SGDHTKAYE 0.029 . Sequence 357 CHILYPGDE 0.013 . Sequence 375 KQQYTLCSA 0.238 . Sequence 374 LKQQYTLCS 0.035 . Sequence 412 QMNDTHPTL 0.028 . Sequence 446 RTVAYTNHT 0.780 *Y* Sequence 415 DTHPTLCIP 0.513 *T* Sequence 490 IVSKYGTTD 0.134 . Sequence 440 AWSITERTV 0.309 . Sequence 633 FNSFYEMWP 0.768 *Y* Sequence 443 ITERTVAYT 0.150 . Sequence 717 EKTGYIVSP 0.980 *Y* Sequence 447 TVAYTNHTV 0.013 . Sequence 735 RIHEYKRQL 0.049 . Sequence 450 YTNHTVLPE 0.063 . Sequence 747 LGIVYRYKK 0.009 . Sequence 477 EIIETIDEE 0.921 *T* Sequence 749 IVYRYKKMK 0.033 . Sequence 492 SKYGTTDTE 0.274 . Sequence 779 AFATYIQAK 0.207 . Sequence 493 KYGTTDTEL 0.367 . Sequence 814 FVPDYNVSV 0.357 . Sequence 495 GTTDTELLK 0.233 . Sequence 917 VFGTYSYDE 0.025 . Sequence 545 KSLETIVDV 0.637 *T* Sequence 919 GTYSYDELM 0.045 . Sequence 553 VEEKTELEE 0.855 *T* Sequence 932 GNEGYGRAD 0.911 *Y* Sequence 638 EMWPTKFQN 0.195 . Sequence 937 GRADYFLVG 0.162 . Sequence 644 FQNKTNGVT 0.031 . Sequence 947 DFPSYIECQ 0.744 *Y* Sequence 648 TNGVTPRRW 0.569 *T* Sequence 958 VDEAYRDQK 0.770 *Y* Sequence 677 WVLNTDKLA 0.471 . Sequence 987 TIHEYAKDI 0.017 . Sequence 715 IREKTGYIV 0.920 *T* ______^______Sequence 757 KEMSTEERA 0.420 . Sequence 778 KAFATYIQA 0.089 . Sequence 790 VKFITDVAA 0.051 . Sequence 795 DVAATVNHD 0.134 . Sequence 834 QHISTAGME 0.075 . Sequence 842 EASGTSNMK 0.158 . Sequence 857 ILIGTLDGA 0.499 . Sequence 916 GVFGTYSYD 0.027 . Sequence 965 QKLWTRMSI 0.007 . Sequence 972 SILNTAGSS 0.033 . Sequence 983 SSDRTIHEY 0.468

Figure 3.4. The predicted phosphorylation sites of the plastidial maize SP protein sequence were analyzed using NetPhos 2.0 Server.

117

3.3.2. Development of recombinant SP

3.3.2.1. PCR

The complete mRNA sequence (3053 bp) of plastidial SP of maize

(GenBank: EU857640.2) was obtained from the National Center for

Biotechnology Information data base (NCBI). Initially, the coding sequence including a part of TP sequence was isolated using the forward primer (SP-F1) 5’

GCGGAGGTGGGGTTCTCCT 3’ and the reverse primer (SP-R1) 5’

GCGAAAGAACCTGATATCCAC 3’ and the purified PCR product was used as the template in next PCR to obtain the 2805 bp of complete mRNA coding sequence which produces plastidial SP with 935 amino acids. Fig 3.5 shows the PCR product of the full length sequence (2805 bp) of SP visualized on a agarose gel.

For the next PCR, the primers were specifically designed for the CloneEZ® PCR

Cloning Kit (GenScript, Cat. No. L00339) with a 15 bp overhang sequence from the vector system pET29a on both forward (SP-F2) and reverse (SP-R2) primers to facilitate the homologous recombination. (Appendix 03 and 04 shows the sequences of all the primers used in the study in PCR and sequence analysis).

118

bp 5000 3000 2805bp

2000

Figure 3.5: The PCR product of complete mRNA coding sequence (2805 bp) of plastidial SP of maize was visualized on a 1% (w/v) agarose gel contained ethidium bromide.

3.3.2.2. Testing the expression level and the synthetic and degradative activity of recombinant SP on glycogen affinity zymogram

The expression level of the cloned gene was qualitatively tested by SDS-

PAGE analysis of produced proteins (Fig. 3.6). Soluble extract of recombinant SP obtained after the culture was induced by 1mM IPTG was run on the gel (30 μg of total protein per well) and compared with equal amounts of soluble proteins obtained from the uninduced cultures (control), the induced Arctic Expression

E.coli cells without the plasmid, induced Arctic Expression E.coli cells only with

119 the plasmid but without the insert (Fig. 3.6A). Induced E.coli cells with the insert

(Lane 1) showed higher level of expression and the immunoblot probed with anti-SP specific antibodies confirmed the higher expression was due to recombinant SP (Fig 3.6B).

The synthetic activity of the recombinant SP was analyzed on 0.1% glycogen affinity zymogram (Fig 3.7A). 90 μg of proteins were run on the zymogram. The soluble recombinant proteins obtained after the cultures were induced by 1 mM IPTG (Lane 1) showed higher activity than the amyloplast lysates (Lane 6). There was no activity observed in the soluble fractions of induced Arctic Express cells without plasmid (Lane 2), uninduced Arctic Express cells with both the plasmid and the insert (Lane 3), and induced Arctic Express cells with the plasmid (Lane 4) or in uninduced Arctic Express cells with the plasmid but without the insert (Lane 5). The immunoblot of the zymogram probed with anti-SP specific antibody recognized the SP in the recombinant soluble fraction (Fig. 3.7B). However, the faint band in lane 3 in uninduced culture in the immunoblot is due to the leaky promoter since there was no band observed in other samples (Fig. 3.7B). Corresponding immunoblots of the native zymogram of SP recombinant proteins showed four distinct bands and may represent the monomeric, dimeric, tetrameric and multimeric (consisting of more than four subunits) configurations of the recombinant SP (Fig. 3.7B).

The synthetic activity and degradative activity of the recombinant protein was qualitatively tested on the zymogram by incubating the zymogram gel in 20 mM of G-1-P and Na2HPO4 as the inorganic phosphate substrate respectively

(Fig. 3.8). Multiple bands on the samples may correspondent to the different

120 multimeric forms (dimeric and tetrameric) of SP. The observation that the activity bands shown in synthetic activity zymogram disappeared in the degradative activity zymogram (3.8D) indicates that the recombinant SP is active in both synthetic and degradative directions in a manner that is similar to the SP presence in the amyloplast lysates (Fig. 3.8).

121

(A) (B)

L 1 2 3 4 5 6 L 1 2 3 4 5 6 kDa 150 100 75

L – Protein marker

1. Recombinant SP obtained after the cultures were induced by 1mM IPTG

2. Uninduced control

3. Only the induced Arctic Expression E-coli cells without the plasmid

4. and 5. Induced Arctic Expression E-coli cells with the plasmid, no insert

6. Amyloplast lysates

Figure 3.6: Over expression of recombinant SP in Arctic express E.coli was analyzed by running the soluble recombinant protein on a 10% SDS gel followed by Coomassie staining (A) and immunoblot analyses by probing with anti-SP specific antibodies (B). 30 μg of proteins were run in each lane. The expression of the recombinant protein was induced by adding the final concentration of 1 mM IPTG (Lane 1), Uninduced cultures (Lane 2), IPTG induced Arctic Express cells without the plasmid (Lane 3), IPTG induced Arctic Express cells with the plasmid but without the insert (Lane 4 and 5) and the amyloplast lysates(Lane 6) are shown. Arrow indicated the expressed SP in lane 1.

122

(A)

(B)

Figure 3.7: The synthetic activity of recombinant SP in a glycogen affinity native zymogram that contained 0.1% glycogen in the gel (A) and corresponding immunoblot of the native zymogram probed with anti-SP specific antibodies (B) are shown. 90 μg of proteins were run in a well and following electrophoresis, the native gel was incubated overnight at 280C with the incubation buffer containing 0.1% glycogen and 20 mM G-1-P in the synthetic direction. The activity bands were visualized by Lugol’s solution and are indicated with arrows (A). Multiple bands which were recognized by SP-specific antibodies on immunoblot are shown by arrows (B).

123

(A) (B) (C) (D)

lysates lysates lysates lysates

SP Recombinant Amyloplast SP Recombinant Amyloplast SP Recombinant Amyloplast SP Recombinant Amyloplast

SP synthetic Immunoblot of SP Immunoblot of SP SP degradative activity zymogram synthetic zymogram activity zymogram synthetic zymogram probed with anti-SP probed with anti-S- antibodies tag antibodies

Figure 3.8: Testing the synthetic and degradative activity of recombinant SP on glycogen affinity native zymogram. The synthetic activity of recombinant SP in glycogen affinity native zymogram (A) and the corresponding immunoblot of the zymogram probed with anti-SP specific antibodies (B), immunoblot probed with anti-S-tag antibodies (C), and degradative activity on zymogram (D) are shown. 30 μg of protein were run in a well and following electrophoresis, the native gel was incubated overnight at 280C with the incubation buffer contained 20 mM G- 1-P.in the synthetic direction (A) and 20 mM sodium phosphate dibasic

(Na2HPO4) in phosphorylitic direction (D). Bands were visualized by Lugol’s solution. Suggested dimeric and multimeric forms of SP and are indicated with arrows.

124

3.3.3. Gel filtration chromatography analysis of recombinant SP

The soluble fraction of the recombinant SP was separated through a

Superdex 200 10/300GL gel permeation column and the fractions collected were analyzed by SDS-PAGE and immunoblotting using peptide specific anti-SP antibodies (Fig. 3.9A). Recombinant SP was eluted in for different peaks and the predicted molecular weights of the eluted SP fractions (based on the elution of the standards) showed the existence of monomeric (112 kDa), dimeric (112 kDa

X 2), tetrameric (112 kDa X 4) and multimeric forms (more than four subunits).

The synthetic activity of the various multimers of recombinant SP was tested on the native zymograms by loading the equal amounts of proteins on the gel (Fig.

3.9B). Activity bands were observed in the dimeric, tetrameric and multimeric forms but no activity was detected in the monomeric form on the zymogram

(Fig. 3.9B).

125

(A)

(B)

(C)

Figure 3.9: Gel filtration chromatography (GPC) analysis of recombinant SP. Recombinant SP soluble fraction was separated by GPC through a Superdex 200 10/300GL gel permeation column. The fractions were run (30 μg of proteins in a well) on SDS-PAGE followed by immunoblot analysis with anti-SP antibodies. Monomeric (112 kDa) dimeric, tetrameric and multimeric forms of SP were detected (A). Fractions containing SP were tested for synthetic activity on a glycogen affinity zymogram (B) and corresponding immunoblot of the zymogram probed with the anti-SP specific antibodies (C). The SP bands correspond to the various SP multimers and are shown by the arrows and the fraction numbers of the bands were shown. The sizes of the known protein standards eluted in the column were indicated in the boxes. AP=amyloplast lysates.

126

3.3.4. Immobilization of recombinant SP on S-Protein Agarose beads

The S-tagged GPC fractions of tetrameric, dimeric and monomeric forms of SP were separately immobilized to S-protein agarose beads following incubation with 0.5 mg/mL of pretreated amyloplast lysates. The success of immobilization of the recombinant tetrameric and dimeric forms to the S- agarose beads was tested by probing immunoblots of washed beads with anti-SP specific and anti S-tag specific antibodies (Fig. 3.10). Both the dimeric and tetrameric SP incubated with both untreated and ATP-treated amyloplast lysates showed very strong immuno-reactive bands. The tetrameric form showed nonspecific binding with the proteins in the amyloplast lysates; however, the level of binding is negligible when compare with the immobilized samples (Fig.

3.10).

To test the protein-protein interactions of the tetrameric and dimeric forms of recombinant SP with major starch biosynthetic enzymes, the beads containing protein complexes were separated on SDS-PAGE gels and immunoblots probed with various peptide-specific antibodies. Interactions were observed between recombinant SP forms only with SSIIa, SBEI and SBEIIb (Fig.

3.11). The tetrameric form of recombinant SP interacted with SSIIa and SBEI when the amyloplast was treated with ATP, but not in the untreated amyloplast lysates or APase treated samples. In contrast, there was no interaction with

SBEIIb and the tetrameric form. In ATP-treated amyloplasts SBEI and SBEIIb interact with the dimeric form but not with the ATP treated SSIIa. SSIIa interacted with the dimeric form of SP in the untreated amyloplast lysates.

Further, the interaction between SBEI and dimeric forms was independent of

127

ATP treatment. The dimeric form of SP showed much stronger interaction with

SBEIIb in ATP-treated sample than in the untreated samples. The APase-treated samples did not show any interaction with any of the enzymes tested. Fig. 3.13 is a schematic diagram summarizing the possible interactions of the recombinant forms of SP with SSIIa, SBEI and SBEIIb enzymes.

128

Tetrameric form of SP Dimeric form of SP L 1 2 3 4 5 6 7 8 L 1 2 3 4 5 6 7 8 kDa 150

100

75 Anti-SP

L 1 2 3 4 5 6 7 8 L 1 2 3 4 5 6 7 8 kDa 150

100

75 Anti-S-Tag

1. Untreated amyloplast lysates (AP) incubated with recombinant SP forms 2. ATP Treated AP incubated with recombinant SP forms 3. APase Treated incubated with recombinant SP forms 4. Beads+ Untreated AP (no recombinant SP forms) 5. Beads+ ATP treated AP (no recombinant SP forms) 6. Beads+ APase treated AP (no recombinant SP forms) 7. SP forms + beads only 8. Amyloplast lysates

Figure 3.10: Immunoblots probed with anti-SP and anti-S-tag peptide specific antibodies to confirm the immobilization of the recombinant GPC fractions by S- Agarose beads. The S-tagged GPC fractions (67.5 μg of protein) were immobilized to S-protein agarose beads after the fractions were incubated with 0.5 mg/mL of untreated and pretreated amyloplast lysates with 1mM ATP, APase. The ATP or APase and untreated amyloplast lysates were incubated with the S-agarose beads without the recombinant fractions as the controls (lane 4- 6). The washed pellets of S-agarose protein bead complexes were subjected to SDS-PAGE and immunoblot analysis. L= protein marker and the size of SP is 112 kDa.

129

Figure 3.11: Immunoblots of the immobilized GPC fractions of the recombinat SP by S-Agarose beads probed with anti-SSIIa, anti-SBEI and anti-SBEIIb peptide specific antibodies. The S-tagged tetrameric and dimeric GPC fractions (67.5 μg of protein) were immobilized to S-protein agarose beads after the fractions were incubated with 0.5 mg/mL of untreated and pretreated amyloplast lysates with 1mM ATP or APase. The ATP, APase and untreated amyloplast lysates were incubated with the S-agarose beads without the recombinant fractions as the controls. The washed pellets of S-agarose protein bead complexes were subjected to SDS-PAGE and immunoblot analysis to test the protein-protein interactions. L= protein marker. The arrows indicate the enzyme SSIIa at 76 kDa, SBEI at 80 kDa, and SBEIIb at 85 kDa.

130

Figure 3.12: Immunoprecipitation of recombinant tetrameric and dimeric forms of SP by peptide specific anti-SP antibodies bound to Protein A- Sepharose beads.

131

Figure 3.13: Schematic diagram summarizing the protein-protein interactions between tetrameric and dimeric forms of recombinant SP with starch biosynthetic enzymes present in the amyloplast lysates.

= Phosphorylation of the enzyme by ATP+plastidial protein kinase

3.3.5. The glucan synthetic and phospholytic activity of recombinant SP

The synthetic activity of the tetrameric, dimeric and monomeric forms of

14 recombinant SP was analyzed in vitro by using [U C]-G-1-P as the substrate.

The transfer of glucosyl units from radio labeled G-1-P to glycogen, amylopectin and maltoheptaose were assayed using 25 mg/mL substrate concentration for

30 minutes and the synthetic activity was calculated as nmol/mg/hr (Fig. 3.14).

The tetrameric form of SP had the highest activity with amylopectin

(928.9612.55), which was approximately 24% greater than with glycogen

(665.1213.56). Synthetic activities were statistically analyzed by Statistix 9 statistics analytical program at (P<0.05) probability using by One-Way ANOVA

132 followed by LSD analysis (F= 247.66, P=0.00001, see appendix 09 for the statistical analysis of the data). There was no significant difference between the activity of the tetrameric form of SP between amylopectin and glycogen substrates. Both glycogen and amylopectin showed significant differences in synthetic activity compared to maltoheptaose for given substrate concentrations. The activity was much lower for the dimeric form in amylopectin

(17.471.0) and glycogen (17.4691.4), compared to the activities of the tetrameric form with these substrates. However, the dimeric form showed slightly higher in synthetic activity (5.030.17) compared to tetrameric form

(2.971.1) with maltoheptaose. Synthetic activity of the dimeric form of SP was not significantly different for glycogen, amylopectin or maltoheptaose substrates

(Fig. 3.13). The tetrameric form of SP with amylopectin and glycogen showed significantly higher phosphorylitic activity at 25 mg/mL substrate concentration compared to maltoheptaose, but no significant difference was observed between amylopectin and glycogen. The variation of the activity of tetrameric SP from synthetic direction to phosphorylitic direction was greater in maltoheptaose (147 fold) compared to amylopectin (2.0 fold) and glycogen (1.1) (Fig. 3.14) (see

Appendix 09 for the analysis of ANOVA).

The Vmax and Km of the tetrameric form of recombinant SP was greater with amylopectin and lower in maltoheptaose in phosphorylitic direction (Table

3.1). Vmax of the tetrameric form was approximately 14.2 times greater than the dimeric form (Table 3.1). The Km value of the dimeric form was approximately 112 times greater than the tetrameric form for amylopectin and about 2.75 times greater for glycogen (Table 3.1).

133

Tetrameric form of Recombinant Dimeric form of SP Recombinant SP

‡ 2000 120 1750 100 1500 1250 80 ‡ + 1000 60 750 * ‡ 40 500 250 20 0 *+ 0 S P S P S P

S P S P S P

Enzyme Activity (nmol/mg/min)Enzyme Activity Enzyme Activity (nmol/mg/min)Enzyme Activity Gly Amy Malto Gly Amy Malto

Figure 3.14: Synthetic and degradative activities of the tetrameric and dimeric forms of recombinant SP in different glucan substrates. The activities were compared at 25 mg/mL substrate concentration in glycogen, amylopectin and maltoheptaose. Significantly different means (at P<0.05) are shown with similar symbols. S= Synthetic direction, P= Phosphorolytic direction

Table 3.1: The Km and Vmax values of dimeric and tetrameric forms of recombinant SP in the phosphorolytic direction. Tetrameric form of Dimeric form of recombinant SP recombinant SP Glucan Substrate Vmax Km Vmax Km (nmol/mg/min) (mg/mL) (nmol/mg/min) (mg/mL)

Glycogen 894.29 0.024 59.52 0.066

Amylopectin 1316.48 0.078 97.86 8.73

Maltoheptaose 497.11 0.0298 - -

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3.4. Discussion

3.4.1. Development of recombinant SP

The investigations presented in this chapter tested the hypothesis that plastidial SP has a role in starch biosynthesis and it is regulated by protein- protein interaction with other starch biosynthetic enzymes. Previous studies confirm the involvement of SP in protein complex formation with other major starch biosynthetic enzymes. SP was involved in the formation of heteromeric protein complexes with SBEI and SBEIIb in a phosphorylation dependent manner in wheat amyloplasts (Tetlow et al. 2004). In the ae mutant, which lacks SBEIIb SP formed complexes with SBEI, SSI, SSIIa and SBEIIa (Liu et al.

2009). However, in the present study the interaction of SP with other starch biosynthetic enzymes in maize amyloplast stroma was not detected by co- immunoprecipitation since the native protein was not immunoprecipated by peptide specific anti-SP antibodies bound to Protein-A sepharose beads (Chapter

2, section 2.3.6). Dimeric and tetrameric configurations of SP may reduce accessibility of the SP antibodies to the epitopes, thus preventing immunoprecipitation of recombinant dimeric and tetrameric forms by SP-specific antibodies and Protein-A sepharose beads (Fig. 3.14). We therefore developed a recombinant SP with an affinity ligand S-tag on the N-terminus of the protein in order to detect protein-protein interactions involving SP.

The complete mRNA sequence (2805 bp) of plastidial SP of wild-type maize without the transit peptide (TP) sequence of 70 amino acids was directly

135 cloned into pET29a expression vector and the complete protein with 935 amino acids was over expressed in Acrtic Express E.coli system.

The amino acid sequence alignment of plastidial SP of maize wild-type endosperm (Zea mays) (SP1, Gene Bank: ACF94692.1) with the TP, Ipomoea batatas tubers (GenBank: AAA63271.1), Oryza sativa endosperm (Japonica type, GenBank: BAG49328.1), Triticum aestivum endosperm (GenBank:

ACC59201.1), Solanum tuberosum tuber (GenBank: CAA52036.1) and the cytosolic form of maize (SP2, Gene Bank: ACF94691.1) showed that C-terminus is highly conserved among the species tested. It further confirmed the previous sequence analysis of Yu et al. (2001) that the peptide sequences of maize SP showed higher identities to potato, sweet potato and spinach but the N-terminus sequence did not align with any other N-terminal sequences.

The L-78 amino acid insertion located in the middle of plastidial SP is a unique characteristic of plastidial SP and is not seen in the cytosolic form of SP

(Young et al. 2006, Yu et al. 2001; Mori et al. 1993). Computational analysis also found that the unique L-80 insertion of the plasitidial form is located at 510-

590 amino acids in maize (Fig 3.2). The exact role of this insertion is not well documented in maize SP. Phosphorylation site prediction analysis indicated that the L-80 insertion consists of 7 serine (Ser) (out of 28) and 2 (out of 7) of the threonine (Thr) residues. These observations are similar to the finding of Young et al. (2006) indicating that there are 10 Ser and 5 Thr residues on L-78 insertion in the plastidial SP in sweet potato tubers and also the serine and threonine residues are intensively involved in the phosphorylation of the enzyme

136

(Young et al. 2006). L-78 insertion of plastidial SP also prevents affinity towards higher molecular branched glucans such as starch and glycogen (Young et al.

2006, Mori et al. 1993). Recombinant form of plastidial SP developed by replacing the L-78 insertion with a cytosolic SP sequence in potato showed the activity of the chimeric protein was five times lesser than the parent type isoform, but its affinity for glycogen was much higher (Mori et al. 1993). In contrast, a higher affinity of the SP to low molecular malto-oligosaccharides

(MOS) is recorded in maize (Yu et al. 2001).

Qualitative analysis of the synthetic activity of the recombinant SP on

0.1% glycogen affinity zymogram (Fig 3.7A) showed catalytic activity of the protein. The lower activity shown in lane 5 (uninduced E.coli cells with the plasmid but without the insert) may be due to the endogenous glucan phosphorylase present in E.coli. However, no such activity was found in induced

E.coli cells with the plasmid but without the insert (lane 4) and the activity level is negligible when compared with the recombinant SP activity. The immunoblot of the zymogram probed with anti-SP specific antibody recognized the SP in recombinant soluble fraction (Fig 3.7B). However, the faint band on the lane 3 in uninduced culture in the immunoblot is due to the leaky promoter since there was no band observed in other samples (Fig 3.7B). Further, the degradative activity of the recombinant SP on zymogram indicates that the protein is also active in degradative direction (Fig. 3.8D).

137

3.4.2. Gel filtration chromatography of recombinant SP

Native SP exists as monomeric, dimeric or tetrameric forms with identical subunits in maize amyloplast stroma (Albrecht et al. 1998; Mu et al. 2001, Liu et al. 2009). These different molecular confirmations remain throughout the development of the endosperm. Immunoblot analysis of soluble fraction of the recombinant SP eluted through the GPC column indicated that the fractions contained monomeric, and high molecular dimeric, tetrameric and multimeric forms. The synthetic activity was detected in the dimeric, tetrameric and multimeric forms of GPC-fractionated recombinant SP. Inactivity shown in the monomeric form was due to the lack of activity of the monomeric form or relatively lower levels of protein are present in the fractions that could not be detected on western blots.

Glycogen phosphorylase in animals and bacteria is homodimeric, and each subunit linked to a pyridoxal phosphate co-factor, which is involved in enzyme catalysis (Buchbinder et al. 2001). Two plastidial phosphorylases (PhoA and

PhoB) in Chlamydomonas reinhardtii which produces starch are shown to function as homodimers containing two 91-kDa (PhoA) subunits and two 110- kDa (PhoB) subunits (Dauvillée et al. 2006). Both of the Chlamydomonas SPs lack the L-80 amino-acid insertion found in higher plant plastidial forms. PhoB is exquisitely sensitive to inhibition by ADP-glucose and has a low affinity for malto-oligosaccharides. PhoA is moderately sensitive to ADP-glucose inhibition and has a high affinity for unbranched malto-oligosaccharides which is similar to the observation in higher plants (Dauvillée et al. 2006; Mu et al. 2001). Further, the mutation in STA4 gene in Chlamydomonas reinhardtii display a significant

138 decrease in amounts of starch with abnormally shaped granules (Dauvillée et al.

2006). Similarly, sh4 (shrunken4) mutant of maize, displays a strong reduction in starch content and this reduction was identified as lack of the SP cofactor pyridoxal-5-phosphate (Tsai and Nelson, 1969). However, external addition of pyridoxal-5-phosphate to the assay system for SP activity of the mutant did not affect the activity (Yu et al. 2001). The product of the sh4 gene is still unknown and this gene may or may not control the supply of pyridoxal phosphate (Yanase et al. 2006; Dauvillée et al. 2006; Burr and Nelson, 1973; Yu et al. 2001).

Formation of multi-subunit configurations of SP, and direct involvement of pyridoxal phosphate in this process in higher plants is yet to be investigated.

The recombinant SP developed in this study consisting of catalytically active dimeric and tetrameric forms were useful in understanding the different biochemical and regulatory mechanisms of these structures.

3.4.3. Investigating protein-protein interactions using immobilized recombinant SP on S-Protein Agarose beads

The SP in maize amyloplast lysates exist as different conformational structures, the tetrameric, dimeric forms (Liu et al. 2009; Mu et al. 2001).

However, the relative availability, quantity or the regulatory mechanisms of these identical subunits in developing maize endosperm are not known. The S- tagged GPC fractions of tetrameric, dimeric and monomeric forms obtained from

GPC analysis were used to analyze the protein- protein interactions of SP with other enzymes. The fractions were separately immobilized to S-protein agarose beads after the fractions were incubated amyloplast lysates. Immunoblot

139 analysis revealed that the tetrameric and dimeric forms were more strongly immobilized to the beads. The monomeric form present in the fractions may be less than the other two forms and not strongly immobilized to agarose beads.

The tetrameric form of recombinant SP interacted with SSIIa and SBEI when the amyloplast extract was treated with 1 mM ATP, but not in the untreated amyloplast lysates or APase treated samples, suggesting a phosphorylation-dependent interaction. In contrast, no interaction was detected with SBEIIb and the tetrameric form. ATP-treated SBEI and SBEIIb strongly interact with the dimeric form. Similar interactions were found in wheat amyloplast lysates, SBEI and SBEIIb interacted with SP in a phosphorylation- dependent manner (Tetlow et al. 2004). However, the multimeric status of SP involved in these interactions in wheat amyloplast lysates is not known. SSIIa interacted with the dimeric form only in the untreated amyloplast lysates.

Further, the interaction between SBEI and the dimeric form of SP was independent of ATP treatment. APase-treated samples did not show any interaction with any of the enzymes tested. The isoforms of the major enzymes involved in starch biosynthesis are regulated by protein phosphorylation, protein-protein interaction in maize amyloplast stroma (Liu et al. 2009; Hennen-

Bierwagen et al. 2008). Experiments in which isolated maize endosperm amyloplasts were incubated with [γ-32P]-ATP provide direct evidence for phosphorylation of SP (Pollack, 2009). The heteromeric complexes of starch synthesis enzymes assembled in the amylose extender (ae) mutant (lacking

SBEIIb) in maize amyloplast stroma was found by Liu et al. (2009). The complex formed by SSI, SSII with SBEIIb in wild-type was replaced by forming SBE1

140 combined with SP in ae mutant (Liu et al. 2009). The assortment of different multimeric forms in the wild-type stroma may be available in different compositions; that may prevent interactions or the level of interactions may be weak and not detectable.

Functional interactions observed between SP and SBE isoforms in rice endosperm suggested the mutual capacities for chain elongation and chain branching (Nakamura et al. 2012). The activity of SP and SBE isoforms also depended on the mutual availability of each group of enzyme and purified SP from rice endosperm could synthesize glucans from G-1-P in the presence of

SBE even without any exogenous glucan primer (Nakamura et al. 2012). In vitro glucan production was higher when SBEI presence compared to SBEIIa or

SBEIIb (Nakamura et al. 2012). Functional and physical interactions between

SBE isoforms and SP (Nakamura et al. 2012; Tetlow et al. 2004; Ball and Morell

2003) and the high affinity towards low molecular malto oligosaccharide (MOS)

(Mu et al. 2001) suggested that SP acts on MOS, which are liberated by the activity of debranching enzymes (DBE) to produce linear maltodextrin (MD) of a length sufficient for a subsequent branching reaction by starch branching enzymes (SBE) (Nakamura et al. 2012; Ball and Morell 2003). Therefore, direct interactions between SP and SBE isoforms, different multimeric forms interact in unique and selective manner, and phosphorylation and dephosphorylation of these multimeric forms may play a major role in starch biosynthesis by controlling the catalytic activity and assembly of protein complexes.

Reduced numbers of Less number of starch granules, with increased granule surface observed in ss4 and ss4/sp mutants in Arabidopsis leaves

141 suggested that SP may be involved in granule initiation in starch biosynthesis process via any kind of functional or physical interaction between SP and SSIV

(Roland et al. 2008; Planchot et al. 2008). Investigating possible interactions of

SP with SSIV was one of the major objectives of this study in order to elucidate the regulation of SP. ATP treated amyloplast lysates showed a weak interaction between SSIV and SP (Chapter 2). Phosphorylation of SP may be a vital requirement for this interaction since SSIV was not regulated by phosphorylation. However, no strong interaction was detected between SSIV and the dimeric and tetrameric forms of recombinant SP. The reason may be due to the small amounts of SSIV that were available in the assays, or recombinant SP forms were not sufficiently phosphorylated, or these proteins do not interact.

Dimeric and tetrameric forms of SP showed higher activities in amylopectin in both synthetic and degradative directions and degradative activity was greater in phosphorylitic direction compared to synthetic direction

(Fig. 3.13). Both multimeric forms show higher activity with highly branched glucan substrates. The Km of the tetrameric form in the phosphorylitic direction was about 2.5 times greater with amylopectin compared to glycogen or maltoheptaose, indicating the lower affinity of the tetrameric form towards highly branched large molecule substrates (Table 3.1). The affinity of dimeric SP for amylopectin was smaller than the tetramer (Km was increased by 111 times and by 3 times in glycogen compared to tetrameric form), indicating that different multimeric forms have different affinity levels for similar substrates.

The reaction of the dimeric form in the phosphorylitic direction was not detected

142 in the given range of substrate concentrations of maltoheptaose that were tested.

Previous work with purified SP from maize amyloplasts recorded that the phosphorylitic reaction was favored over the synthetic reaction when malto- oligosaccharides were used as the substrate (Mu et al. 2001). The Vmax and Km of SP in maize amyloplast lysates recorded in this study was approximately 7 and 5.8 times lower than with purified SP in phosphorolytic reaction (Mu et al.

2001). Unlike the purified form of SP, the activity of SP present in amyloplast lysates is regulated by other starch biosynthetic enzymes, and metabolites such as Pi, G-1-P and ADP-glucose present in the lysates (Tiessen et al. 2011; Mu et al. 2001; Schupp and Ziegler, 2004; Matheson and Richardson, 1978). For instance, G-1-P and Pi present in amyloplast lysates and high Pi/G-1-P ratios are thought to control SP activity (Mu et al. 2001; Schupp and Ziegler, 2004).

However, according to the findings of Hwang et al. (2010), the incorporation of

[U14C]-G-1-P into starch was only partially affected by the concentration of Pi in rice. Even under physiological G-1-P substrate levels (0.2 mM) and a 50-fold higher level of Pi in vitro than the normal physiological level, the Pho1 from of rice was able to carry out the biosynthetic reaction. Partially purified recombinant tetrameric and dimeric forms produced in this study had 11.5 times greater and 0.88 times less Vmax compared to purified-SP by Mu et al. (2001) in phosphorolytic direction in amylopectin. The activity of SP in recombinant multimers may be changed by desalting of the extracts which was not carried out in the experiments.

143

The preference of SP for different α-glucans has been recorded in many plant species (Schupp and Ziegler, 2004; Mu et al. 2001; Yu et al. 2001; Young et al. 2006; Mori et al. 1993). Differentiating from maize SP, in sweet potato tubers plastidial SP showed a low binding affinity toward starch and a high affinity toward low molecular weight linear MOS (Young et al. 2006). In contrast, the cytosolic isoform, has a high affinity towards highly branched polyglucan amylopectin (Young et al. 2006). The synthetic activity of SP to amylopectin

(Km =0.13) is higher when compared to the highly branched glycogen

(Km=0.45) in maize (Yu et al. 2001), in potato tubers (Liddle et al. 1961) and in spinach leaves (Shimomura et al. 1982). In sweet potato tubers, the unique L-

78 amino acid peptide insertion located in the middle of plastidial form of SP, appears to block the binding site of SP to high molecular weight α-glucans

(Young et al. 2006). However, it was not observed in maize amyloplast SP whether the L-78 insertion influences the kinetics of the enzyme. In fact there is no clear evidence for cleavage of L-78 in plastidial isoforms of SP from cereals.

In this chapter experiments were carried out to elucidate the regulation of

SP in maize amyloplasts. SP is a stromal enzyme and remains active throughout the endosperm development and exists as homodimeric or homotetrameric configurations throughout the developmental stages. This study suggested that the tetrameric and dimeric forms have different catalytic activities, and may be involved in starch biosynthesis by being regulated differently from each other.

The synthetic and phosphorolytic activity assays showed that the SP multimers are variously active in both directions. SP showed greater activities with amylopectin compared to glycogen and maltoheptaose in both synthetic and

144 phosphorylitic directions. ATP-treated SP showed higher activities in both directions in amylopectin substrate indicating that ATP may be involved in regulating the SP through protein phosphorylation. However, the protein-protein interactions could not be detected by co-immunoprecipitation as the native SP could not be immunoprecipitated by SP-antibodies and Protein-A sepharose beads. This may be due to lack of accessibility of the epitopes, in the dimeric and tetrameric configurations. Therefore, the development of S-tagged recombinant SP was used for analyzing protein-protein interactions of SP.

145

CHAPTER 4

146

Biochemical Investigation of the Regulation of Starch Synthase IV in Maize Endosperm

4.1. Introduction

The glucan synthases catalyze the elongation of glucan chains by transferring a glucosyl moiety to the non-reducing end of an α-(1-4)-linked glucan primer. Glucan synthases are found in both eukaryotes and prokaryotes but the most intensively studied enzyme in this class is glycogen synthase which is responsible for α-glucan elongation of glycogen (Szydlowski et al. 2011; Ball and Morell, 2003; Roach, 2002; Cao et al. 1999; Denyer et al. 1995; Madsen,

1991; Preiss, 1988; Preiss, 1984). Glycogen is a water-soluble polyglucan that is produced in mammals, fungi, bacteria; cyanobacteria and archaebacteria (Ball and Morell, 2003; Roach, 2002; Furukawa et al. 1993; Furukawa et al. 1990). In contrast, starch is a water-insoluble polyglucan produce in plants, green algae, and some cyanobacteria (Nakamura et al. 2005; Ball and Morell, 2003). One of the principle differences between glycogen and starch synthesis is the nucleotide sugar substrate of the glucan synthases involved in biosynthesis. UDP-glucose is the glucan donor for glycogen synthesis (Leloir et al. 1961) and ADP-glucose is the substrate of starch synthesis (Nakamura et al. 2005; Ball and Morell, 2003;

Roach, 2002). Structurally, glycogen is distinct from starch in that glycogen is water-soluble, and possesses a higher degree of branching (10%), and has a more open structure that expands in a globular fashion (Ball and Morell, 2003;

Roach, 2002). Starch is characterized by clustered branch points (about 6% of branching frequency) creating a water-insoluble granule (Manners, 1989).

Several isoforms of starch synthases and branching enzymes are found in plants

147 whereas glycogen synthase and glucan branching enzyme each exist as a single isoforms (Fujita et al. 2011; Ball and Morell, 2003)

The starch synthesized in higher plants consists of two types of glucose polymers, amylose and the amylopectin. Amylose is a sparsely branched linear molecule found to be about 1000 to 50000 glucose units, whereas highly branched amylopectin has 105–106 glucose units. Both amylose and amylopectin are elongated by the starch synthases (SS) by transferring the α-D-glucose units from ADP-glucose, the precursor of the starch biosynthesis to the non reducing end of the glucan chain (Leloir et al. 1961). Five major isoforms of starch synthases (SS) have been recorded in higher plants; SSI, SSII, SSIII

SSIV and GBSS (Tetlow, 2011; Ball and Morell, 2003). GBSS is essential for amylose synthesis and is exclusively bound to the starch granule (Nakamura et al. 1993; Sano, 1984; Echt and Schwartz, 1981). SSI, SSII, SSIII, and SSIV isoforms are thought to be responsible for amylopectin synthesis (Dian et al.

2005; Denyer et al. 1999; Gao et al. 1998; Denyer et al. 1995). Mutant analysis and biochemical studies have shown that each class of SS has a distinct role in the synthesis of amylopectin (Nakamura, 2002; Fontaine et al. 1993; Morell et al. 2003). SSI is responsible for producing DP= 8-12 glucan chains (Commuri and Keeling, 2001), SSII and SSIII respectively produce 12-25 (Zhang et al.

2004; Morell et al. 2003) and DP= 25-40 or greater glucan chains in amylopectin (Tomlinson and Denyer, 2003). However, there is little information available about the function/role of SSIV. The role and the regulation of the

SSIV in storage starch biosynthesis are largely unknown. The different isoforms of starch biosynthetic enzymes are differentially expressed at different stages of

148 endosperm development in cereals (Dian et al. 2005). The SSIIa, SSIII-1 and

SBEIIa are expressed in early stage and SSI, SSII-3, SSIII-2 and SBEIIb are expressed in the middle stage of the grain filling and GBBSSI, SSIV and SBE1 are differentially expressed at the later stage of the grain filling in cereals (Liu et al. 2009; Fujita et al. 2006; Dian et al. 2005; Morell et al. 1997; Gao et al.

1996).

Sequence analysis revealed that there are some similarities and differences of the SSIV in different plant species (Leterrier et al. 2008, see figure 4.1). The predicted protein sequence of SSIV in maize endosperm is 104 kDa in size and has a highly conserved C-terminal region compared with other

SSs. The C-terminus contains the catalytic and the starch-binding domains of

SSs (Cao et al. 1999). In common with other SSs (Fig. 1.5 and Fig. 4.1), the N- terminal region of SSIV is different from other SS isoforms (Leterrier et al.

2008), (Fig. 4.2). In addition, two coiled-coil domains were found in the SSIV- specific region from amino acids 1–405, which are thought to be involved in protein-protein interactions (Leterrier et al. 2008; Jody et al. 2004), (Fig. 4.2).

14-3-3-protein recognition sites [R/KXXpSXP and R/KXXXpSP, Muslin et al.,

(1996)] are also found in the N-terminal region of SSIV and may be involved in the regulation of the enzyme [14-3-3 proteins are commonly linked to binding with various signaling proteins such as kinases and phosphatases and act as

‘adaptor proteins’ in various phosphorylation-dependent protein-protein interactions (Comparot et al, 2003)], (Fig. 4.2). Expression of SSIV is tissue- dependent and found to be highest in non-endosperm tissues such as in leaf, embryo and roots in wheat and the level of expression in the endosperm was

149 relatively low independent from the regulation of the circadian clock. Therefore, the transcript accumulation levels did not coincide with the period of high carbon flux to starch in the wheat endosperm (Leterrier et al. 2008).

SSIV is exclusively localized in the amyloplast stroma (Leterrier et al.

2008; Roldan et al. 2007). Two different genes, the OsSSIV-1 was expressed in the endosperm and OsSSIV-2 was expressed in leaves in rice (Dian et al. 2005).

In addition, the cDNA sequence of SSIV expressed in developing wheat seedling is similar to rice SSIV-2 and, shares a similar exon-intron arrangement

(Leterrier et al. 2008). These findings suggest that two different SSIV isoforms may be responsible in transient and storage starch biosynthesis. No such different isoforms of SSIV have been identified in maize. Further, the SSIV protein in Arabidopsis thaliana (112.99 kDa in size, Roldan et al. 2007) shows

87% intron sequence identity to rice (OsSSIV-2 in leaves, accession number

AY373258) (Leterrier et al. 2008).

150

CLUSTAL 2.1 multiple sequence alignment

Ta ------MACS------AAAGVEATALLSPRCPAPSPPDGRSRRRLALASGTR 40 Os ------MAC------LAAGAEAAPLLFRRRLAPSPVAAR--RRLLVSCRAR 37 Zm PHPPRLPMSCS------AAAGAEATALLIR-SAAPSTIVGR--HRLAMSRRTS 90 At KGSPKPILSINSGLQSNNDEESDLENGSADSVPSLKSDAEKGSSIHGSIDMNHADENLEK 120 :: ...::.. * *. . .

Ta HRSLRAAAQRPHKSATGAD--PLYNNRANVRSDEAS------VSAEKERQRKYNDGDGI 91 Os RRGLRLVAQSAGSRGCGVVGAPGCDYWVNMQRDEAS------VSSDKERQEKYGDENGI 90 Zm RRNLRTGVHPHQKSAPSAN----HRNRASIQRDRAS------ASIDEEQKQMSEDENGL 139 At KDDIQTTEVTRRKSKTAKKKGESIHATIDIGHDDGKNLDNITVPEVAKALSLNKSEGEQI 180 : .:: . . .: * .. . : . : : : CC Ta SNLKLEDLVGMIQNTEKNILLLNQARLQAMEHADKVLKEKEALQRKINILETRLSETDEQ 151 Os SNLQLEDLIQMIQNTEKNIMLLNQARLQALEHVETVLKEKEDLQRKLKILETRLSETDAR 150 Zm LDIQLEDLVGMIQNTQKNILLLNQARLQALERADKILKEKETLQQKINILEMKLSETGKQ 199 At SDGQFGELMTMIRSAEKNILRLDEARATALDDLNKILSDKEALQGEINVLEMKLSETDER 240 : :: :*: **:.::***: *::** *:: :.:*.:** ** ::::** :****. :

Ta HKLSSEGNFS----DS------PLALELGILKEE--NILLKEDIEF 185 Os LKLSAEGQFGTEINDS------LPVLELDDIKEENMETLLKDDIQF 190 Zm SVLSSEVKSD------EESLEFDVVKEE--NMLLKDEMNF 231 At IKTAAQEKAHVELLEEQLEKLRHEMISPIESDGYVLALSKELETLKLE--NLSLRNDIEM 298 ::: : *: :* * : *::::::

Ta FKTKLIEVAEIEEGIFKLEKERALLDASLRELESRFIAAQADTMKLGPR----DAWWEKV 241 Os LKTMLIEVAETENSIFTLEKERALLDASLRELESRFIDAQADMLKSDPRQY--DAWWEKV 248 Zm LKGKLIEITETEESLFKLEKECALLNASLRELECTSTSAQSDVLKLGPLQQ--DAWWEKV 289 At LKSELDSVKDTGERVVVLEKECSGLESSVKDLESKLSVSQEDVSQLSTLKIECTDLWAKV 358 :* * .: : : :. **** : *::*:::**. :* * : .. * **

Ta EKLEDLLETTANQVEHAAVILDHNHDLQDRLDNLEASLQAANISKFSCS----LVDLLQQ 297 Os ENLGDLLETATNKVENAAMVLGRNHDLEDKVDKLEASLAEANISKFSCY----FVDLLQE 304 Zm ENLEDLLDSTANQVEHASLTLDGYRDFQDKVDKLKASLGTTNVSEFCLY----LVDILQQ 345 At ETLQLLLDRATKQAEQAVIVLQQNQDLRNKVDKIEESLKEANVYKESSEKIQQYNELMQH 418 *.* **: ::::.*:* : * :*:.:::*::: ** :*: : . :::*.

Ta KVKLVEDRFQACNSEMHSQIELYEHSIVEFHDTLSKLIEESEKRSLENFTGNMPSELWSK 357 Os KIKSVEERFQVCNHEMHSQIELYENSIAEFHDILSKLVEETEKRSLEHSASSMPSELWSR 364 Zm RVKSVEERFQACNHEMHSQIELYEHSIVEFHGTLSKLINESEKKSMEHYAEGMPSEFWSR 405 At KVTLLEERLEKSDAEIFSYVQLYQESIKEFQETLESLKEESKKKSRDEPVDDMPWDYWSR 478 ::. :*:*:: .: *:.* ::**:.** **: *..* :*::*:* :. . .** : **:

Ta ISLLIDGWLLEKKIAYNDASMLREMVRKRDSRLREAYLSYRGTENRDVMDSFLKMALPGT 417 Os ISLLIDGWLLEKRISYNDANTLREMVRKRDSCLREAYLSCRGMKDREIVDNFLKITLPGT 424 Zm ISLLIDGWSLEKKISINDASMLREMAWKRDNRLREAYLSSRGMEERELIDSFLKMALPGT 465 At LLLTVDGWLLEKKIASNDADLLRDMVWKKDRRIHDTYIDVKDKNERDAISAFLKLVSSPT 538 : * :*** ***:*: ***. **:*. *:* ::::*:. :. ::*: :. ***:. . *

Ta SSGLHIAHIAAEMAPVAKVGGLADVISGLGKALQKKGHLVEIILPKYDCMQVDQVSNLKV 477 Os SSGLHIIHIAAEMAPVAKVGGLADVISGLGKALQKKGHLVEIILPKYDCMQNDQVNNLKV 484 Zm SSGLHIVHIAAEMAPVAKVGGLADVISGLGKALQKKGHLVEIILPKYDCMQHNQINNLKV 525 At SSGLYVVHIAAEMAPVAKVGGLGDVVAGLGKALQRKGHLVEIILPKYDCMQYDRVRDLRA 598 ****:: ***************.**::*******:**************** ::: :*:.

Ta LDVLVQSYFEGNMFNNKIWTGTVEGLPVYFIEPQHPAMFFSRAQYYGEHDDFKRFSYFSR 537 Os LDVVVQSYFEGNLFNNKIWTGTVEGLPVYFIEPQHPAKFFWRAQYYGEHDDFKRFAYFSR 544 Zm LDVVVKSYFEGNMFANKIWTGTVEGLPVYFIEPQHPGKFFWRAQYYGEHDDFKRFSYFSR 585 At LDTVVESYFDGKLYKNKIWIGTVEGLPVHFIEPQHPSKFFWRGQFYGEQDDFRRFSYFSR 658 **.:*:***:*::: **** ********:*******. ** *.*:***:***:**:****

Ta AALELLYQSGKKVDIIHCHDWQTAFVAPLYWDVYANLGFNSARICFTCHNFEYQGTAPAR 597 Os AALELLYQSQKKIDIIHCHDWQTAFVAPLYWEAYANLGFNSARICFTCHNFEYQGAAPAQ 604 Zm VALELLYQSGKKVDIIHCHDWQTAFVAPLYWDVYANLGFNSARICFTCHNFEYQGIAPAQ 645 At AALELLLQSGKKPDIIHCHDWQTAFVAPLYWDLYAPKGLDSARICFTCHNFEYQGTASAS 718

.***** ** ** ******************: ** *::*************** *.*

151

Ta DLAWCGLDVEHLDRPDRMRDNSHG-RINAVKGAIVYSNIVTTVSPTYALEVR-SEGGRGL 655 Os DLACCGLDVQQLDREDRMRDNSHG-RINVVKGAIVYSNIVTTVSPTYALEVR-SEGGRGL 662 Zm DLAYCGLDVDHLDRPDRMRDNSHG-RINVVKGAVVYSNIVTTVSPTYAQEVR-SEGGRGL 703 At ELGSCGLDVNQLNRPDRMQDHSSGDRVNPVKGAIIFSNIVTTVSPTYAQEVRTAEGGKGL 778 :*. *****::*:* ***:*:* * *:* ****:::************ *** :***:**

Ta QDTLKVHSRKFLGILNGIDTDTWNPSTDRYLKVQYNAKDLQGKAANKAALREQLNLASAY 715 Os QDSLKLHSRKFVGILNGIDTDTWNPSTDRHLKVQYNANDLQGKAANKAALRKQLNLSSTN 722 Zm QDTLKVHSKKFVGILNGIDTDTWNPSTDRFLKVQYSANDLYGKSANKAALRKQLKLASTQ 763 At HSTLNFHSKKFIGILNGIDTDSWNPATDPFLKAQFNAKDLQGKEENKHALRKQLGLSSAE 838 :.:*:.**:**:*********:***:** .**.*:.*:** ** ** ***:** *:*:

Ta PSQPLVGCITRLVAQKGVHLIRRAIYKTAELGGQFVLLGSSPVPEIQREFEGIADHFQNN 775 Os ASQPLVGCITRLVPQKGVHLIRHAIYKTAELGGQFVLLGSSPVPHIQREFEGIADHFQNN 782 Zm ASQPLVGCITRLVPQKGVHLIRHAIYKITELGGQFVLLGSSPVQHIQREFEGIADQFQNN 823 At SRRPLVGCITRLVPQKGVHLIRHAIYRTLELGGQFVLLGSSPVPHIQREFEGIEQQFKSH 898 . :**********.********:***: ************** .******** ::*:.:

Ta NNIRLILKYDDALSHCIYAASDMFVVPSIFEPCGLTQMIAMRYGSVPIVRKTGGLNDSVF 835 Os NNIRLLLKYDDSLSHWIYAASDMFIVPSMFEPCGLTQMIAMRYGSVPIVRKTGGLNDSVF 842 Zm NNVRLLLKYDDALAHMIFAASDMFIVPSMFEPCGLTQMVAMRYGSVPVVRRTGGLNDSVF 883 At DHVRLLLKYDEALSHTIYAASDLFIIPSIFEPCGLTQMIAMRYGSIPIARKTGGLNDSVF 958 :::**:****::*:* *:****:*::**:*********:******:*:.*:*********

Ta DFDDETIPMEVRNGFTFVKADEQGLSSAMERAFNCYTRKPEVWKQLVQKDMTIDFSWDTS 895 Os DFDDETIPKELRNGFTFVHPDEKALSGAMERAFNYYNRKPEVWKQLVQKDMRIDFSWASS 902 Zm DLDDETIPMEVRNGFTFLKADEQDFGNALERAFNYYHRKPEVWKQLVQKDMKIDFSWDTS 943 At DIDDDTIPTQFQNGFTFQTADEQGFNYALERAFNHYKKDEEKWMRLVEKVMSIDFSWGSS 1018 *:**:*** :.:***** .**: :. *:***** * :. * * :**:* * ***** :*

Ta ASQYEDIYQKAVARARAVA--- 914 Os ASQYEDIYQRAVARARAAA--- 921 Zm VSQYEEIYQKTATRARAAA--- 962 At ATQYEELYTRSVSRARAVPNRT 1040 .:***::* ::.:****..

Figure 4.1: Amino acid sequence alignment of SSIV in different plant species. Ta- Triticum asetivum (GenBank: DQ400416.1), At- Arabidipsis thaliana (GenBank: FW301560.1), Os- Oryza sativa (GenBank: FB702573.1), Zm- Zea mays (GenBank: AAC197339). The epitope for the peptide specific anti-SSIV antibodies of maize is highlighted in red. The coiled-coil domain (CC) and the conserved catalytic domains in the C–terminal region (K-V-G-G-L and K-T-G-G- K) are shown in blue boxes.

152

14-3-3 14-3-3

Figure 4.2: A schematic diagram showing the major domains found within the predicted amino acid sequence of SSIV in wheat endosperm. The starch catalytic domain (GT-5) and glycosyltranferase domain (GT-1) characteristic of the SS family are shown. Predicted 14-3-3 recognition sites and the coiled-coil domains (blue boxes and 'CC' respectively), as well as the two highly conserved KVGGL and KTGGL domains are also shown (Leterrier et al. 2008).

Although the involvement of SSIV in glucan chain length elongation is not clear, the growth rate in the mutant alleles of ss4 in Arabidopsis thaliana was decreased without changing total SS activity (Roldan et al. 2007). Further, the starch content was deceased by 35-40% in the mutant lines while the size of silique, number of seeds per silique and germination ratios remained unchanged

(Roldan et al. 2007). Interestingly, the total activity of starch phosphorylase

(SP) was increased by 1.4–2-fold in both cytosolic and plastidial forms in

Arabidopsis ssiv mutants (Roldan et al. 2007). More importantly, the amylose/amylopectin ratio, or the structure of the starch were not altered in the ss4 mutants, the starch granule surface area was increased by 1.5 times and by

4 times in ss4/sp double mutants indicating the increase in granule size

(Planchot, et al. 2008). In contrast, the number of granules per chloroplast

153 decreased to 2-3 in ss4 single mutants, where as the wild-type contains contained 4–5 starch granules per chloroplast. Interestingly, the double mutants of ssiv/sp had 1-2 granules per chloroplast (Planchot, et al. 2008). These observations suggested that the SSIV potentially interacts (either functionally or physically) with SP, and both are involved in the priming of the starch granule

(Planchot, et al. 2008; Roldan et al. 2007). The mechanism of starch granule initiation is largely unknown (D’Hulst et al. 2010; D’Hulst and Merida, 2012).

The homologous double mutants of starch synthases produced in

Arabidopsis thaliana (ss1/ss4, ss2/ss4 and ss3/ss4) are helpful in understanding the interactive role of SS in starch biosynthesis (Szydlowski et al. 2009). Starch accumulation deceased in ss1/ss4 and ss2/ss4 double mutants equal to the sum of the decreased starch levels in their respective single mutant lines. However starch accumulation in the single mutants of ss4 and ss3 were recorded as

122% (Zhang et al. 2005) and 62% (Rolden et al. 2007), respectively compared to their wild- types at the end of 12h light period. However, the double mutant of ss3/ss4 did not accumulate any measurable amounts of starch, irrespective of light conditions (Szydlowski et al. 2009). Therefore, the presence of either SSIII or SSIV appears to be a crucial requirement in transient starch biosynthesis

(Szydlowski et al. 2009). In addition, the significant increase in the activity of SP in the ss3/ss4 double mutants suggested the existing of alternative SP-mediated starch biosynthetic pathway using hexose phosphates as glycosyl donors

(Szydlowski et al. 2009; Fettke et al. 2010).

The investigations discussed in this chapter tested the hypothesis that

SSIV is involved in storage starch biosynthesis in maize amyloplasts and that

154 the enzyme is regulated by protein phosphorylation and protein-protein interactions. The cellular localization and biochemical analyses were performed to characterize and understand the regulatory mechanism of the enzyme.

Recent evidence from Arabidopsis thaliana suggested that SP and SSIV may physically and/or functionally interact and may be involved in priming the starch granule. The possible interactions of SSIV specifically with SP and with other starch biosynthetic enzymes were tested in maize amyloplast stroma.

155

4.2. Materials and Methods

4.2.1. Analysis of the localization of SSIV in the plastid

To investigate the localization of SSIV in the amyloplast, amyloplasts were isolated and the soluble and granule bound proteins, and plastid envelop membrane proteins were separated from 22 DAA (days after anthesis) old maize endosperms as described earlier in chapter 2. The presence of SSIV and other

SS isoforms, SSI, SSII, and SSIII in the amyloplast stroma and the granule was determined by running the protein extracts on 10% SDS gels and the immunoblotted proteins were identified using peptide-specific anti-maize antibodies. The purified SSIV antibody generated using the synthetic peptide

ANHRNRASIQRDRASASI from the first bleed serum developed in rabbit was used after dilution by 1:800 in 1.5% BSA (antibodies were purified as described in chapter 2). The procedures for SDS-PAGE and immunoblot analysis were as described in chapter 2.

4.2.2. Determination of the protein expression of SSIV in developing endosperm

The equal amounts of proteins from the amyloplast lysates extracted from the maize kernels at 12, 15, 17, 22 DAA were run on 10% SDS gels. Following the electrophoresis, the immunoblots were probed with peptide specific SSIV antibodies.

156

4.2.3. Determination of SSIV catalytic activity by zymogram analysis

Zymogram analysis was performed to estimate the activity of SSIV and other SS isoforms of amyloplast stroma following incubation of the lysates with

ATP or APase to respectively phosphorylate and dephosphorylate amyloplast proteins.

SS zymograms were carried out according to the methods described by

(Tetlow et al. 2004). 90 μg of proteins were run in a well after gels were prepared as native 5% (w/v) polyacrylamide gels in 375 mM TRIS-HCl, pH 8.8, and 10 mg of the α-amylase inhibitor Acarbose (Bayer, ‘Prandase’) and 0.3%

(w/v) rabbit liver glycogen (type III, Sigma-Aldrich). The gel was run using

0.25M Tris, 192 mM glycine running buffer without SDS at 120V for 1.5hr in the cold room. After electrophoresis, the gel was incubated for 48–72 h in a buffer containing 50 mM glycylglycine, pH 9.0, 100 mM (NH4)2SO4, 20 mM DTT, 5 mM

-1 MgCl2, 0.5 mg mL BSA, and 4 mM ADP-glucose.

4.2.4. Substrate-affinity electrophoresis

Affinity electrophoresis was carried out as described earlier by Commuri and Keeling (2001) using different glucan substrates at various concentrations; amylopectin, glycogen and maltoheptaose (at 0, 5, 10, 25 mg/mL concentrations) in the native gels. Amyloplast lysates (22 DAA) were run on the gel at a protein content of 30 µg/mL per well. The migration distances of specific enzyme were measured after immunoblotting. Affinity electrophoresis served as a means of measuring protein-glucan interactions, and the dissociation constants (Kd) were calculated from the retardation of the electrophoretic

157 mobility of enzyme/protein by the substrate contained in the supporting medium.

4.2.5. Gel filtration chromatography (GPC)

GPC analysis was performed as described in Chapter 2, section 2.2.2.4.3.

4.2.6. Co-Immunoprecipitation of SSIV

In order to identify protein-protein interactions of SSIV and other starch biosynthetic enzymes, co-imunoprecipitation was performed with amyloplast lysates of 22 DAA using the methods previously described in Chapter 2, section

2.2.2.5.7.4. using peptide specific anti-SSIV antibodies.

4.2.7. Phosphorylation of SSIV using -32P-ATP

Phosphorylation of SSIV was investigated by incubating 400 μL of amyloplast lysate with 0.5 uCi of -32P-ATP in a final concentration of 1 mM ATP on a rotator for an hour at 250C and then the SSIV was immunoprecipitated by using SSIV specific antibodies bound to Protein-A sepharose beads following the procedure described in section 4.2.4 in Chapter 2. Non-specifically bound proteins were removed by washing the remaining pellet for eight times each with 1 mL phosphate buffered saline (PBS), followed by three similar washes with 10 mM HEPES/KOH, pH 7.5 buffer (at 100 g, 1 min centrifugation).

Following washing, the immunoprecipitated pellet was boiled in 2X SDS loading buffer for 8 min and separated by SDS-PAGE. Following electrophoresis, proteins in the gel were transferred to nitrocellulose membranes exposed to X-ray film

158 for two weeks at -800C. The phosphorylation of SSIV was detected by alignment of X-ray film with the developed immunoblot, which was probed with anti-SSIV specific antibodies.

159

4.3. Results

4.3.1. Testing the specificity of peptide specific anti-SSIV antibodies

The SSIV isoform in maize is predicted to be 104 kDa based on its amino acid sequence. The SSIV-specific antibody (ANHRNRASIQRDRASASI) was derived against amino acids located at position 55-72 at the N-terminal end of full length amino acid sequence of maize SSIV (909 amino acids, see figure 4.1)

(Accession number - EU99036.1). Immunoblots of the amyloplast lysates run on

SDS-PAGE were probed with purified SSIV antibodies and pre-immune serum to detect the specificity of the purified antibodies in detecting SSIV (Fig. 4.3A). The purified anti-SSIV specific antibodies were subjected to a series of dilutions and the optimal concentration of antibodies required to detect SSIV in amyloplast lysate was 1:800 dilution (Fig. 4.3B).

4.3.2. Localization of SSIV

Localization of SSIV in maize amyloplast was investigated by immunodetection using the peptide-specific antibodies to SSIV. Analysis of the proteins extracted from the wild-type amyloplast stroma and the loosely-bound proteins from the starch granule at 22 DAA confirmed that SSIV is localized only in the amyloplast stroma, while SSI and SSII, and in some cases SSIII can be seen in both amyloplast stroma and as granule-associated proteins (Fig. 4.4).

160

(A)

kDa 150

100

(B)

MW MW MW MW

Figure 4.3: Immunoblots of amyloplst proteins probed with purified SSIV- specific antibodies (A). Purified anti-SSIV specific antibodies were diluted to 1:800, 1:1000, 1:2000, and 1:5000 in 1.5% BSA to determine the optimal concentration of the antibodies required to detect SSIV (B).

161

Figure 4.4: Immunodetection of SSI, SSII, SSIII and SSIV in stroma and starch granules of wild-type maize amyloplasts at 22 DAA. Amyloplast lysates (25 μg proteins) were separated on 10% acrylamide gels, electroblotted onto nitrocellulose membranes, and developed with peptide-specific anti-maize antibodies. The expected mass (predicted from the amino acid sequence) of each protein is given below the respective immunoblot.

4.3.3. Determination of the expression of SSIV in developing endosperm

Testing of equal amounts of proteins from the amyloplast lysates extracted from the maize kernels at 12, 15, 17, 22 DAA with the peptide specific

SSIV antibodies showed that the SSIV protein is expressed in the later stages of endosperm development (Fig. 4.5).

162

Figure 4.5: Immunodetection of SSIV at different stages of endosperm development in maize wild-type amyloplasts. Amyloplast lysates from 12, 15, 17, and 22 old endosperms were run (25 μg proteins per well) in SDS-PAGE and immunoblot was developed by the peptide specific anti-SSIV antibodies.

4.3.4. Determination of the affinity of the SSIV in amyloplast lysates to different α-glucan substrates

The affinity of SSIV in amyloplast lysates for α-glucans was established by affinity electrophoresis (Fig 4.6A). The amyloplast lysates (approximately 30 μg proteins) were subjected to native PAGE in the presence of different concentrations (0, 0.5, 1, 2.5 mg/mL) of amylopectin, glycogen and maltoheptaose (see Fig 4.6A). The relative migration (Rm) and then dissociation constant (Kd) of the SSIV were calculated from the plot of the graph developed by 1/Rm vs. substrate concentration as described by Commuri and Keeling,

(2001) (Fig 4.6B). The SSIV showed a relatively higher Kd value in glycogen

(2.5 mg/mL) followed by maltoheptaose (1.5 mg/mL) and the amylopectin (1.0 mg/mL) (Fig. 5.4B) (Table 4.1).

163

(A)

Amylopectin (mg/mL) Maltoheptaose (mg/mL) Glycogen (mg/mL)

0 0.5 1 2.5 0 0.5 1 2.5 0 0.5 1 2.5

Figure 4.6A: A representative western blot of the native zymogram gel showing the mobility of SIIV in different glucan substrates used to determine the relative mobility of the SSIV in amyloplast lysates. The relative mobility of SSIV was determined by the transferring the native zymogram to nitrocellulose membranes and probing with anti-SSIV antibodies. The mean relative mobility (Rm) was determined as the ratio of the migration of the activity band and the migration of the dye from three different experiments.

164

(B) Glycogen 3.00 y = 0.4885x + 1.2983

2.00

1/Rm 1.00

0.00 -3 -2.5 -2 -1.5 -1 -0.5 0 0.5 1 1.5 2 2.5 3 Substrate Concentration (mg/mL)

Amylopectin

6.00 y = 1.0576x + 1.1474

4.00

1/Rm 2.00

0.00 -2.5 -2 -1.5 -1 -0.5 0 0.5 1 1.5 2 2.5 Substrate Concentration (mg/mL)

Maltoheptaose

4 y = 0.7772x + 1.1909

3 2 1/Rm 1 0 -2.5 -2 -1.5 -1 -0.5 0 0.5 1 1.5 2 2.5 3 3.5 Substrate Concentration (mg/mL)

Figure 4.6B: Plots of the reciprocal of the relative mobility (1/Rm) of maize SSIV against the concentration of different glucan substrates. The dissociation constant (Kd) of SSIV is shown as the intersect at the X-axis.

165

Table 4.1: Comparison of Kd values of maize SSIV (from present study) with SSI, SSIIa and SP in different glucan substrates. SSI, SSIIa and SP values were estimated by Commuri and Keeling, (2001).

Glucan Kd values (mg/mL) Substrate

SSI SSIIa SP SSIV

Amylopectin 0.20.04 0.240.01 0.020.01 1.00.01 Starch 0.490.01 0.490.01 0.080.01 - Glycogen - - - 2.50.02 Maltoheptaose - - - 1.50.7

4.3.5. Investigating the regulation of SSIV by phosphorylation using -

32P-ATP

Possible phosphorylation of SSIV was investigated by pre-incubating amyloplast lysates with -32P-ATP, immunoprecipitating SSIV with peptide- specific antibodies and analyzing the immunoblots by autoradiography. Figure

4.5 shows the developed nitrocellulose membrane of immunoprecipitated SSIV after treatment of amyloplast lysates with -32P-ATP (4.7A) and the autoradiograph developed from the same nitrocellulose membrane (4.7B). The immunoblot developed by anti-SSIV antibodies showed that the SSIV was successfully immunoprecipitated, no SSIV band was detected in the remaining supernatant after the SSIV was immunoprecipitated (Fig 4.7A). However, the autoradiograph did not show any radioactivity corresponding to SSIV indicating that SSIV was not phosphorylated under these conditions (Fig 4.7B). (Apendix

06 shows the predicted phosphorylation sites of maize SSIV)

166

(A) (B)

Figure 4.7: SSIV from amyloplast lysates was immunoprecipitated following incubation of amyloplast lysates with -32P-ATP. 400 μL of amyloplast lysates (approximately 1 mg/ mL) were treated with final concentration of 0.5 uCi of - 32P-ATP + 1 mM ATP mixture on a rotator for in hour. SSIV was immunoprecipitated by using anti-SSIV specific antibodies. After the non- specifically bound proteins were removed, the pellet was boiled in 1X SDS loading buffer for 8 min and run the SDS-PAGE. The proteins in the gel were transferred to nitrocellulose membranes and developed with anti-SSIV antibodies (A) and the autoradiograph was developed from the same membrane after the proteins were transferred to the X-ray film for two weeks at -800C (B). The phosphorylation of SSIV was tested by aligning the X-ray film with the developed immunoblot with anti-SSIV specific antibodies. Arrows indicate the location of SSIV.

167

4.3.6. Determination of the activity of ATP or APase treated SSIV on zymogram

ATP-treated or APase-treated SS activity was measured qualitatively by an in-gel activity assay. Maize amyloplast lysates were pre-incubated with 1mM

ATP, and SS activity was detected on zymogram and compared with the untreated amyloplast lysates (Fig. 4.8A). The SS activity banding profile in untreated amyloplast lysates was changed equally in ATP or ATP+PI

(PI=phosphatase inhibitor) treated samples. PI was added to inhibit the activity of endogenously available protein phosphatases. No difference in the banding pattern was observed between untreated, PI treated and APase treated samples.

Immunoblot analysis of the SS activity zymogram for SSI and SSII activities showed that the samples treated with ATP (with or without PI) became more mobile and therefore, showed less affinity to the given glycogen concentration (0.3%) than in untreated, PI treated and APase treated samples.

However, no clear band corresponding to SSI was seen in APase treated samples (Fig. 4.8A). The peptide-specific SSIII antibodies did not recognize the

SSIII in any treatment (Fig. 4.8B). The synthetic activity corresponding to SSIV was reduced when treated with APase and the activities were higher in ATP treated samples when compared with untreated controls (Fig. 4.8B). However, the mobility of SSIV indicated by immunoblots was similar in all treatments (Fig.

4.8B). SSIV in amyloplast lysates was removed by immunoprecipitation with anti-SSIV specific antibodies. SSIV immunoprecipitation was used in conjugation with zymogram analysis to understand the mobility of the enzyme and the changing of overall SS activity profile in the absence of SSIV. The zymogram

168 lacking SSIV showed loss of a major activity band (as indicated by the arrow) on the zymogram (Fig 4.8C) in addition to at least two other minor (unidentified) bands of SS activity.

169

(A)

(B)

Figure 4.8: Zymogram analysis of SS activity in amyloplast lysates of wild-type maize endosperm at 22 DAA. Amyloplast lysates were separated (90 μg protein per well) on a native 5% acrylamide gels containing 0.3% (w/v) glycogen, and developed for 48 h at in a buffer containing 4 mM ADP-glucose. SS activities were visualized by staining with Lugol’s solution. Identical gels were electroblotted onto nitrocellulose membranes and probed with peptide specific anti-SSI, SSIIa (A), SSIII and SSIV peptide specific antibodies (B).

170

(C)

1 2

1. Amyloplast lysates after removal of SSIV by immunoprecipitation

2. Amyloplast lysates with SSIV

Figure 4.8C: The activity of SS in the amyloplast lysates in the absence of SSIV. The native SSIV was removed by immunoprecipitation with anti-SSIV specific antibodies bound to Protein A-sepharose beads and the remaining supernatant was run with amyloplast lysates to compare the relative position of SSIV and to detect the change of SS activity profile of other starch synthases (C).

4.3.7. Gel filtration chromatography of SSIV

The amyloplast lysates at 22 DAA from wild-type maize were treated with

ATP and APase and separated through a Superdex 200 10/300GL gel permeation column. The fractions were subjected to SDS-PAGE followed by immunoblot analysis to identify the SSIV eluted fractions. SSIV eluted in fraction numbers

29/30 in all treatments of amyloplast lysates. Approximate molecular weight of the fraction that SSIV eluted was determined by eluting the standard proteins with known molecular weights from the same column which is approximately at

100 kDa (Fig. 4.9).

171

440 kDa 232kDa 100kDa Fraction Numbers L 15 16 17 18 19 20 21 22 23 24 25 26 2 28 29 30 31 32 33 34 35 36 37 38 39 40 C L kDa

150

100 SSIV-Untreated Control 75

150 SSIV- ATP 100 Treated 75

150 100 SSIV- APase 75 Treated

Figure 4.9: Gel filtration chromatography analysis of SSIV in amyloplast lysates. 450 μg of total protein in a volume of 500 µL from each treatment was separated by size exclusion chromatography (GPC) through a Superdex 200 10/300GL gel permeation column. The fraction numbers from 16 to 41 were run on SDS-PAGE followed by immunoblot analysis using peptide specific anti SSIV antibodies. The SSIV bands are shown by the arrows at 104 kDa. The column was calibrated by protein standards with known molecular weights and predicted molecular weights of the fractions are indicated in boxes. L= protein marker, C=amyloplast lysates before loaded in the column. Arrows indicate the location of SSIV.

172

4.5.8. Detection of protein-protein interactions of SSIV by co- immunoprecipitation

To investigate the protein-protein interaction of SSIV with other starch biosynthetic enzymes, the co-immunoprecipitation was performed with maize wild-type amyloplast lysates at 22 DAA. The SSIV antibodies (30 mg/mL) were used to immunoprecipitate the native SSIV protein from amyloplast lysates (1 mL) using Protein-A Sepharose beads. Figure 4.10 shows immunoblots of immunoprecipitated SSIV probed with SSIV (Fig. 4.10A) and other peptide- specific starch biosynthetic enzymes antibodies of SSI, SSIIa, SSIII, SBEI,

SBEIIb, ISOI and SP (Fig. 4.10B).

SSIV in amyloplast lysates was completely immunoprecipitated, since no

SSIV was detected in the remaining supernatant (Fig 4.10A). There is no non- specific binding to the beads, and only the purified SSIV antibodies were bound to the beads since no band was observed in the immunoprecipitation carried out by using pre-immune serum (Fig 4.10A). When the immunoblots were incubated with SSI, SSII and SSIII, no bands were detected from SSIV immunoprecipitated beads (Lane 1 in Fig. 4.10B) and the enzyme levels showed in supernatants remained same after the pull down. Similarly, SSIV immunoblots probed with SBEI, SBEIIb and ISOI antibodies showed no bands

(Fig. 4.10B). The SSIV immunoblot probed with anti-SP specific antibodies showed no clear interaction of SSIV with SP (Fig. 4.10B). The faint band observed in SSIV-pulldown beads may be from non-specific bounding of SP to the beads. Therefore, no clear protein-protein interactions were detected recorded between SSIV and other starch biosynthetic enzymes tested under

173 these conditions (Fig 4.10). In addition, co-immunoprecipitation experiments were performed with amyloplast lysates treated with 1 mM ATP or 30U APase.

No interactions between SSIV and other starch biosynthetic enzymes were detected but a weak interaction was detected with SP when amyloplast lysates were treated with ATP (Fig. 4.11). (Appendix 07 shows the Co- immunoprecipitation of stromal proteins from wild-type maize amyloplasts using peptide specific anti-SBEIIb antibodies to investigate the protein-protein interactions of SBEIIb with SSIV and SP. No interaction was detected between

SBEIIa and SSIV or SBEIIa and SP).

174

(A)

Figure 4.10A: Immunoprecipitation of stromal SSIV from wild-type maize amyloplasts using peptide specific anti-SSIV antibodies to investigate the protein-protein interactions. 1 ml amyloplast lysates (1 mg/mL) prepared from wild-type maize endosperm at 22 DAA were incubated with peptide-specific anti- SSIV antibodies (30 mg/mL final concentration) at room temperature for 1 hr, and then immunoprecipitated with Protein-A-Sepharose beads. The washed Protein-A-Sepharose-antibody-antigen complexes were boiled in 200 μL 1XSDS- loading buffer and 30 μL was loaded onto 10% SDS gels. Immunoblotted membrane was developed with maize anti-SSIV antisera (A). Arrow indicates the immunoprecipitation of SSIV using SSIV specific antibodies. The IgG is approximately showed at 50 kDa as a large thick band.

175

(B)

Figure 4.10B: Co-Immunoprecipitation of of stromal proteins from wild-type maize amyloplasts using peptide specific anti-SSIV antibodies to investigate the protein-protein interactions. SSIV in maize amyloplast lysates was immunoprecipitated by peptide-specific anti-SSIV antibodies (30 mg/mL final concentration) with Protein-A-Sepharose beads (Fig 4.10A) and the washed Protein-A-Sepharose-antibody-antigen complexes were boiled in 200 μL 1XSDS- loading buffer and 30 μL was loaded onto 10% SDS gels. Immunoblotted membranes were developed with various anti-maize antisera as shown. Arrow indicates the expected position of different starch biosynthetic enzymes in the Protein-A-Sepharose-antibody-antigen complexes. The MW of the enzymes are; SSI at 74 kDa, SSIIa at 85 kDa, SBEI at 80 kDa, SBEIIb at 85 kDa, SP at 112 kDa and, Iso-1 at 80 kDa. The IgG is approximately showed at 50 kDa as a large thick band.

176

L 1 2 3 4 5 6 7 8 L 1 2 3 4 5 6 7 8 kDa kDa 150 150 100 100 75 75

Anti- SSIV Anti- SBEI

L 1 2 3 4 5 6 7 8 L 1 2 3 4 5 6 7 8 kDa kDa 150 150 100 100 75 75

Anti- SSI Anti- SBEIIb

L 1 2 3 4 5 6 7 8 L 1 2 3 4 5 6 7 8 kDa kDa 150 150 100 100 75 75

Anti- SSII Anti- SP

L. Protein marker

1. SSIV Co-IP+ATP-treated amyloplast lysates in beads

2. SSIV Preimmune Co-IP+ATP in beads 3. SSIV Co-IP+ATP supernatant 4. SSIV Co-IP+APase-treated amyloplast lysates in beads 5. SSIV Preimmune Co-IP+APase in beads 6. ATP treated amyloplast lysates (No SSIV antibody) in beads 7. APase treated amyloplast lysates (No SSIV antibody) in beads 8. Amyloplast lysate

Figure 4.11: Co-immunoprecipitation of ATP or APase treated stromal proteins from wild-type maize amyloplasts using peptide specific anti-SSIV antibodies to investigate the protein-protein interactions of SSIV with other starch biosynthetic enzymes. 1 ml amyloplast lysates (1 mg/mL) prepared from wild- type maize endosperm at 22 DAA were incubated by adding 1mM ATP and APase (25 unit/ml) for 1 hr and incubated further with peptide-specific anti-SSIV antibodies (30 mg/mL final concentration) at room temperature for 1 hr. The SSIV was immunoprecipitated with Protein-A-Sepharose beads. The washed Protein-A-Sepharose-antibody-antigen complexes were boiled in 200 μL 1XSDS- loading buffer and 30 μL was loaded onto 10% SDS gels. Immunoblotted membranes were developed with various anti-maize antisera as shown. Arrows indicate the immunoprecipitation of SSIV using SSIV specific antibodies and the enzymes at; SSI at 74 kDa, SSIIa at 85 kDa, SBEI at 80 kDa, SBEIIb at 85 kDa and SP at 112 kDa. The IgG is seen at 50 kDa as a large thick band.

177

4. 4. Discussion

Maize SSIV (Accession number – EU599036.1) is the most recently identified isoform of starch synthases and 104 kDa in size (Yan et al. 2008).

Among the isoforms of starch synthases (SS), GBSS is essential for amylose synthesis and is exclusively bound to the starch granule where as SSI, SSII, and

SSIII isoforms are found to be responsible for amylopectin biosynthesis (Ball and Morell, 2003). All isoforms are unique and probably play a distinct role in the synthesis of amylopectin. SSI is responsible for producing DP 8-12 glucan chains (Nakamura, 2002), SSII and SSIII respectively produce DP=12-25

(Zhang et al. 2004; Morell et al. 2003) and DP 25-40 or greater glucan chains in amylopectin (Tomlinson and Denyer, 2003). However, the biochemical role of

SSIV in storage starch biosynthesis remains unclear. The investigations discussed in Chapter 4 are based on the hypotheses that SSIV in maize regulates starch synthesis through the interactions between other starch synthesis related enzymes by the formation of phosphorylation-dependent protein complexes. This study also tested the hypothesis that the SSIV and SP proteins interact. To investigate the role and regulation of SSIV, its localization, and various biochemical characterizations were carried out.

Immunodetection of SSIV indicated that the enzyme is exclusively localized in the amyloplast stroma of the wild-type maize endosperm as similarly observed in wheat endosperm by Leterrier et al. (2008) (Fig 4.4). SSIV was not detected as a granule bound protein in the starch granules. SSI and SSIIa isoforms are found both in the stroma and as granule bound proteins (Grimaud et al. 2008; Mu-foster et al. 1996; Rahman et al. 1995). However, the

178 antibodies were not able to detect the SSIII in the granule. The granule association of the SSIV was investigated mostly in the Arabidopsis thaliana chloroplast where transient starch is synthesized (Roldan et al. 2007, Szydlowski et al. 2009). SSIV is thought to be a vital requirement to determine the correct number of starch granules found in chloroplasts of Arabidopsis leaves and has been suggested to be involved in granule initiation (Szydlowski et al. 2009,

Planchot, et al. 2008, Roldan et al. 2007; D’Hulst and Merida 2012). However, loss of SSIV does not completely prevent starch granule formation in chloroplasts, suggesting that other factors may be involved in initiation process other than SSIV. Mutation in both SSIV and SSIII completely prevent starch biosynthesis indicating a mutual requirement for SSIII and SSIV in starch biosynthesis in the chloroplasts (Szydlowski et al. 2009). To investigate the process of granule initiation by SSIV, the specific localization of SSIV in the chloroplast was investigated by the florescence microscopic techniques

(Szydlowski et al. 2009). The results indicated that the SSIV has a plastidial localization and is present among the grana in the central part of the chloroplast, as well as in the grana-free peripheral part of the chloroplast.

Further, SSIV is not uniformly distributed within the stroma but was mainly located in specific regions at the boundary of starch granules (Szydlowski et al.

2009). Immunoblot analysis indicated that no SSIV was found inside the granules in Arabidopsis leaves (Szydlowski et al. 2009).

In this study, SSIV was shown to be expressed at later stages of endosperm development (Fig. 4.5). Immunodetection of proteins from amyloplast lysates extracted from maize kernels at 12, 15, 17, 22 DAA showed

179 that SSIV is expressed at greater levels in the later stage of endosperm development (Fig. 4.5). In rice endosperm, a greater level of SSIV-1 expression was found after 14 DAP (Dian et al. 2005). SSIIa, and SSIII are expressed in early stage (8 DAA) (Dian et al. 2005), and SSI in maize is expressed in the middle stage (16-20 DAA) of endosperm development (Cao et al. 1999), and studies in other plants indicate that different SS isoforms are expressed at different developmental stages (Dian et al. 2005). In chapter 2, it was reported that SP is actively expressed thoughout the various developmental stages in maize endosperm. Since the later stage of endosperm development shows higher levels of SSIV expression, SSIV may have other catalytic and/or regulatory roles in starch biosynthesis other than the proposed function of granule initiation. This idea is supported by the sequence analysis of SSIV in maize endosperm. The highly conserved C-terminal region of SSs has the catalytic and the starch-binding domains (Cao et al. 1999) (Fig. 1.5). The C- terminal domain of SSIV is conserved with other SS isoforms, but the N-terminal domain of SSIV protein is unique in cereal endosperms (Fig. 1.5); SSIV in wheat

(at 1-405 specific region), has two coiled-coil domains, which are thought to be involved in protein-protein interactions (Leterrier et al. 2008; Jody et al. 2004).

The 14-3-3-proteins are commonly linked to binding with various signaling proteins such as kinases and phosphatases (Comparot et al, 2003) and the N- terminus of SSIV in wheat has recognition sites for 14-3-3 proteins (Leterrier et al. 2008), which are conserved in other SS isoforms in barley (Alexander and

Morris, 2006) and Arabidopsis (Sehnke et al. 2001). Arabidopsis chloroplast

SSIII contains a conserved phosphoserine binding motif (RYGSIP), identifying it

180 as a putative target for binding with 14-3-3 proteins (Sehnke et al. 2001).

Moreover, GBSSI, SSI, SSII, and SBEIIa were shown to interact with 14-3-3 proteins in wheat amyloplasts (Alexander and Morris, 2006).

Determination of the affinity of the SSs in amyloplast lysates for different

α-glucan substrates was important characteristic to discriminate between the different SS isoforms (Commuri and Keeling, 2001). The substrate-enzyme dissociation constant (Kd) is inversely proportional to the affinity of the enzyme to the substrate. The affinity of SSIV towards different glucan substrates in different concentrations was evaluated in terms of relative migration (Rm) and the dissociation constant (Kd) (Fig. 4.6A/B). The results indicated that the SSIV has relatively higher affinity to amylopectin (Kd=1.0 mg/ml) compared to glycogen (Kd=2.5 mg/ml) and maltoheptaose (Kd=1.5 mg/ml) (Fig 4.6B). In previous studies, the affinity of SSI and SSII was found to be higher in amylopectin compared to starch (Kd=0.2 and 0.49 mg/ml respectively)

(Commuri and Keeling, 2001) (Table 4.1). Deletion of the N-terminal arm of maize SSI did not affect the Kd value suggesting that the starch-affnity domain of SSI is probably associated with, or close to the catalytic domain, at the C- terminus (Cao et al. 1999; Leterrier et al. 2008). SSI and SSII elongate different lengths of glucan chains, but share similar affinities towards amylopectin (Cao et al. 1999). Affinity of SSIII (Km=4.28 mM) and SSIV (Km=0.96 mM) for ADP- glucose was varied in Arabidopsis chloroplasts suggesting that the substrate binding capacity may be different in different isoforms of SSs (Valdez et al.

2008). Phylogenic analysis showed that the SSI, SSII, and GBSSI have distinct evolutionary origins compared with SSIIIs and SSIV, which have similar

181 evolutionary origins (Leterrier et al. 2008). However, the valine residue in the common K-V-G-G-L substrate binding domain in evolutionary related SSIII and

SSIV may be different in primer/substrate binding capacities than the other SS isoforms (Leterrier et al. 2008). A slight reduction in the number of shorter glucan chains (DP 7-10) in the starch of Arabidopsis SSIV mutants compared to the wild-type indicated that SSIV may involved in producing shorter glucan chains (Roldan et al. 2007).

Protein phosphorylation has been shown to play an important role in the regulation of enzymes involved in storage starch biosynthesis (Liu et al. 2009;

Hennen –Bierwagen et al. 2008; Tetlow et al. 2008; Tetlow et al. 2004). The effect of protein phosphorylation on the activity of SSIV was investigated by native affinity zymograms (Fig. 4.8A/B). Zymogram analysis of the activity of

SSIV indicated that the mobility of the protein was not altered following treatment with ATP or APase. However, the activity of SSIV (based on zymogram analysis) was reduced when treated with APase compared to ATP- treated and untreated samples (Fig 4.8B). SSIV was not found to be phosphorylated when the protein was tested with pre-incubated amyloplast lysates with -32P-ATP (Fig 4.7). Therefore, the reduction in the activity by APase treatment may be due to the indirect effect of dephosphorylation of another protein(s) that causes alterations in protein folding or has other regulatory effects on SSIV. Protein phosphorylation was identified as a mechanism for regulating starch biosynthesis in developing wheat endosperm by Tetlow et al.

(2004); and starch branching enzymes, SBEI, SBEIIa and SBEIIb and also SP in amyloplast stroma were phosphorylated and further involved in protein-protein

182 interactions forming phosphorylation-dependent multi sub-unit complexes. In wheat endosperm amyloplasts, protein phosphorylation enhanced the activity of

SBEIIb, whereas dephosphorylation using alkaline phosphatase (APase) reduced its catalytic activity (Tetlow et al. 2004). The granule bound SS isoform of SSIIa was also found to be phosphorylated (Tetlow et al. 2004). In maize amyloplast stroma, two functional protein complexes; one containing SSI, SSII and another containing SSII, SSIII, SBEIIa, and SBEIIb were identified confirming the phosphorylation-dependent physical interactions between SSs and SBEs

(Hennen –Bierwagen et al. 2008; Liu et al. 2009). In zymogram analysis (Fig.

4.8A/B), SSI and SSII had less mobility in the gel than SSIV and formed dense activity band in ATP-treated samples, which could be due to the formation of protein complexes. In wheat and maize endosperms, SSIIa can be phosphorylated and a high molecular weight functional protein complex consisting of SS isoforms (SSI, SSIIa, SSIII) and SBEs (SBEIIa and SBEIIb) formed, which showed higher affinity towards glucan substrate than the respective monomers (Liu et al. 2009; Hennen-Bierwagen et al. 2008; Tetlow et al. 2008). GPC analysis showed no difference in the elution of SSIV when amyloplast lysates were treated with ATP or APase (compared to untreated samples) and SSIV eluted in fractions suggestive of a monomeric protein (Fig.

4.9).

Mutant analysis in Arabidopsis suggests that SSIV in chloroplasts is involved in starch granule formation since distruption of this enzyme resulted in lower numbers of starch granules per chloroplast, with increased granule sizes

(Roland et al. 2008; Planchot et al. 2008). This work suggested a possibility of a

183 functional or physical interaction between SP and SSIV (Roland et al. 2008;

Planchot et al. 2008). In co-immunoprecipitation experiments, amyloplast lysates treated with 1 mM ATP detected interaction between SSIV and SP (Fig.

4.11). Since SSIV was not phosphorylated by ATP under given experimental conditions (Fig. 4.7), phosphorylation of SP may be required to drive this interaction (Fig. 4.11). No protein-protein interaction was found between SSIV and SSI, SSIIa, SSIII, SBEI, SBEIIb, or ISOI in amyloplast lysates under these experimental conditions. (Fig. 4.10B).

Recent work on the investigation of transient starch biosynthesis in

Arabidopsis thaliana suggests that SP and SSIV may interact, and may be involved in priming the starch granule (Roldan et al. 2007; Planchot, et al.

2008; (Szydlowski et al. 2009; Zhang et al. 2005). The presence of either SSIII or SSIV is recognized as a vital requirement in producing transient starches

(Szydlowski et al. 2009). However, given the expression of SSIV throughout endosperm development it is possible that SSIV also plays a role in starch biosynthesis, despite its lack of interaction with other major SSs or SBEs. SSIV may be involved in protein-protein interactions with proteins which we could not detect by co-immunoprecipitaion.

184

General Discussion

The aim of the investigations presented in this thesis were to test the hypothesis that the plastidial form of starch phosphorylase (SP) in cereal endosperm is involved in starch synthesis by its direct interaction with other enzymes of the pathway. This study also tested the hypothesis that these protein-protein interactions are regulated by protein phosphorylation. A biochemical approach was employed in order to address these questions. In this study, maize was used as an example of a cereal; maize is a widely grown crop in Ontario/North America and its endosperm produces high yields of starch.

Maize has been used as a model plant in many starch biosynthetic studies and efficient extraction procedures of amyloplasts, and peptide-specific antibodies of major starch biosynthetic enzymes of maize have been developed by our laboratory.

SP is the second most abundant enzyme present in maize amyloplasts next to SBEIIb (Yu et al. 2001). In the localization analysis, SP is found only in the amyloplast stroma of the wild-type maize endosperm (section 2.3.1 in the thesis and Yu et al. 2001), which was similarly observed in wild-type wheat

(Tetlow et al. 2004) and rice (Satoh et al. 2008) amyloplasts. Interestingly, the ae- mutant of maize endosperm lacking SBEIIb in the plastid, SP is not only found in the stroma but also in the starch granule as a granule-bound protein, thought to be a result of its association within a multi sub-unit protein complex formed by interaction with other starch biosynthetic enzymes (Liu et al. 2009;

Grimaud et al. 2008). Further, in the amyloplast stroma of the same ae- mutant,

SP was shown to form larger multi sub-unit complexes with SBEI, SBEIIa, SSI

185 and SSIIa (Liu et al. 2009). In the ae- mutant, lacking SBEIIb, increased amounts of granule bound SBEI, SBEIIa, SSIII, and SP are found without affecting SSI or SSIIa (Grimaud et al. 2008). These observations suggest a functional role for SP in starch biosynthesis, at least in the absence of SBEIIb. It was suggested that the presence of SP in the amyloplast stroma could compensate for the activity of missing isoforms of major starch biosynthetic enzyme (SBEIIb) in the ae- mutant (Liu et al. 2009). In wild-type maize, SP is not bound to the granule but closely associated with the granule surfaces, as are some other enzymes e.g. SSI, SBEI and SBEIIb (Fig. 2.2). In contrast, SSII was comparatively abundant at the granule surface and very little of this enzyme is detected in the stroma. Recent evidence suggests that SSIIa is a central component of the trimeric protein complex involved in amylopectin cluster synthesis, and directs it self and SSI, and SBEIIb into the starch granule (Liu et al. 2012). As Tickle et al. (2009) suggested, in wheat amyloplasts, SP could act directly on the surface of the starch granule in a degradative manner, where SP modifies the granule structure in a phosphorolytic manner to produce G-1-P which can be recycled back to produce starch via AGPase. However, more investigations are required to analyze this hypothesis.

In the wild-type maize amyloplast, SP remains active in the synthetic direction throughout the various developmental stages observed from 12 to 24

DAA (days after anthesis) in maize amyloplast (Chapter 2, section 2.3.2). This observation was parallel to the observations in other storage starch producing plants such as in wheat, rice and in potato tubers; suggesting that the SP has a certain involvement in the starch synthesis process in plants (Tickle et al. 2009;

186

Satoh et al. 2008; Schupp and Ziegler, 2004; Yu et al. 2001; Brisson et al.

1989; Tsai and Nelson, 1968), despite earlier suggestions that SP had a primarily degradative role (Preiss, 1982; Preiss, 1984). Early studies of starch synthesis suggested that SP was the enzyme responsible for glucan elongation

(Leloir, 1964). However, the fact that α-glucan phosphorylase (EC 2.4.1.1) found in animals, fungi, and prokaryotes plays a major role in glucan catabolism

(Alonso-Casajús et al. 2006; Ball and Morell, 2003; Newgard et al. 1989; Preiss,

1984), led many researchers to believe that SP had an essentially degradative role in plant cells. In rice endosperm, zymogram analysis of mutants lacking SP showed no change in the activities of DBE isofoms (isoamylase and pullulanase),

SBE isoforms (SBEI, SBEIIa, and SBEIIb), and SS isoforms (SSI and SSIIIa) but a reduction in total starch content was observed in the rice endosperm forming a shrunken phenotype (Satoh et al. 2008). The starch content per grain in mutants lacking SP was even less than in the shrunken 2 mutants of rice, lacking the AGPase large subunit (Satoh et al. 2008). Mutants of rice endosperms lacking SP grown at 300C produced about 6% of the shrunken phenotypes, the starch content was similar in the wild-type. Percentages of shrunken phenotype was increased in SP mutant plants grown at 250C and 200C by 35-39% and 66% respectively with a severe reduction in starch accumulation suggesting that SP may play an important role in starch biosynthesis at fluctuating and/or adverse temperature conditions (Satoh et al.

2008). Further, the reduced starch content produced by mutants lacking SP and the fact that SP is actively expressed in early stages of endosperm development

187 in rice endosperm suggest that SP is essential for the early steps of starch biosynthesis in rice endosperm (Satoh et al. 2008).

Peptide specific anti-SP antibodies recognized the plastidial SP in amyloplasts but not in chloroplasts. This may be due to reduced levels of SP in chloroplasts, or the chloroplastic SP may have different amino acid sequences in the region where epitopes were designed (Chapter 2, Fig. 2.1). In rice mutants lacking plastidial SP; the endosperm had severely reduced levels of starch and had a shrunken phenotype (Satoh et al. 2008), and in Arabidopsis leaves lacking

SP in chloroplasts, no significant change in the total accumulation of starch was observed compared to their wild-types (Zeeman et al. 2004), suggesting a divergent role of plastidial SP present in storage starch biosynthesis compared to transient starch biosynthesis in chloroplasts.

Recent research confirmed that SP in cereal endosperms is regulated by protein phosphorylation as similarly observed in some other isoforms of the major starch biosynthetic enzymes (Liu et al. 2009; Pollack, 2009; Hennen-

Bierwagen et al. 2008; Grimaud et al. 2008; Tetlow et al. 2008; Tetlow et al.

2004). Phosphorylation of SP may promote the formation of protein-protein interactions (Liu et al. 2009; Hennen-Bierwagen et al. 2008; Grimaud et al.

2008; Tetlow et al. 2004). SP in wheat endosperm was shown to be involved in the formation of protein complexes with SBEI and SBEIIb (Tetlow et al. 2004).

From the research presented in this thesis, the mobility of SP on phosphate affinity SDS-PAGE using Phos-TagTM ligand-acrylamide gel showed no alteration following treatment in ATP compared to untreated SP in the SDS-PAGE gel

(Chapter 2, Fig. 2.6). Treatment of amyloplasts with 1 mM ATP [under

188 conditions previously determined to cause phosphorylation of SP by Pollack,

(2009)] or APase (known to cause non-specific protein dephosphorylation) did not alter the catalytic activity of SP (Chapter 2, Fig. 2.5). Phosphorylation of SP therefore, may have a role in complex formation, either with other enzymes of starch synthesis (Chapter 3 section 3.3.4. and Chapter 4 section 4.5.8) or in the formation of SP multimers, but does not appear to play a role in regulating its catalytic activity.

In the present study, GPC analysis confirmed that the SP exists in the amyloplast stroma mainly as tetrameric and dimeric forms throughout the developmental stages (both multimeric states were observed at 15-35 DAA) in maize endosperm (Fig. 2.8). These conformational structures of SP are found to be as a natural molecular characteristic of SP, which has previously been observed in higher plants (Liu et al. 2009; Mu et al. 2001; Brisson et al. 1989) and the dimeric forms are observed in bacteria (Dauvillee et al. 2006) and yeast

(Tanabe et al. 1987). In the GPC analysis, the elution profile of the ATP-treated and APase treated native SP did not drastically change from the untreated amyloplast lysates (Fig. 2.8) suggesting that the formation of the homodimeric or homotetrameric forms of SP is probably not controlled by protein phosphorylation (Chapter 2, Fig. 2.8).

It was previously reported that SP from wheat endosperm amyloplasts formed protein complexes with SBEI and SBEIIb in a phosphorylation-dependent manner (Tetlow et al. 2004). In the maize ae- mutant lacking SBEIIb, SP was shown to interact with different proteins. The complex in ae- contained SSI,

SSIIa and SBEI and SP. In this complex it was suggested SBEI and SP in some

189 way compliment the loss of SBEIIb in the mutant (Liu et al. 2009). In addition

SP, which is part of the novel protein complex, was found as a granule-bound protein, reinforcing the fact that protein complex components become granule bound by an as yet unknown mechanism (Liu et al. 2009; Grimaud et al. 2008).

The multimeric status of the SP in the wild-type wheat endosperm complex, and the complex in ae- mutant endosperm is not known.

In this study, peptide specific anti-maize SP antibodies were used to immunoprecipitate the native SP from the wild-type maize amyloplast stroma using Protein-A Sepharose beads to investigate possible protein-protein interactions (Chapter 2, section 2.3.6). It was not possible to immunoprecipitate the native SP using the Protein-A Sepharose beads (Fig 2.10) and consequently we were unable to employ the antibodies in immunoprecipitation and co- immunoprecipitation experiments. The reason for the inability of the peptide- specific antibodies to recognize the native protein is unclear, but it is possible that the native SP in someway shields the epitope, irrespective of the multimeric state of the protein. Therefore, an S-tagged recombinant SP was developed by over expressing the full length mRNA sequence (3053 bp) of plastidial maize SP in Artic Express E.coli cells after cloning in pET29a expression vectors (Chapter

3).

GPC was a useful tool for separating the amyloplast lysates or cell extracts and in identification of major starch biosynthetic enzymes as monomers or in complexes in fractionated extracts with predicted molecular weights

(Hennen-Bierwagen et al. 2008; Tetlow et al. 2208; Liu et al. 2009).

Fractionation of recombinant SP extracts by GPC partially purified the

190 recombinant SP and enabled us to identify different multimeric forms of recombinant SP (Fig. 3.9A). Greater amounts of recombinant SP was aggregated

(2000 kDa) and found to be active, including tetrameric, dimeric and monomeric forms of SP (Fig. 3.9B/C). Dimeric and tetrameric forms of active recombinant S-tagged SP separated by GPC were immobilized by S-Protein

Agarose beads, and used as affinity ligands to isolate and detect amyloplast proteins which interact with SP (Fig. 3.10). The various pull down assays that were carried out with recombinant SP and amyloplast lysates indicated that certain starch biosynthetic enzymes specifically interacted with the dimeric and tetrameric forms of SP in a phosphorylation-dependent manner (Figs. 3.12,

3.13). Many of the protein-protein interactions previously observed in cereal endosperm amyloplasts have also been shown to be phosphorylation dependent

(Liu et al. 2009; Grimaud et al. 2008; Hennen-Bierwagen et al. 2008; Tetlow et al. 2008; Tetlow et al. 2004). SBEI directly interacted with both tetrameric and dimeric forms of SP and the SBEIIb interacted only with the dimeric forms of SP when plastid lysates were pre-treated with 1 mM ATP. Weak interactions between SSIIa and SP were observed; unlike the SBE-SP interactions, no interactions between SP and SSIIa have been observed previously. Unlike the

SP-SBE interactiions, previous experiments involving immunoprecipitation of

SSIIa have not detected SP as an interacting partner. The ATP-dependence of some of the protein-protein interactions suggest a phosphorylation dependent mechanism of complex assembly. In other complexes studied, some of the components are directly phosphorylated (Liu et al. 2009). Other than the SP, previous research had already confirmed that SSIIa, SBEI and SBEIIb are

191 regulated by protein phosphorylation (Liu et al. 2009; Tetlow et al. 2008; Tetlow et al. 2004).

Glucan phosphorylases found in both prokaryotic and eukaryotic systems exist as dimers or tetramers of identical subunits (Dauvillee et al. 2006; Mu et al. 2001; Brisson et al. 1989; Tanabe et al. 1987). Both dimeric and tetrameric configurations of SP have been observed in maize amyloplasts lysates (Mu et al.

2201; Liu et al. 2009). In addition to SP, SBEIIa and SBEIIb have been found to be associated as homodimers (Tetlow et al. 2008). However, based on the elution profiles from GPC analysis, it was not clear that the interactions found between the homodimeric forms of SP were with monomers or homodimeric forms of SBEIIb. Although the precise roles of the various protein-protein interactions in amyloplasts is not clear, it is possible that some of the interactions with SP and other enzymes regulate SP activity by controlling the multimeric status of the protein. Different multimeric states of SP may have variable affinities for other proteins, which may be controlled and regulated by protein phosphorylation. The relative competition of different multimeric forms of SP and other proteins for each other is an area for future study. For example, homodimeric forms of SBEIIb interacting with SP may prevent the interactions between the tetrameric forms of SP.

The protein-protein interactions is the fact that may enzymes of the pathway are differentially expressed throughout endosperm development. In maize endosperm, SSIIa, SSIII and SBEIIa are expressed in early stages of development (approximately 8-15 DAA) and SSI, SSIIb and SBEIIb are expressed in the middle stage (approximately 16-24 DAA) and GBBSSI, SSIV

192 and SBE1 are expressed at the later stage (over 24 DAA) of the grain filling (Liu et al. 2009; Zhang et al. 2004; Mu et al. 2001; Mu-Forster et al. 1996).

As discussed in previous studies, SP has various potential functions in starch biosynthesis. SP showed a higher capacity to synthesize longer linear glucans from small MOS than SSIIa (Satoh et al. 2008). A possible function of

SP was suggested by Nakamura et al. (2012) and Satoh et al. (2008) based on the ‘starch trimming model’ (Ball and Morell 2003), whereby small malto dextrins produced by the activity of DBE provide a substrate for SP to produce linear glucan chains, which in turn serve as the substrates for SBE to form branched glucans in the starch initiation process.

Functional interactions between SP and SBE isoforms were observed in rice endosperm. Purified SP from rice endosperm synthesized glucans from G-1-

P in the presence of SBE without any exogenous glucan primer and glucan production was higher when SBEI was present compared to SBEIIa or SBEIIb

(Nakamura et al. 2012). Activities of SP and SBE were dependent on the mutual availability SP and SBE and showed mutual capacities for chain elongation and chain branching (Nakamura et al. 2012). These observations further support the function of SP proposed by Satoh et al. (2008). In contrast, according to the proposed functions of SP suggested by Tickle et al. (2009), SP may play a degradative role by directly acting on the starch granule to produce G-1-P, or may degrade the MOS which are produced by DBE reaction to produce G-1-P and supplying the substrate for AGPase for starch biosynthesis. The presence of catalytically active SP thoughout the grain filling period of maize endosperm, and the interaction of different multimeric forms of SP with SBE insoforms

193 support a synthetic role for SP in starch biosynthesis in maize endosperm as suggested by Satoh et al. (2008) and Nakamura et al. (2012) in rice. Low G-1-P concentrations, and high Pi/G-1-P ratios are considered as the controlling mechanism of SP activity in glucan synthesis (Tiessen et al. 2011; Schupp and

Ziegler, 2004; Mu et al. 2001; Matheson and Richardson, 1978). Plastidial and cytosolic SP activities in degradative direction were reduced by 80% and 20% respectively when Pi was added in vitro (Mu et al. 2001) suggesting that Pi regulates degradative activity of plastidial SP more than cytosolic SP. Low levels of G-1-P and a 50-fold excess of Pi in vitro were able to sustain the SP biosynthetic reaction (Hwang et al. 2010), suggesting that, plastidial SP preferentially carries out starch biosynthesis over degradation of starch.

The leaves of Arabidopsis ss4 mutants (where transient starch is synthesized) showed reductions in granule number and increased granule size

(1.4-2 fold) (Roldan et al. 2007) and the double mutants of ss4 and sp further increased the granule size by 4-fold (Planchot et al. 2008) compared with the wild-type plants, suggesting the possibility that SSIV and SP may form functional protein-protein interactions and are in some way involved in granule initiation in chloroplasts. One of the major hypotheses tested in the study was to investigate the possible interactions between SSIV and SP. In co- immunoprecipitation experiments conducted by using peptide-specific anti-SSIV antibodies, in ATP-treated amyloplasts lysates SP weakly interacted with SSIV

(Chapter 4, section 4. section 4.8). Since there was no evidence for SSIV phosphorylation (Chapter 4, section 4.5.), the ATP-dependent interaction observed may be due to phosphorylation of SP or other as yet unidentified

194 factors. Since the reciprocal interactions using S-tagged recombinant SP did not show any interactions with SSIV, the results with the SSIV co- immunoprecipitation experiment should be treated with caution. It is possible that SP and SSIV interact weakly, and/or transiently in vivo and under these experimental conditions the interaction is not observed consistently. In the S- tagged SP studies, the total protein (0.5 mg/mL) of the amyloplast lysates were comparatively lower than in the co-immunoprecipitation analysis (1.0 mg/mL) so that the amount of available SSIV may be limited, and below detectable levels in these interactions. Also, the recombinant forms of SP may not be phosphorylated as efficiently as the native form, leading to less stable interactions. The phosphorylation status of the recombinant SP following ATP- treatment of amyloplast lysates was not examined. The interaction found in the study between SP and SSIV may have significance in relation to our understanding of the initiation of the starch granule. In addition, SP was the only protein which interacted with SSIV indicating a high specificity towards SP.

Activity and/or the affinity of the SSIV required to initiate the priming of granule initiation may be regulated by the interactions with SP.

To elucidate both the synthetic and the degradative activities of the recombinant tetrameric and dimeric forms of SP, they were tested in glucan substrates of maltoheptaose, glycogen and amylopectin and at 25 mg/mL concentration, both multimeric states are active in both synthetic and phosphorylitic directions (Fig. 3.13). The higher activities of both multimeric forms of SP with high molecular weight amylopectin followed by glycogen and maltoheptaose were observed in both synthetic and phosphorolytic direction,

195 and was similar to previous findings in maize (Yu et al. 2001), potato (Liddle et al. 1961) and spinach leaves (Shimomura et al. 1982). Bacterial SP has a tetrameric configuration, and also shows a higher activity in starch than in maltopentaose in both directions (Weinhäusel et al. 1997). The Km values indicate the affinity level of SP towards different glucan substrates in phosphorolytic direction (Table 3.1). In tetrameric SP, the higher Vmax showed with amylopectin also showed a higher Km (lower affinity) compared to maltoheptaose which had a lower Vmax but a lower Km (higher affinity), which was similarly observed in both synthetic and degradative directions by Mu et al.

(2001), and suggests higher affinity of enzyme to the substrate not essentially increased the activity of SP (Table 3.1).

The variation in the activity of tetrameric SP from synthetic direction to phosphorylitic direction was greater in maltoheptaose (147 fold) compared to amylopectin (2.1 fold) and glycogen (1.1 fold) (Table 3.1) indicating the preference of SP for low molecular MOS in degradative directions. This has also been observed by Mu et al. (2001). However, the higher activities of SP forms with highly branched amylopectin conflicts with the proposed function of SP in the suggested model proposed by Satoh et al. (2008) and Nakamura et al.

(2012). In the model, during discontinuous synthesis of starch granules, the short glucan chains released from pre-amylopectin by the action of debranching enzymes are converted to longer glucan chains by SP.

In potato tuber (plastidial SP) and leaf (cytosolic SP) were defined as low affinity (SP-L) and high affinity (SP-H) isoforms respectively according to the

196 affinities showed to both amylopectin and glycogen in synthetic direction (Mori et al. 1993) (Table 1). The proposed function of the L-78 insertion located in the middle of the plastidial SP, which was not observed in cytosolic SP (Yu et al.

2001; Albrecht et al. 1998; Nakano and Fukui, 1986) is to obstruct the binding affinity of plastidial SP to large, highly branched starch compared to glycogen

(Young et al. 2006; Albrecht et al. 1998). Very little is known about the regulatory mechanism of SP-specific L-78 insertion existing in the plastidial form of SP and no evidence for L-78 cleavage or the function of the insertion is available for maize. In the sweet potato tuber enzyme, serine residues located in

L-78 insertion are phosphorylated and are thought to then target the L-78 peptide for proteolytic cleavage (Young et al. 2006).

The results presented in this thesis demonstrate that SP is catalytically active in dimeric and tetrameric forms throughout the endosperm development and is involved in protein-protein interactions with the major starch biosynthetic enzymes. Some of the interactions were enhanced by pre-treatment with ATP and SP has previously been shown to be phosphorylated (Pollock, 2009; Liu et al. 2009; Grimaud et al. 2008; Tetlow et al. 2004) suggesting phosphorylation of SP may control, in some as yet unknown manner, protein-protein interactions. For future directions, investigating the glucan priming and glucan synthesizing capacities of different dimeric and tetrameric forms and their regulation by G-1-P or Pi in vitro would be essential in further understanding the function of SP. Fig. 5.1 illustrates the proposed functions dimeric and tetrameric isoforms of SP in starch biosynthesis; phosphorylation of SP and SBE enzymes facilitate the formation of protein-protein interactions between these enzymes

197 and between SP and SSIV. Interaction between SP and SBE may regulate and activate SBE to in turn facilitate interactions with starch synthases in the amyloplast. Another potential function for SP is in starch granule initiation by interacting with SSIV (Fig. 4.11).

Figure 5.1: Schematic diagram illustrating the proposed functions of dimeric and tetrameric forms of plastidial SP. Phosphorylation of SP and SBE facilitate the formation of protein-protein interactions; phosphorylated SBEI interacts with both dimeric and tetrameric forms of SP while phosphorylated SSIIa interacts with the tetrameric form of SP and phosphorylated SBEIIb interacts with dimeric SP forms and may regulate and activate the branching enzymes to facilitate interactions with starch synthases in the amyloplast while SP remains in the stroma. A second function of SP may be in starch granule initiation by interacting with SSIV. Phosphorylated proteins are denoted by the P symbol.

198

This research provides further insight into our growing understanding of the coordinated activities of different enzymes associated in starch synthesis through protein-protein interactions and complex formation in developing maize endosperm. The protein-protein protein interactions and the complexes formed in amyloplasts are suggested to be a vital requirement in synthesizing starches with different morphological characteristics by modulating granule fine structure.

Understanding the basis of these modulations is essential for rational manipulation of starch in crops. Application of starch in food and non-food industries depends on different structural and functional properties of starch which can be modified with the knowledge of its genetic manipulations. This research provides information to understand the basics of starch biosynthesis to develop models in developing modify polymer structures of starch.

199

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Appendixes

Appendix 01:

Synthetic activity of plastidial SP in amyloplast lysates (22 DAA) was slightly reduced with the absence of SSIIa. Protein-protein interactions between both dimeric and tetrameric forms of SP with SSIIa may have affected for the activity of SP. There was no different in the synthetic activity of SP when SSIV was removed.

(A) (B) (C) (D)

Synthetic activity of plastidial SP in amyloplast lysates (22 DAA) in the absence of SSIIa (A) and SSIV (C) was tested in non-denaturing affinity native zymogram containing 0.1% glycogen in the gel. Immunoblot of the zymogram gels (A and C) were probed by peptide specific anti-SP (B and D) antibodies. SSIIa and SSIV in amyloplast lysates were removed by immunoprecipitating the proteins using anti-SSIIa and anti-SSIV antibodies bound to Protein-A sepharose beads. The supernatants obtained after immunoprecipitation of stromal SSIIa and SSIV were used (90 µg/mL per well) in zymogram analysis.

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Appendix 02:

(A) (B)

Immunoprecipitation of stromal SP from maize amyloplasts (22 DAA) in the absence of SSIIa was tested using peptide specific anti-SP antibodies following immunoprecipitation with anti-SSIIa antibodies. 1 ml amyloplast lysates (1 mg/mL) were incubated with peptide-specific anti-SSIIa (15 mg/mL final concentration) at room temperature for 1 hr, and then immunoprecipitated with Protein-A-Sepharose beads. The supernatants were obtained after the beads bound to SSIIa were centrifuged at 13,000 rpm for 5min at 40C. Supernatant was used (1 mg/mL) immunoprecipitate SP by anti-SP antibodies (15 mg/mL final concentration). Washed Protein-A-Sepharose-antibody-antigen complexes were boiled in 200 μL 1XSDS-loading buffer and 30 μL was loaded onto 10% SDS gels. Immunoblotted membranes were developed with SSIIa (A) and SP (B) anti-maize antisera.

The results showed that SP was not immunoprecipitated by anti-SP antibodies bound to Protein A-sepharose beads after removing the SSIIa present in the amyloplast lysates, suggesting that the SSIIa is not preventing the binding of SP to anti-SP antibodies bound to Protein A-sepharose beads.

219

Appendix 03:

Primers used in PCR to isolate the complete coding sequence of plastidial SP of maize endosperm. SP-F1 and SP-R1 primers were designed with the part of transit peptide sequence and 15 bp overhangs from pET29a vector are underlined in SP-F2 and SP-R2 primers.

Primer Primer sequence Annealing Name temperature SP-F1 5’ GCGGAGGTGGGGTTCTCCT 3’ 640C

0 SP-R1 5’ GCGAAAGAACCTGATATCCAC 3’ 62 C SP-F2 5’GGTTCCATGGCTGATTCAGCGCGCAGCG 3’ 460C

0 SP-R2 5’GAATTCGGATCCGATCTAGGGAAGGATGGC 3’ 48 C

Appendix 04:

Primers used in sequence analysis of the isolated plastidial SP sequenced cloned into pET29a vector. In addition to SP-F2 AND SP-R2 primers (see appendix 03), T7 promoter and T7 terminator universal primers and F1, F2, F3 primeres were used. The mRNA complete coding sequence of plastidial SP of maize endosperm from NCBI was used to design the primers.

Primer Primer sequence Annealing Location in Name temperature original sequence T7- 5′ TAA TAC GAC TCA CTA TAG GG 3′ 480C - promoter

T7- - 5’ GCTAGTTATTGCTCAGCGG 3’ 760C terminator

F1 5’ GGAACCAGATGCTGCCCTG 3’ 620C 393-411 bp

F2 5’ GTTGCAGTGCAGATGAATGAC 3’ 680C 1006-1026 bp

F3 5’ GGTGTAGCTGAAATTCACAGTG 3’ 680C 1636-1657 bp

220

Appendix 05:

Following is the alignment comparison of the predicted amino acid sequence of plastidial maize SP obtained from NCBI with the amino acid sequence of the recombinant SP produced in the study. Amino acid sequence of recombinant SP was derived from the mRNA sequence of PCR product of the full length sequence (2805 bp) of SP (except transit peptide). Arrow shows thw change in amino acid sequence of recombinant SP from the predicted sequence.

(http://www.ch.embnet.org/software/LALIGN_form.html)

(A) ./wwwtmp/.25133.1.seq predicted SP (NCBI) 849 bp - 849 aa (B) ./wwwtmp/.25133.2.seq Recombinant SP 724 bp - 724 aa using matrix file: BL50 (15/-5), gap-open/ext: -14/-4 E(limit) 0.05 99.6% identity in 706 aa overlap (73-778:1-706); score: 4614 E(10000): 0

80 90 100 110 120 130 Predicted TLNYPAWGYGLRYEYGLFKQIITKDGQEEIAENWLEMGYPWEVVRNDVSYPVKFYGKVVE :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: Recombinant TLNYPAWGYGLRYEYGLFKQIITKDGQEEIAENWLEMGYPWEVVRNDVSYPVKFYGKVVE 10 20 30 40 50 60

140 150 160 170 180 190 Predicted GTDGRKHWIGGENIKAVAHDVPIPGYKTRTTNNLRLWSTTVPAQDFDLAAFNSGDHTKAY :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: Recombinant GTDGRKHWIGGENIKAVAHDVPIPGYKTRTTNNLRLWSTTVPAQDFDLAAFNSGDHTKAY 70 80 90 100 110 120

200 210 220 230 240 250 Predicted EAHLNAKKICHILYPGDESLEGKVLRLKQQYTLCSASLQDIIARFESRAGESLNWEDFPS :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: Recombinant EAHLNAKKICHILYPGDESLEGKVLRLKQQYTLCSASLQDIIARFESRAGESLNWEDFPS 130 140 150 160 170 180

260 270 280 290 300 310 Predicted KVAVQMNDTHPTLCIPELMRILMDVKGLSWSEAWSITERTVAYTNHTVLPEALEKWSLDI :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: Recombinant KVAVQMNDTHPTLCIPELMRILMDVKGLSWSEAWSITERTVAYTNHTVLPEALEKWSLDI 190 200 210 220 230 240

320 330 340 350 360 370 Predicted MQKLLPRHVEIIETIDEELINNIVSKYGTTDTELLKKKLKEMRILDNVDLPASISQLFVK :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: Recombinant MQKLLPRHVEIIETIDEELINNIVSKYGTTDTELLKKKLKEMRILDNVDLPASISQLFVK 250 260 270 280 290 300

221

380 390 400 410 420 430 Predicted PKDKKESPAKSKQKLLVKSLETIVDVEEKTELEEEAEVLSEIEEEKLESEEVEAEEESSE :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: Recombinant PKDKKESPAKSKQKLLVKSLETIVDVEEKTELEEEAEVLSEIEEEKLESEEVEAEEESSE 310 320 330 340 350 360

440 450 460 470 480 490 Predicted DELDPFVKSDPKLPRVVRMANLCVVGGHSVNGVAEIHSEIVKQDVFNSFYEMWPTKFQNK :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: Recombinant DELDPFVKSDPKLPRVVRMANLCVVGGHSVNGVAEIHSEIVKQDVFNSFYEMWPTKFQNK 370 380 390 400 410 420

500 510 520 530 540 550 Predicted TNGVTPRRWIRFCNPALSALISKWIGSDDWVLNTDKLAELKKFADNEDLHSEWRAAKKAN :::::: ::::::::::::::::::::::::::::::::::::::::::::::::::::: Recombinant TNGVTPXRWIRFCNPALSALISKWIGSDDWVLNTDKLAELKKFADNEDLHSEWRAAKKAN 430 440 450 460 470 480

560 570 580 590 600 610 Predicted KMKVVSLIREKTGYIVSPDAMFDVQVKRIHEYKRQLLNILGIVYRYKKMKEMSTEERAKS :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: Recombinant KMKVVSLIREKTGYIVSPDAMFDVQVKRIHEYKRQLLNILGIVYRYKKMKEMSTEERAKS 490 500 510 520 530 540

620 630 640 650 660 670 Predicted FVPRVCIFGGKAFATYIQAKRIVKFITDVAATVNHDSDIGDLLKVVFVPDYNVSVAEALI :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: Recombinant FVPRVCIFGGKAFATYIQAKRIVKFITDVAATVNHDSDIGDLLKVVFVPDYNVSVAEALI 550 560 570 580 590 600

680 690 700 710 720 730 Predicted PASELSQHISTAGMEASGTSNMKFAMNGCILIGTLDGANVEIREEVGEENFFLFGAEAHE :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: Recombinant PASELSQHISTAGMEASGTSNMKFAMNGCILIGTLDGANVEIREEVGEENFFLFGAEAHE 610 620 630 640 650 660

740 750 760 770 Predicted IAGLRKERAEGKFVPDPRFEEVKEFVRSGVFGTYSYDELMGSLEGN ::::::::::::::::::::::::::::::::::::::: ::: :: Recombinant IAGLRKERAEGKFVPDPRFEEVKEFVRSGVFGTYSYDELXGSLXGN 670 680 690 700

222

Appendix 06:

Predicted phosphorylation sites of maize SSIV was analyzed by NetPhos 2.0 server.

Phosphorylation sites predicted: Ser: 37 Thr: 7 Tyr: 9

Serine predictions

Name Pos Context Score Pred ______v______Sequence 21 PAHTSTPLF 0.010 . Sequence 667 MRDNSHGRI 0.987 *S* Sequence 38 DAAASSSTP 0.520 *S* Sequence 681 AVVYSNIVT 0.025 . Sequence 39 AAASSSTPF 0.193 . Sequence 688 VTTVSPTYA 0.545 *S* Sequence 40 AASSSTPFT 0.213 . Sequence 697 QEVRSEGGR 0.658 *S* Sequence 55 RLPMSCSAA 0.580 *S* Sequence 711 LKVHSKKFV 0.981 *S* Sequence 57 PMSCSAAAG 0.003 . Sequence 729 TWNPSTDRF 0.293 . Sequence 71 LLIRSAAPS 0.007 . Sequence 739 KVQYSANDL 0.758 *S* Sequence 75 SAAPSTIVG 0.979 *S* Sequence 747 LYGKSANKA 0.009 . Sequence 86 RLAMSRRTS 0.840 *S* Sequence 761 LKLASTQAS 0.017 . Sequence 90 SRRTSRRNL 0.998 *S* Sequence 765 STQASQPLV 0.009 . Sequence 104 PHQKSAPSA 0.010 . Sequence 803 VLLGSSPVQ 0.009 . Sequence 107 KSAPSANHR 0.013 . Sequence 804 LLGSSPVQH 0.231 . Sequence 115 RNRASIQRD 0.883 *S* Sequence 844 IFAASDMFI 0.179 . Sequence 122 RDRASASID 0.940 *S* Sequence 851 FIVPSMFEP 0.541 *S* Sequence 124 RASASIDEE 0.947 *S* Sequence 868 MRYGSVPVV 0.245 . Sequence 133 QKQMSEDEN 0.995 *S* Sequence 881 GLNDSVFDL 0.978 *S* Sequence 194 EMKLSETGK 0.027 . Sequence 939 KIDFSWDTS 0.886 *S* Sequence 200 TGKQSVLSS 0.622 *S* Sequence 943 SWDTSVSQY 0.862 *S* Sequence 203 QSVLSSEVK 0.974 *S* Sequence 945 DTSVSQYEE 0.904 *S* Sequence 204 SVLSSEVKS 0.687 *S* ______^______Sequence 208 SEVKSDEES 0.997 *S* Sequence 212 SDEESLEFD 0.987 *S* Sequence 245 ETEESLFKL 0.603 *S* Sequence 259 LLNASLREL 0.964 *S* Sequence 267 LECTSTSAQ 0.420 . Sequence 269 CTSTSAQSD 0.096 . Sequence 272 TSAQSDVLK 0.116 . Sequence 298 DLLDSTANQ 0.421 . Sequence 307 VEHASLTLD 0.007 . Sequence 326 KLKASLGTT 0.228 . Sequence 333 TTNVSEFCL 0.132 . Sequence 349 QRVKSVEER 0.997 *S* Sequence 363 HEMHSQIEL 0.947 *S* Sequence 371 LYEHSIVEF 0.115 . Sequence 380 HGTLSKLIN 0.028 . Sequence 386 LINESEKKS 0.953 *S* Sequence 390 SEKKSMEHY 0.988 *S* Sequence 400 EGMPSEFWS 0.540 *S* Sequence 404 SEFWSRISL 0.038 . Sequence 407 WSRISLLID 0.040 . Sequence 414 IDGWSLEKK 0.047 . Sequence 420 EKKISINDA 0.966 *S* Sequence 425 INDASMLRE 0.275 . Sequence 444 EAYLSSRGM 0.981 *S* Sequence 445 AYLSSRGME 0.033 . Sequence 456 ELIDSFLKM 0.024 . Sequence 466 LPGTSSGLH 0.043 . Sequence 467 PGTSSGLHI 0.007 . Sequence 492 ADVISGLGK 0.005 . Sequence 532 VVVKSYFEG 0.915 *S* Sequence 581 FKRFSYFSR 0.944 *S* Sequence 584 FSYFSRVAL 0.778 *S* Sequence 594 LLYQSGKKV 0.610 *S* Sequence 626 LGFNSARIC 0.004 .

223

Threonine predictions Tyrosine predictions

Name Pos Context Score Pred Name Pos Context Score Pred ______v______v______Sequence 9 RPRPTARAR 0.972 *T* Sequence 313 TLDGYRDFQ 0.547 *Y* Sequence 20 DPAHTSTPL 0.593 *T* Sequence 338 EFCLYLVDI 0.017 . Sequence 22 AHTSTPLFP 0.060 . Sequence 368 QIELYEHSI 0.100 . Sequence 27 PLFPTAAHA 0.027 . Sequence 394 SMEHYAEGM 0.964 *Y* Sequence 41 ASSSTPFTL 0.159 . Sequence 442 LREAYLSSR 0.287 . Sequence 44 STPFTLQPH 0.041 . Sequence 512 ILPKYDCMQ 0.513 *Y* Sequence 65 GAEATALLI 0.022 . Sequence 533 VVKSYFEGN 0.026 . Sequence 76 AAPSTIVGR 0.375 . Sequence 554 GLPVYFIEP 0.046 . Sequence 89 MSRRTSRRN 0.960 *T* Sequence 570 WRAQYYGEH 0.012 . Sequence 96 RNLRTGVHP 0.035 . Sequence 571 RAQYYGEHD 0.409 . Sequence 154 MIQNTQKNI 0.269 . Sequence 582 KRFSYFSRV 0.045 . Sequence 181 KEKETLQQK 0.067 . Sequence 592 LELLYQSGK 0.494 . Sequence 196 KLSETGKQS 0.274 . Sequence 615 VAPLYWDVY 0.886 *Y* Sequence 240 LIEITETEE 0.376 . Sequence 619 YWDVYANLG 0.973 *Y* Sequence 242 EITETEESL 0.204 . Sequence 638 HNFEYQGIA 0.701 *Y* Sequence 266 ELECTSTSA 0.177 . Sequence 649 QDLAYCGLD 0.208 . Sequence 268 ECTSTSAQS 0.127 . Sequence 680 GAVVYSNIV 0.136 . Sequence 299 LLDSTANQV 0.017 . Sequence 691 VSPTYAQEV 0.467 . Sequence 309 HASLTLDGY 0.440 . Sequence 738 LKVQYSAND 0.261 . Sequence 329 ASLGTTNVS 0.061 . Sequence 744 ANDLYGKSA 0.941 *Y* Sequence 330 SLGTTNVSE 0.134 . Sequence 789 RHAIYKITE 0.229 . Sequence 378 EFHGTLSKL 0.481 . Sequence 832 LLLKYDDAL 0.081 . Sequence 465 ALPGTSSGL 0.103 . Sequence 866 VAMRYGSVP 0.123 . Sequence 545 NKIWTGTVE 0.134 . Sequence 918 RAFNYYHRK 0.028 . Sequence 547 IWTGTVEGL 0.564 *T* Sequence 919 AFNYYHRKP 0.320 . Sequence 608 HDWQTAFVA 0.583 *T* Sequence 947 SVSQYEEIY 0.904 *Y* Sequence 632 RICFTCHNF 0.035 . Sequence 951 YEEIYQKTA 0.983 *Y* Sequence 685 SNIVTTVSP 0.101 . ______^______Sequence 686 NIVTTVSPT 0.803 *T* Sequence 690 TVSPTYAQE 0.013 . Sequence 706 GLQDTLKVH 0.697 *T* Sequence 723 NGIDTDTWN 0.228 . Sequence 725 IDTDTWNPS 0.239 .

Sequence 730 WNPSTDRFL 0.182 .

Sequence 762 KLASTQASQ 0.027 . Sequence 773 VGCITRLVP 0.032 . Sequence 792 IYKITELGG 0.021 . Sequence 859 PCGLTQMVA 0.406 . Sequence 875 VVRRTGGLN 0.109 . Sequence 889 LDDETIPME 0.037 . Sequence 899 RNGFTFLKA 0.025 . Sequence 942 FSWDTSVSQ 0.423 . Sequence 954 IYQKTATRA 0.148 . Sequence 956 QKTATRARA 0.066 . ______^______

224

Appendix 07:

SBEIIb Co-IP SBEIIb Co-IP SBEIIb Co-IP Probed with anti-SBEIIb Probed with anti-SP Probed with anti-SSIV

L 1 2 3 4 5 6 L 1 2 3 4 5 6 kDa L 1 2 3 4 5 6 kDa kDa 150 150 150 100 100 100 75 75 75 50 50 50

L. Protein marker 1. SBEIIb Co-IP in protein A-Sepharose beads 2. SBEIIb Co-IP Pre Immune in protein A-Sepharose beads 3. SBEIIb Co-IP-supernatant 4. SBEIIb Co-IP- Pre Immune supernatant 5. Protein A-Sepharose beads + amyloplast lysates 6. Amyloplast lysates

Co-immunoprecipitation of stromal proteins from wild-type maize amyloplasts using peptide specific anti-SBEIIb antibodies to investigate the protein-protein interactions between SBEIIb, SSIV and SP. 1 ml amyloplast lysates (1 mg/mL) prepared from wild-type maize endosperm at 22 DAA were incubated with peptide-specific anti-SSIV antibodies (15 mg/mL final concentration) at room temperature for 1 hr, and then immunoprecipitated with Protein-A-Sepharose beads. The washed Protein-A-Sepharose-antibody-antigen complexes were boiled in 200 μL 1XSDS-loading buffer and 30 μL loaded onto 10% SDS gels. Immunoblotted membranes were developed with anti-maize SBEIIb, SSIV and SP antisera.

225

Appendix 08:

All the data were analysed using Statistix 09 statistical program.

ONE-WAY ANOVA of the synthetic activity of SP of amyloplast lysates in different glucans One-Way AOV for: V001 V002 V003 V004 V005 V006 V007 V008 V009

Source DF SS MS F P Between 8 39542.8 4942.86 82.74 0.028 Within 18 1075.3 59.74 Total 26 40618.2

Grand Mean 76.055 CV 10.16

Homogeneity of Variances F P Levene's Test 1.28 0.3152 O'Brien's Test 0.57 0.7911 Brown and Forsythe Test 0.30 0.9570

Welch's Test for Mean Differences Source DF F P Between 8.0 93.27 0.0008 Within 7.4

Component of variance for between groups 1627.71 Effective cell size 3.0

Variable Mean V001 100.53 (Glycogen-Untreated) V002 114.45 (Glycogen-ATP-treated) V003 37.43 (Glycogen-APase-treated) V004 69.08 (Maltoheptaose-Untreated) V005 60.01 (Maltoheptaose-ATP-treated) V006 27.35 (Maltoheptaose-APase-treated) V007 99.43 (Amylopectin-Untreated) V008 143.34 (Amylopectin-ATP-treated) V009 32.88 (Amylopectin-APase-treated) Observations per Mean 3 Standard Error of a Mean 4.4624 Std Error (Diff of 2 Means) 6.3108

LSD All-Pairwise Comparisons Test

Variable Mean Homogeneous Groups V008 143.34 A V002 114.45 B V001 100.53 C V007 99.427 C V004 69.077 D V005 60.013 D V003 37.430 E V009 32.877 E V006 27.353 E

Alpha 0.05 Standard Error for Comparison 6.3108 226

Critical T Value 2.101 Critical Value for Comparison 13.259 There are 5 groups (A, B, etc.) in which the means are not significantly different from one another.

Statistix ONE-WAY ANOVA of the phosphorolytic activity of SP of amyloplast lysates in different glucans

One-Way AOV for: V001 V002 V003 V004 V005 V006

Source DF SS MS F P Between 5 3672.08 734.416 35.57 0.0004 Within 12 247.76 20.647 Total 17 3919.84

Grand Mean 34.566 CV 13.15

Homogeneity of Variances F P Levene's Test 1.84 0.1790 O'Brien's Test 0.82 0.5593 Brown and Forsythe Test 0.40 0.8397

Welch's Test for Mean Differences Source DF F P Between 5.0 26.05 0.0008 Within 5.5

Component of variance for between groups 237.923 Effective cell size 3.0

Variable Mean V001 24.493 (Maltoheptaose-Untreated) V002 27.040 (Maltoheptaose-ATP-treated) V003 16.640 (Maltoheptaose-APase-treated) V004 46.913 (Amylopectin-Untreated) V005 58.873 (Amylopectin-ATP-treated) V006 33.433 (Amylopectin-APase-treated) Observations per Mean 3 Standard Error of a Mean 2.6234 Std Error (Diff of 2 Means) 3.7101

LSD All-Pairwise Comparisons Test

Variable Mean Homogeneous Groups V005 58.873 A V004 46.913 B V006 33.433 C V002 27.040 CD V001 24.493 DE V003 16.640 E

Alpha 0.05 Standard Error for Comparison 3.7101 Critical T Value 2.179 Critical Value for Comparison 8.0836 There are 5 groups (A, B, etc.) in which the means are not significantly different from one another.

227

Statistix ONE-WAY ANOVA of the synthetic and phosphorolytic activity of recomb tetrameric form of SP in different glucans

One-Way AOV for: V001 V002 V003 V004 V005 V006

Source DF SS MS F P Between 5 5359282 1071856 247.66 0.0000 Within 12 51935 4328 Total 17 5411217

Grand Mean 766.23 CV 8.59

Homogeneity of Variances F P Levene's Test 3.39 0.0385 O'Brien's Test 1.51 0.2587 Brown and Forsythe Test 1.37 0.3014

Welch's Test for Mean Differences Source DF F P Between 5.0 793.69 0.0011 Within 4.7

Component of variance for between groups 355843 Effective cell size 3.0

Variable Mean V001 665.11 (Glycogen-Tetrameric form) V002 762.1 (Glycogen-Dimeric form) V003 928.9 (Amylopectin-Tetrameric form) V004 1796.7 (Amylopectin-Dimeric form) V005 3.0 (Maltoheptaose-Tetrameric form) V006 441.7 (Maltoheptaose-Dimeric form) Observations per Mean 3 Standard Error of a Mean 37.982 Std Error (Diff of 2 Means) 53.715 LSD All-Pairwise Comparisons Test

Variable Mean Homogeneous Groups V004 1796.7 A V003 928.90 B V002 762.08 C V001 665.13 C V006 441.68 D V005 2.9500 E

Alpha 0.05 Standard Error for Comparison 53.715 Critical T Value 2.179 Critical Value for Comparison 117.03 There are 5 groups (A, B, etc.) in which the means are not significantly different from one another.

228

Appendix 09:

1. Chemical composition of the phosphotase inhibitor cocktail (PI, G-

Biosciences, trade name Phosphatase ArrestTM, Catalog number 788-

450).

Phosphotase inhibitor cocktail (G-Bioscience) has five phosphatase inhibitors, target serine/threonine specific and tyrosine specific and dual specificity phosphatises. The solution is 100x strength containing NaF, Na orthovanadate, Na pyrophosphate, beta glycerophosphate and Na molybdate.

10uL per mL of the amyloplast sample (with total protein conc.>1mg/mL) was used in the experiments.

2. Chemical composition of the protease inhibitor cocktail (PI, G-

Biosciences, trade name Photease ArrestTM, Catalog number 786-322).

ProteCEASE™ is a superior general protease inhibitor cocktail that is suitable for purification from mammalian, plant, bacteria and yeast samples. The cocktail contains both irreversible and reversible protease inhibitors to inhibit serine, cysteine and other proteases. EDTA is an optional component for inhibiting metalloproteases. ProteCEASE™ has been specifically developed for large scale preparative applications.

229

Appendix 10:

Representative graph illustrating the elution profile of amyloplast lysates ran on Superdex 200 10/300GL gel permeation column. (Protein Content= 1.0 mg/mL, Loaded Volume 0.5 mL)

AP 22DAYS RENUKA001:10_UV AP 22DAYS RENUKA001:10_Fractions AP 22DAYS RENUKA001:10_Logbook

mAU

10.0

8.0

6.0

4.0

2.0

0.0

-2.0 A10 A11 A12 A13 A14 A15 B1 B2 B3 B4 B5 B6 B7 B8 B9 B10 B11 6.0 7.0 8.0 9.0 10.0 11.0 12.0 ml

Fraction Number

230

Representative graph illustrating the elution profile of recombinant SP ran on Superdex 200 10/300GL gel permeation column.

(Protein Content= 6.75 mg/mL, Loaded Volume 0.5 mL)

sp rrecomb16may2012:10_UV sp rrecomb16may2012:10_Fractions sp rrecomb16may2012:10_Logbook mAU

140

120

100

0 A1 A3 A5 A7 A9 A11 A13 A15 B2 B4 B6 B8 B10 B12 B14 C1 C3 C5 C7C8 0.0 5.0 10.0 15.0 ml Fraction Number

231