REGULATION of MAIZE (Zea Mays L.) STARCH SYNTHASE Iia by PROTEIN PHOSPHORYLATION

Regulation of Maize (Zea mays L.) Starch Synthase IIa by

Protein Phosphorylation

Usha P. Rayirath

A Thesis

presented to The University of Guelph

In partial fulfillment of requirements

for the degree of Doctor of Philosophy in Molecular and Cellular Biology

Guelph, Ontario, Canada

REGULATION OF MAIZE (Zea mays L.) STARCH SYNTHASE IIa BY PROTEIN PHOSPHORYLATION

Usha P. Rayirath Advisor: Dr. Michael J. Emes University of Guelph, 2014

Starch is a significant carbohydrate reserve in plants and has enormous use in both food and non-food industries. Biosynthesis of storage starch in maize (Zea mays L.) occurs in the amyloplasts of the developing endosperm, through the coordinated actions of several enzymes, including ADP-glucose pyrophosphorylase (AGPase), starch synthases (SS), starch branching enzymes (SBE) and debranching enzymes (DBE).

Starch synthase IIa (SSIIa) catalyzes the synthesis of intermediate glucan chains (DP 12-

25) and plays a significant role in starch biosynthesis. Previous studies indicate that in cereal endosperm, protein phosphorylation plays a major role in regulating the formation of functional multi-enzyme complexes between SSs and SBEs during starch biosynthesis and that SSIIa forms the core of such a functional protein complex, with SSI and SBEIIb.

The present study investigated the specific role of protein phosphorylation on the regulation of SSIIa, in the amyloplasts of developing maize endosperm, during starch biosynthesis. In vitro phosphorylation of stromal SSIIa in maize amyloplasts was detected by phosphate affinity Mn2+ Phos-tagTM gel electrophoresis, Pro-Q® Diamond phospho-protein gel staining, and by autoradiography following radio labelling with γ-

[32-P] ATP. The results indicated that granule bound SSIIa exists in the phosphorylated state. In vitro phosphorylation of recombinant maize SSIIa and immunopurified SSIIa

ii occurred only in the presence of amyloplast lysates and could be inhibited by protein kinase inhibitors, suggesting the presence of one or more protein kinase(s) in amyloplasts. ATP caused marked shifts in the electrophoretic mobility of SSIIa in non- denaturing polyacrylamide gels, and also in phosphate affinity (Phos-tag) gels, further suggesting the role of post-translational protein phosphorylation in regulating maize

SSIIa. Protein phosphorylation significantly enhanced (12-fold), and dephosphorylation substantially reduced the catalytic activity (Vmax) of maize SSIIa, whereas its dissociation constant (Kd) and affinity for amylopectin was not affected. Depending on the phosphorylation status, stromal maize SSIIa existed in two distinct protein complexes, a

LMW (260 kDa) protein complex was formed with SSI and SBEIIb under conditions favouring phosphorylation; whereas under conditions favouring dephosphorylation, this

260 kDa complex of SSIIa-SSI-SBEIIb possibly associated with other starch synthesizing enzymes and/or itself to form HMW complexes of 670 kDa or more.

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Acknowledgements

I humbly bow my head before the God Almighty, who blessed me with the will power and courage to complete this endeavor. I submit this small venture before God, for

His grace in providing me with the health and strength throughout the study.

Firstly, I express my profound gratitude to my supervisors Dr. Michael J. Emes, and Dr. Ian J. Tetlow, for their excellent guidance, valuable suggestions, constructive criticism, constant encouragement and above all, for their patience, understanding and whole-hearted co-operation rendered throughout the course of my PhD program.

I wish to extend my sincere gratitude to my advisory committee members, Dr.

Annette Nassuth, and Dr. Bernard Grodzinski, for their time, help and constant support rendered during my study. I also extend my sincere thanks to the members of the examination committee, Dr. Ravi Chibbar (University of Saskatchewan) (External), Dr.

Andrew Bendall (Chair), Dr. Annette Nassuth and Dr. Nina Jones.

The funding support from NSERC Discovery Grant to Dr. M. Emes is greatly acknowledged. I also deeply acknowledge the Alexander Graham Bell Canada Graduate

Scholarship (Doctoral) from NSERC, the Ontario Graduate Scholarship (declined) from

Govt. of Ontario, and Deans’ Tri-council Scholarship from the University of Guelph, provided to me to conduct my PhD studies.

My sincere thanks go to the past and present members of Emes/Tetlow lab, at the

Department of Molecular and Cellular Biology, University of Guelph for their help, support and constant encouragement. I am especially grateful to Dr. Amina

Makhmoudova, for her timely and constant help and advice, whenever I needed. Special thanks to Drs. Nadya Romanova, Wendy Allan and Fushan Liu, for their help and support. I am very much grateful to my seniors, Dr. Renuka Subasinghe and Dr. Zaheer

Ahmed for their enormous help and support given to me, whenever needed. I am equally thankful to my other lab mates, Lily Navanosky (especially for her immense help with recombinant protein work), Jenelle Patterson (especially for her help with radioactive experiments, and during thesis preparation), Qianru Zhao (Ruby), Sarah Massey and You

Wang.

My special thanks to all the staff members of the Department of Molecular and

Cellular Biology, New Science Complex, for their help and encouragement during the entire course of study. I express my sincere thanks to all the fellow graduate students in the department for sharing experiences, and developing valuable friendships.

I am in dearth of words to express my gratitude and indebtedness to my loving husband for his constant support, understanding and love in all my endeavors. Words can’t express my boundless love to my son, for his understanding, sacrifice and support given to me during this endeavor. Last, but not least, I owe my profound gratitude to my father, mother, brother and in-laws, relatives and friends for their boundless affection, constant prayers, moral support, and unfailing inspiration, throughout my career.

Affectionately Dedicated to my Loving Family

To my loving husband, my son and my loving parents and in laws and all who were understanding and supportive helping me to accomplish this…

Table of Contents

Abstract ii

Acknowledgements iv

Dedication v

Table of Contents vii

List of Figures xiv

List of Tables xviii

List of Abbreviations xix

CHAPTER 1: GENERAL INTRODUCTION 2

1. Introduction 2

1.1 Starch 2

1.2 Molecular structure and composition of Starch 3

1.2.1 Starch granule 3

1.2.2 Starch composition- Amylose and Amylopectin 5

1.3 Biosynthesis of starch 8

1.3.1 Starch biosynthetic enzymes 9

1.3.1.1 ADP-glucose pyrophosphorylase (AGPase EC 2.7.7.27) 9

1.3.1.2 Starch synthases (SSs, EC 2.4.1.21) 12

1.3.1.2.1 Granule bound starch synthases (GBSS) 16

1.3.1.2.2 Starch synthase I (SSI) 17

1.3.1.2.3 Starch synthase II (SSII) 19

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1.3.1.2.4 Starch synthase III (SSIII) 21

1.3.1.2.5 Starch synthase IV (SSIV) 23

1.3.1.3 Starch Branching Enzymes (SBEs, EC 2.4.2.18) 24

1.3.1.3.1.Starch Branching Enzyme I (SBEI) 25

1.3.1.3.2 Starch Branching Enzyme II (SBEII) 26

1.3.1.4 Starch Debranching Enzymes (DBEs, EC 3.2.1.41 and EC 3.2.1.68) 27

1.3.1.5 Starch Phosphorylase (SP,EC 2.4.1.1) 29

1.3.1.6 Disproportionating enzyme (D-enzyme, EC 2.4.1.25) 31

1.4 Regulation of starch biosynthetic enzymes 32

1.4.1 Regulation at the transcriptional level 32

1.4.2 Allosteric regulation and redox modulation 34

1.4.3 Protein phosphorylation 35

1.4.3.1 Protein phosphorylation – a universal post-translational regulatory 35 mechanism

1.4.3.2 Protein phosphorylation and plant metabolism 39

1.4.3.3 Carbohydrate metabolism is regulated by protein phosphorylation – 41 from bacterial glycogen to plant

1.4.4 Phosphorylation dependent protein-protein interactions in starch 47 biosynthesis

1.4.5 Regulation of starch turnover by protein phosphorylation 52

1.5 Importance of starch synthase IIa (SSIIa) in starch biosynthesis 55

1.6 Hypothesis and Objectives of the study 58

CHAPTER 2: INVESTIGATION OF PROTEIN PHOSPHORYLATION 60 OF STARCH SYNTHASE IIa IN MAIZE ENDOPSERM

2.1 Introduction 60

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2.2 Materials and Methods 67

2.2.1 Materials 67

2.2.1.1 Plant materials 67

2.2.1.2 Chemicals 67

2.2.2 Methods 67

2.2.2.1 Isolation of amyloplasts from maize endosperms 67

2.2.2.2 Preparation of maize whole cell extracts 68

2.2.2.3 Isolation of starch granule bound proteins 69

2.2.3 Proteomic analysis 70

2.2.3.1 Quantification of proteins 70

2.2.3.2 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS- 70 PAGE)

2.2.3.3. Coomassie blue staining 71

2.2.4 Immunological techniques 72

2.2.4.1 Preparation of peptides and antisera 72

2.2.4.2 Purification of polyclonal maize antibodies 72

2.2.4.3 Immunoblot analysis 73

2.2.4.4 Immunopurification of SSIIa from maize amyloplast lysates 74

2.2.5 Detection of phosphorylation of SSIIa in maize endosperm 75

2.2.5.1 Phosphorylation and dephosphorylation of SSIIa in maize amyloplast 75 stroma

2.2.5.2 Phos-tag TM phosphate affinity acrylamide gel electrophoresis 76

2.2.5.3 Pro-Q diamond phospho-protein gel staining 79

2.2.5.4 In vitro phosphorylation of maize amyloplasts using γ- [32-P] ATP 80

2.2.5.5 Autoradiography 80

2.2.5.6 Expression and purification of recombinant maize SSIIa in Escherichia 81 coli

2.2.5.7 Immobilization of recombinant maize SSIIa on S-protein agarose beads 83 and pull down assay

2.2.5.8 In vitro phosphorylation of S-tag immobilized recombinant SSIIa using 83 γ- [32-P] ATP and autoradiography

2.3 Results 85

2.3.1 Detection of starch synthase IIa in the amyloplast stroma and in starch 85 granules

2.3.2 Optimization of Phos-tag TM phosphate affinity acrylamide gel 86 electrophoresis

2.3.3 Detection of in vitro phosphorylation of SSIIa in the amyloplast stroma 89 using Phos-tag TM phospho-protein affinity acrylamide gel electrophoresis

2.3.4 Comparison of the phosphorylation status of maize SSIIa in amyloplast 90 stroma and starch granule

2.3.5 Detection of phosphorylation of maize SSIIa using Pro-Q diamond gel 92 staining

2.3.6 Detection of phosphorylation of maize SSIIa by γ- [32-P] ATP labeling 93 and autoradiography

2.3.7 The effect of protein kinase inhibitors on phosphorylation of SSIIa 94

2.3.8 Phosphorylation of recombinant maize SSIIa 96

2.3.8.1 Expression of recombinant SSIIa in E coli cell lysates 96

2.3.8.2 Immobilization of recombinant maize SSIIa on S-protein Agarose beads 98

2.3.8.3 In vitro phosphorylation of recombinant maize SSIIa using γ- [32-P] 100 ATP labeling and autoradiography

2.4 Discussion 102

CHAPTER 3: THE EFFECT OF PROTEIN PHOSPHORYLATION ON REGULATION OF MAIZE SSIIA 114

3.1 Introduction 114

3.2 Materials and Methods 119

3.2.1 Zymogram analysis of SS activity 119

3.2.2 Effect of ATP concentration and duration of incubation on the mobility of 121 maize SSIIa during non-denaturing PAGE

3.2.3 Substrate affinity electrophoresis 122

3.2.4 Preparation of radio-labelled ADP-[U-14C] glucose as substrate for starch 123 synthase

3.2.4.1 Expression and purification of E coli recombinant AGPase (adenosine 123 5′-diphosphate glucose pyrophosphorylase)

3.2.4.2 Synthesis of ADP- [U14C] Glucose using recombinant AGPase 124

3.2.5 Determining the effect of protein phosphorylation on the catalytic activity 125 of SSIIa in maize amyloplast stroma

3.3 Results 127

3.3.1 Effect of ATP and APase on the activity of native SSIIa as visualized in 127 the SS activity zymogram

3.3.2 Effect of ATP concentration on the mobility of native maize SSIIa 131

3.3.3 Effect of substrate and ATP on the electrophoretic mobility of maize SSIIa 133 in non-denaturing polyacrylamide gels

3.3.4 Specificity of nucleotide phosphate-induced mobility shift of stromal 134 maize SSIIa in non-denaturing gels

3.3.5 Effect of protein kinase inhibitors on ATP-induced polyacrylamide gel 137 mobility shift of maize SSIIa

3.3.6 Effect of phosphatase inhibitors on the mobility of maize SSIIa in non- 139 denaturing gels

3.3.7 Glucan binding properties of maize SSIIa under conditions of 140

xi phosphorylation and dephosphorylation

3.3.8 Preparation of radio-labelled ADP-[U-14C] glucose 144

3.3.8.1 Production and purification of recombinant AGPase in E coli 144

3.3.8.2 Synthesis of ADP- [U14C] Glucose from recombinant AGPase 147

3.3.9 Activity of immunopurified SSIIa from maize amyloplast stroma under 149 conditions of phosphorylation and dephosphorylation

3.3.10 Kinetics of immunopurified SSIIa under conditions of phosphorylation 152 and dephosphorylation

3.4 Discussion 158

CHAPTER 4: THE EFFECT OF PROTEIN PHOSPHORYLATION ON THE INTERACTIONS OF MAIZE SSIIa WITH OTHER STARCH 170 BIOSYNTHETIC ENZYMES

4.1 Introduction 170

4.2. Materials and Methods 178

4.2.1 Immunoprecipitation of SSIIa from maize amyloplast lysates 178

4.2.2 Chemical cross-linking to detect protein – protein interactions of SSIIa 179

4.2.3 Gel permeation chromatography (GPC) 179

4.2.4 Phospho-protein affinity (Phos-tagTM) acrylamide gel electrophoresis 180

4.2.5 Substrate affinity electrophoresis of GPC fractions 180

4.2.6 Co-immunoprecipitation of SSIIa in the HMW and LMW GPC fractions 181 with other major starch biosynthetic enzymes

4.3 Results

4.3.1 Determining the effects of protein -phosphorylation on protein–protein 181 interactions of SSIIa

4.3.2 Phosphorylation dependence of protein-protein interactions between SSIIa 183 and other starch biosynthetic enzymes

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4.3.3. Analysis of the phosphorylation -dependent interactions of SSIIa with 186 other starch biosynthetic enzymes by Gel Permeation Chromatography (GPC)

4.3.4 Phosphorylation status of SSIIa in the GPC fractions 189

4.3.5 Glucan binding properties of HMW and LMW fractions containing SSIIa 190

4.3.6 Relative electrophoretic mobility of SSI, SBEIIb and SSIIa in the HMW 194 and LMW fractions following non-denaturing PAGE

4.3.7 Protein –protein interactions of SSIIa following GPC of amyloplast lysates 198

4.3.8 Detection of protein –protein interactions of SSIIa with other major starch 202 biosynthetic enzymes by chemical cross-linking

4.4 Discussion 204

CHAPTER 5: GENERAL DISCUSSION 216

5.1 Post-translational modification of maize SSIIa by protein phosphorylation 216

5.2 Regulation of the catalytic functions of maize SSIIa by protein 219 phosphorylation

5.3 Interactions of SSIIa with other starch biosynthetic enzymes is regulated by 223 protein phosphorylation

5.4 Future directions of research 227

REFERENCES 230

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List of Figures

CHAPTER I

Figure 1.1 Diagrammatic representation of a starch granule showing the crystalline and 5 amorphous lamellae

Figure 1.2 Components of starch- amylose and amylopectin 7

Figure 1.3 Outline of the starch biosynthetic pathway showing the major groups of 8 enzymes involved

Figure 1.4 Comparison of amino-acid domains in different SS isoforms 16

Figure 1.5 Glycogen metabolism- steps involved in the synthesis and degradation of 45 glycogen

Figure 1.6 Schematic diagram showing the bi-cyclic cascades involving major enzymatic modifications in the control of glycogen metabolism in mammalian muscle 46 tissue

CHAPTER II

Figure 2.1: Proposed model (Liu et al., 2012b) demonstrating the critical role of SSIIa in the granule deposition of the major amylopectin synthesizing enzymes SSI, SSIIa 65 and SBEIIb in maize amyloplasts

Figure 2.2 Sequence structure of Novagen pET29b expression vector containing a 15 82 amino acid S-tag on the N-terminus with a thrombin digestion site, and a T7 promoter

Figure 2.3: The presence of SSIIa in maize seed whole cell extract, amyloplast stroma 85 and in the starch granule

Figure 2.4: Optimization of Phos-tag TM phospho protein affinity gel electrophoresis 87

Figure 2.5: Mobility of SSIIa in Phos-tag acrylamide gel electrophoresis 88

Figure 2.6: Detection of in vitro phosphorylation of SSIIa in amyloplast stroma 90

Figure 2.7: Phosphorylation state of SSIIa in amyloplast stroma and starch granules 91

Figure 2.8: In vitro phosphorylation of immunopurified SSIIa from wild type maize 93

xiv amyloplast stroma detected using Pro-Q Diamond phospho-protein gel staining

Figure 2.9: Auto radiography of immunopurified SSIIa from wild type maize 94 amyloplast stroma following radiolabelling with γ- [32-P] ATP

Figure 2.10: Phosphorylation state of stromal SSIIa in maize amyloplast lysates is 95 inhibited by protein kinase inhibitors

Figure 2.11: Expression of recombinant maize SSIIa in E coli 97

Figure 2.12: Immobilization of recombinant maize SSIIa to S- protein agarose beads 99

Figure 2.13: In vitro phosphorylation of recombinant maize SSIIa 101

Figure 2.14: Principle and application of Phos-tag TM acrylamide gel electrophoresis 104

Figure 2.15: Identification of potential phosphorylation sites within maize SSIIa 111 protein

CHAPTER III

Figure 3.1 Effects of dephosphorylation on SBE activity from amyloplast stroma and 118 chloroplasts of wheat (taken from Tetlow et al., 2004a)

Figure 3.2: Effect of ATP on SS activity and mobility of maize SSIIa in non- 129 denaturing gels in the presence of different glucan primers

Figure 3.3: Effect of ATP on SS activity and mobility of maize SSIIa and SSI in non- 130 denaturing PAGE

Figure 3.4: Effect of ATP concentration on the mobility of native, maize SSIIa 131 following non-denaturing PAGE

Figure 3.5: Time-dependence of ATP-induced, mobility-shift of maize SSIIa 132

Figure 3.6: Effect of glucan substrate on electrophoretic mobility of phosphorylated 134 and dephosphorylated forms of maize SSIIa

Figure 3.7: Effect of mono-, di- and tri-nucleotide phosphates on the mobility of maize 136 SSIIa in non-denaturing PAGE

Figure 3.8: Effect of protein kinase inhibitors and ATP concentration on the 138 electrophoretic mobility of maize SSIIa

Figure 3.9: Effect of protein kinase inhibitors on the ATP- induced, mobility shift of 138 maize SSIIa

Figure 3.10: Effect of phosphatase inhibitors on the electrophoretic mobility of SSIIa 140 from maize amyloplast lysates

Figure 3.11: Determination of dissociation constant of maize SSIIa for amylopectin 142 and glycogen by affinity gel electrophoresis

Figure 3.12: Effect of phosphorylation on the dissociation constant (K ) of maize d 143 SSIIa for (A) amylopectin and (B) glycogen

Figure 3.13: Expression and purification of recombinant AGPase from E coli 146

Figure 3.14: Synthesis of ADP-[U-14C] glucose from [U-14C] glucose -1-phosphate 148

Figure 3.15: Immunopurification of maize SSIIa from amyloplast stroma 149

Figure 3.16: Effect of protein phosphorylation on the catalytic activity of 151 immunopurified maize stromal SSIIa

Figure 3.17: Michaelis-Menten kinetics of immunopurified maize SSIIa for (A) amylopectin and (B) ADP-glucose under conditions of phosphorylation and 154 dephosphorylation

Figure 3.18: Effect of protein phosphorylation on the K and V of SSIIa for m max 156 amylopectin

Figure 3.19: Effect of protein phosphorylation on the K and V of SSIIa for ADP- m max 157 glucose

CHAPTER IV

Figure 4.1: Protein–protein interactions between the major starch biosynthetic 175 enzymes during starch granule formation in cereal endosperms

Figure 4.2: Immunoprecipitation of stromal SSIIa from wild type maize amyloplasts 183 using peptide specific anti- SSIIa antibodies

Figure 4.3: Co-immunoprecipitation of SSIIa with other starch biosynthetic enzymes 185

Figure 4.4: Calibration of GPC for the determination of molecular weight 186

Figure 4.5: GPC separation of amyloplast stromal proteins under conditions of 188 phosphorylation and dephosphorylation

Figure 4.6: Phosphorylation state of SSIIa in the HMW and LMW fractions obtained 190 by GPC separation of maize amyloplast lysates pretreated with ATP and APase

xvi

Figure 4.7: Substrate affinity electrophoresis of HMW (670 kDa) and LMW (260 kDa) 192 forms of maize SSIIa

Figure 4.8: Dissociation constants of HMW (670 kDa) and LMW (260 kDa) forms of 193 SSIIa

Figure 4.9: Co-migration of SSI and SBEIIb relative to SSIIa from HMW (670 kDa) 197 and LMW (260 kDa) GPC fractions on non-denaturing gels

Figure 4.10: Protein-protein interactions of SSIIa from the designated HMW (670 200 kDa) and LMW (260 kDa) GPC fractions with other starch biosynthetic enzymes

Figure 4.11: Cross-linking of stromal proteins in wild type maize amyloplasts under 203 conditions of phosphorylation and dephosphorylation

Figure 4.12: Proposed interactions of SSIIa with other starch biosynthetic enzymes 213

xvii

List of Tables

CHAPTER 2

Table 2.1: Composition of the stacking and resolving gels used for SDS-PAGE 71

Table 2.2: The synthetic peptide sequences derived from the amino acid 72 sequences of various starch biosynthetic enzyme isoforms in maize

Table 2.3: The composition of Phos-Tag TM phosphate affinity acrylamide gel 78

CHAPTER 3

Table 3.1 Kinetics of recombinant maize SSIIa expressed in Escherichia coli 116

Table 3.2: Composition of non –denaturing starch synthase zymogram gels 121 containing different glucan substrates

Table 3.3 Dissociation constants (K ) of maize stromal SSIIa for corn d 144 amylopectin under phosphorylating and dephosphorylating conditions

Table 3.4 Kinetic constants of immunopurified maize SSIIa under conditions 155 favouring protein phosphorylation or dephosphorylation

CHAPTER 4

Table 4.1 The dissociation (Kd) constants of HMW and LMW forms of SSIIa for corn amylopectin following GPC separation of wild type maize amyloplast 194 lysate

xviii

List of Abbreviations ae amylose extender ADP adenosine diphosphate AGPase ADP-glucose pyrophosphorylase AGP-L AGPase large subunit AGP-S AGPase small subunit AMP adenosine monophosphate AP amyloplasts APase alkaline phosphatase ATP adenosine triphosphate BCIP/NBT bromo-4-chloro-3-indonyl phosphate/nitro blue tetrazolium BS3 Bis-(sulfosuccinimidyl) suberate BSA bovine serum albumin Bt Brittle CAM Crassulacean acid metabolism CBM carbohydrate binding module cDNA complementary DNA CE crude extract CRC colorectal cancer CSK chloroplast sensor kinase CTP cytidine triphosphate DAA days after anthesis DBE debranching enzyme D-enzyme disproportionating enzyme DMSO dimethylsulphoxide DP degree of polymerization DPA days post anthesis DTT Dithiothreitol

xix

DU dull E.coli Escherichia coli e.g. example EC enzyme commission EDTA ethylenediaminetetraacetic acid Fd Ferredoxin FSBA 5'-fluorosulfonylbenzoyl-5'-adenosine FTR ferredoxin: thioredoxin reductase G-1-P glucose-1-phosphate G-6-P glucose-6-phosphate GBSS granule bound starch synthase GDP guanosine diphosphate GMP guanosine monophosphate GP glycogen phosphorylase GPC gel filtration chromatography GS glycogen synthase GSK glycogen synthase kinase GT glucosyl transferase GTP guanosine triphosphate GWD glucan water dikinase HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid IPTG isopropyl-3-D-thiogalactopyranoside ISO/ISA isoamylase K252a staurosporine

Kd dissociation constant kDa kilo Dalton LB Luria Bertani LHC light harvesting complex MAP mitogen activated protein

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 NR nitrate reductase OCL outer chain length OD optimal density PAGE polyacrylamide gel electrophoresis PBS phosphate buffered saline PCR polymerase chain reaction PEPC poly enol pyruvate carboxylase 3-PGA 3- phosphoglycerate Pho1 plastidial starch phosphorylase Pho2 cytosolic starch phosphorylase PI phosphatase inhibitor (cocktail) Pi inorganic phosphate PK protein kinase PP protein phosphatase PPi inorganic pyrophosphate PS Photosystem psi pressure per square inch PWD phospho glucan water dikinase RB rupturing buffer RCF relative centrifugal force

Rm relative migration Rpm rotations per minute RS resistant starch

xxi

SBD starch binding domain SBE starch branching enzyme SDS sodium dodecyl sulfate SNF sucrose non-fermenting SP starch phosphorylase SPS sucrose phosphate synthase SS starch synthase su Sugary TAK thylakoid associated kinase TBS tris buffered saline TEMED tetramethylethylenediamine TLC thin layer chromatography Tris tris (hydroxymethyl) aminomethane Trx Thioredoxin UDP uridine diphosphate UTP uridine triphosphate v/v volume/volume viz., Namely w/v weight/volume Wx waxy mutant

xxii

CHAPTER 1

CHAPTER 1: GENERAL INTRODUCTION

1. Introduction

1.1 Starch

Starch is the most significant storage carbohydrate synthesized in plants, and is the most important dietary energy source for humans, forming up to 80% of the total calorific intake (Tetlow, 2011). In plants, starch is synthesized in specialized subcellular organelles called plastids enabling cells to store high energy in the form of carbohydrate.

Starches perform different functions in different tissue types. Thus, transient starches produced in leaf chloroplasts during the day (short term storage) are broken down and the expected degradation products (glucose/maltose) are used to form sucrose in the cytosol and utilized as the carbon source for providing energy for non-photosynthetic metabolism at night or translocated to sink tissues. Storage starches meant for long term storage in tuberous tissues (e.g. potato tuber) and in endosperms of developing seeds are synthesised in non-photosynthetic plastids called amyloplasts. They serve as long term energy reserve to be accessed as environmental challenges dictate and also during germination and growth (Tetlow, 2006; 2011).

Storage starches produced in the endosperms of cereals such as wheat, maize and rice make up to 90% of the total world starch market (Burrell, 2003). Among the cereal crops, maize (Zea mays L.) is predominant in contributing significantly to the world’s starch production, with its global share of starch accounting for about ˃80%

(International Starch Institute, 2004). In addition to starch being a major caloric component of the diet, there is increasing recognition that specific starches have human

2 health benefits. Resistant starch (RS) is a component of dietary starch that escapes digestion in the small intestine and reaches the lower bowel (colon) undigested and behaves similar to dietary fibre, helping to reduce obesity and colorectal cancer (CRC), a common tumour resulting in 8% of cancer deaths in Western countries (Keenan et al.,

2006; Le Leu et al., 2007; Clarke et al., 2008). Recent studies have shown that high amylose maize starch (that belongs to the second group of RS of the four groups, viz.,

RS2 consisting of native starch granules highly resistant to α- amylases) significantly reduces intestinal cancer in rats (Le Leu et al., 2007; Clarke et al., 2008). Being a cheap, natural and renewable raw material, starch is also exploited widely in industrial applications and as a source of energy for biofuels (Burrell, 2003; Shigechi et al., 2004;

Tetlow, 2006). Some of the physical and chemical properties of starch determining its suitability for various uses in agri-food and industrial sectors can be generated and or altered through chemical and physical manipulations or by enzyme modifications

(Wurzburg, 1986; Jobling, 2004; Xie and Liu, 2004; Tetlow, 2011). However, the knowledge and understanding of the basic biosynthetic pathway and the regulatory mechanisms involved in the synthesis of various functional starch forms is an essential prerequisite for rational manipulation of the quality and quantity of starch produced by crop plants.

1.2 Molecular structure and composition of starch

1.2.1 Starch granule

Starch exists as a water insoluble granule composed of two glucosyl polymers arranged in a hierarchical order within the granule viz.; amylose and amylopectin (see section 1.2.2). The unique semi-crystalline structure of amylopectin differs from

3 glycogen, its counterpart found in bacterial and animal systems. Glycogen exists as an open structure easily accessible to water due to its globular shape consisting of homogenously branched glucan polymers (Blennow et al., 2002; Roach, 2002; Kötting et al., 2009). The structure of the starch granule is mostly determined by the nature and packing of amylopectin molecules within (Myers et al., 2000) as well as the proportion of amylose to amylopectin. The polymodal glucan chain distribution of amylopectin allows the condensing of shorter glucan chains and subsequently generates the well packed left- handed double helices (6 to 7.5nm in length) which form the crystalline lamella (Fig 1.1).

The regular branching points coupling these parallel helices form the amorphous lamella

(3nm in length) of the granule (French, 1984; Hizukuri, 1986). This highly condensed and organized structure allows compaction of large amounts of glucose molecules

(Maddelein et al., 1994) and limits access of water, contributing to the insolubility of starch and thus making it a biologically beneficial storage material.

In cereals such as wheat, oats and barley, the packing of starch granules exhibit a bimodal distribution (round and lens shaped) with two types of granules; viz., A-type granules that are large size-lens shaped and B type granules which are small size- spherical. In maize (polyhedral), the granules are not bimodal and measure between 2-

30µm (Jobling, 2004).

Figure 1.1 Diagrammatic representation of a starch granule showing the crystalline and amorphous lamellae. (a). Amorphous and semi crystalline regions. (b) & (c). Semi crystalline growth rings within the granule, showing the alternating crystalline and amorphous lamellae. (Derived from Tetlow, 2006)

1.2.2 Starch composition- Amylose and Amylopectin

Starch granules consist of two glucan polymers called amylose and amylopectin. The proportion of these polymers within the granule is largely regulated genetically. Under normal conditions, 75% of a starch granule is composed of amylopectin and 25% is composed of amylose. Amylose consists of unbranched or less

5 branched chains (< 1%) of glucose residues (100-10,000 glucosyl units) connected by α

(1-4) glucosidic bonds (Ball et al., 1998). Amylopectin is a highly branched (4-6%) glucan polymer with distinctive α (1-6) linkages between glucose residues (105-106 glucosyl units) connected by α (1-4) glucosidic bonds (Detherage et al., 1955; Ball et al.,

1998) (Fig 1.2) with a specific spatial distribution (Gallant et al., 1997). In starch granules, the ratio between these two glucan polymers varies with the genotype depending on the environmental conditions (Denyer et al., 2001).

The enzymes involved in the starch biosynthetic pathway and their properties determine the number of glucosyl units in the linear α (1-4) chains and the relative position of α (1-6) branch linkage. Thus approximately, there is one branch point for every 20 glucose residues within the amylopectin molecule (Manners, 1989). Amylose is thought to occupy the amorphous, non-crystalline regions of the starch granule in a single helical form (Jane et al., 1992).

(a)

(b)

Figure 1.2 Components of starch- amylose and amylopectin (a) Amylose is a relatively linear, sparsely branched glucan polymer containing α (1-4) linkages (b) Amylopectin is a highly branched glucan polymer containing both α (1-4) and α (1-6) linkages (Taken from Ball et al., 1998). = reducing end

1.3 Biosynthesis of starch

Biosynthesis of starch is achieved through the concerted and coordinated action of several starch biosynthetic enzymes in the plastid. The various enzymes involved in this biosynthetic pathway catalyze specific reactions and exist in different biologically active isoforms in all starch synthesizing organelles (Martin and Smith, 1995; Myers et al.,

2000; Nakamura, 2002; James et al., 2003). The four major groups of starch biosynthetic enzymes include ADP-glucose pyrophosphorylase (AGPase), five isoforms of starch synthase (SS), three isoforms of starch branching enzyme (SBE) and two isoforms of debranching enzyme (DBE) (Fig. 1.3).

Figure 1.3 Outline of the starch biosynthetic pathway showing the major groups of enzymes involved.

1.3.1 Starch biosynthetic enzymes

1.3.1.1. ADP-glucose pyrophosphorylase (AGPase EC 2.7.7.27)

In higher plants, the soluble precursor and the substrate for the different classes of starch synthase is ADP-glucose. The enzyme AGPase, (Adenosine 5’-disphosphate glucose pyrophosphorylase) catalyses the first committed step of starch synthesis by generating the soluble substrate, ADP- glucose, from glucose-1-phosphate and ATP

(Ghosh and Preiss,1966; Preiss, 1988; Fu et al.,1998; Myers et al., 2000; and Tetlow,

2006). This reversible reaction of is shown below.

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

AGPase exists in all starch- synthesizing tissues in higher plant species (Copeland and Preiss, 1981; Sowokinos and Preiss, 1982; Morell et al. 1987; Lin et al. 1988; Okita et al. 1990) as a hetero-tetrameric protein, containing two large regulatory subunits

(AGP-L) and two small (AGP-S) catalytic subunits encoded by at least two different genes (Preiss and Sivak, 1996; Johnson et al., 2003). In wheat endosperm, the large subunits and the small subunits of AGPase are 58 and 55 kDa, respectively (Tetlow et al.,

2003) whereas the size of the large and small subunits of AGPase in spinach leaves and potato tubers range about 54-55kDa and 50-51kDa respectively (Sowokinos and Preiss,

1982; Morell et al. 1987; Okita et al. 1990). In maize, the shrunken2 (sh2) and brittle2

(bt2) genes encode for the large and small subunits of AGPase (Bae et al. 1990; Bhave et al. 1990). The N-terminus of the small subunit of AGPase in potato provides heat stability to the protein and regulates its catalytic activity (Ballicora et al. 1995). In contrast, in bacterial systems like Escherichia coli and Salmonella typhimurium AGPase

9 shows a homo-tetrameric structure having two subunits of 200kDa and 48kDa in size, respectively (Preiss,1988).

Based on the genetic and biochemical evidence, AGPases from monocots exist in two distinct forms, localized in the cytosol and in the plastid (Okita et al., 1990; Denyer et al. 1996; Tetlow et al. 2003; Tiessen et al. 2011). In cereal endosperm, AGPase is predominantly localized in the cytosol; for example 65-95% of the AGPase is found in the cytosolic form in wheat and maize respectively (Denyer et al. 1996; Tetlow et al.

2003; Geigenberger, 2011). In contrast to the above condition, in dicotyledons AGPase is exclusively present in the plastid, representing 98% of the total AGPase activity in the cell (Thorbjørnsen et al. 1996; Tiessen et al. 2011). However, in the case of plastidic

AGPase, the large and small subunits sizes are slightly smaller compared to those of the cytosolic form (Beckles et al. 2001; Tetlow et al. 2003). The predominance of cytosolic

AGPase in cereal endosperms necessitates having a specialized nucleotide sugar transporter for ADP-glucose to enter the amyloplast during storage starch biosynthesis. In maize, a specialized nucleotide sugar transporter, the ADP-glucose/ADP transporter, encoded by the brittle1 (Bt1) gene is located at the inner amyloplast envelope and appears to fulfill this necessity (Sullivan et al., 1991). Thus the ADP-glucose synthesised in the cytosol traverses the membrane into the plastid for starch biosynthesis (Cao et al., 1995;

Hannah, 2007) in exchange for ADP or AMP. In wheat endosperm, adenylates ADP and

AMP act as counter-exchange substrates and mediate ADP-glucose transport into the amyloplasts (Bowsher et al. 2007).

Most plant AGPases are subject to allosteric regulation by the metabolites involved in the plant carbon metabolism. AGPase is allosterically activated by 3-

10 phosphoglycerate (3-PGA), the first intermediate in the Calvin cycle, and inhibited by inorganic phosphate (Pi) (Ghosh and Preiss, 1966; Neuhaus and Stitt, 1990; Tetlow et al.,

2003) and this mode of regulation of AGPase by 3PGA and Pi has been demonstrated in cereal endosperms and potato tubers (Sowokinos and Preiss, 1982; Tiessen et al. 2003;

Tetlow et al. 2003). The ratio of the allosteric regulators 3-PGA and Pi (3PGA/Pi) is also an important factor in regulating AGPase activity. For example, in wheat endosperm, the plastidial AGPase was found less sensitive to 3-PGA as compared to potato tubers

(Hylton and Smith, 1992; Ballicora et al., 1995; Gomez-Casati and Iglesias, 2002; Tetlow et al., 2003). Therefore, the degree to which AGPase activity changes also varies with species (Ballicora et al., 1995; Gomez-Casati and Iglesias, 2002; Tetlow et al., 2003) and also between the different combinations of its large and small subunits (Fritzius et al.,

2001). The activity of wheat endosperm AGPase was also found to be inhibited by adenosine diphosphate (ADP) and fructose-1,6-bisphosphate and the inhibition was reversed by adding 3-PGA and fructose-6-phosphate (Gomez-Casati and Iglesias, 2002).

In photosynthetic tissues, during day time the levels of Pi decreases as it is utilized as a substrate in ATP synthesis during photophosphorylation and hence the ratio between

3PGA to Pi increases (Buchanan et al., 2000) and subsequently affects the activity of plastidial AGPase.

AGPase activity is also influenced by post-translational redox modulation in several plant species (Fu et al., 1998; Tiessen et al., 2002). This type of post translational redox modulation occurs through the inter-conversion of monomeric and dimeric forms of the two catalytic subunits (small subunits) of the enzyme via formation of reversible disulfide-bridges between them (Fu et al., 1998; Tiessen et al., 2002; Hendriks et al.,

2003) depending on the sugar concentration and light (Hendriks et al., 2003).

Monomerization is correlated with light and higher sucrose levels and causes activation of the enzyme, while dimerization due to intermolecular cysteine bridge formation occurs under dark conditions and leads to deactivation (Tiessen et al., 2002; Hendriks et al.,

2003). Redox activation of AGPase by light signals resembles the light activation of enzymes in the Calvin cycle (Geigenberger, 2011). The reducing power from photosynthetic electron transport reduces ferredoxin (Fd) and the reducing equivalents are transferred by ferredoxin: thioredoxin reductase (FTR) to Thioredoxin (Trx) f and m

(in the pathway) which activate AGPase through the reduction of regulatory disulphides

(Schürmann and Buchanan, 2008; Geigenberger, 2011). Thus the reducing agent, dithiothreitol, (DTT) caused a 4 fold increase in the activity of recombinant AGPase from potato (Sowokinos and Preiss, 1982) whereas exposure to oxidized thioredoxin partially inactivated the enzyme by dimerization (Fu et al. 1998). Sucrose and glucose signalling involved in the post translational redox regulation of AGPase are thought to occur through the activity of respective SNF-1-related protein kinases (SnRK1) and hexokinase respectively (Tiessen et al., 2003).

1.3.1.2. Starch synthases (SSs, EC 2.4.1.21)

Starch synthases are a group of glucosyltransferases, (GTs) that constitute a large family of enzymes which are involved in the biosynthesis of oligosaccharides, polysaccharides, and glycol-conjugates (Taniguchi et al., 2002) and catalyze glycosidic bond formation using sugar donors containing a nucleoside phosphate or a lipid phosphate leaving group (Lairson et al., 2008). Other glycosyl transferases include mammalian glycogen synthase, bacterial glycogen synthase etc. In higher plants, starch

12 synthases catalyze the transfer of the glucosyl moiety of ADP-glucose to the non- reducing end of an - (14)-linked glucan primer generating amylose and amylopectin.

Having the highest number of isoforms among the enzymes in the starch biosynthetic pathway (Fujita et al., 2011), SSs fall into two groups; those involved in amylose biosynthesis, the granule bound starch synthases (GBSS); and those which synthesize amylopectin, the soluble SSs (Echt and Schwartz, 1981; Sano, 1984; Nakamura et al.

1993). There are five major isoforms of SSs which have been categorized based on their sequences that are highly conserved through dicots and monocots (Ball and Morell,

2003). The five classes of SS identified are GBSS, SSI, SSII, SSIII and SSIV and are encoded by at least five different genes. The second group of starch synthases, the soluble

SSs (SSI, SSII, SSIII, and SSIV) are exclusively involved in amylopectin synthesis and their intra-plastidic location varies between tissues and plant species and their expression varies with developmental stages. Analysis of the expression pattern of SSI (responsible for synthesizing shorter glucan chains of DP ≤ 10) indicated that SSI is expressed during storage starch synthesis in cereal endosperms and tubers (Mu-Forster et al., 1996; Abel et al., 1996; Kossmann et al., 1999) and during transient starch synthesis in the leaves

(Kossmann et al., 1999; Delvalle et al., 2005). The relative contribution of SSI towards total SS activity was 70% in japonica rice lacking SSII (Nakamura et al., 2005; Fujita et al., 2006); while in potato SSIII contributed >70% of the total SS activity during starch biosynthesis (Abel et al., 1996) demonstrating how the expression and concentration of the SS isoforms varies between plant species. Similarly, the expression and relative abundance of SSI in developing wheat endosperm, varied with developmental stages.

During the post anthesis period, SSI transcripts accumulated more during 5-10 days-post-

13 anthesis, (DPA) than that at later stages (15-25DPA) (Peng et al., 2001). Again, during the active phase of endosperm development, SSI concentration in the starch granules remained stable, while the concentration of soluble SSI increased from 10-15 DPA; whereas, during later stages (15-25DPA), the granule associated SSI increased compared to the soluble fractions ( Peng et al., 2001). Of the two isoforms of SSII present in plants,

SSIIa predominates in cereal endosperms, while SSIIb is mostly confined to the vegetative and photosynthetic tissues (Harn et al., 1998; Morell et al., 2003). In wheat,

SSII mRNA expression was detected in the leaves and pre-anthesis florets and also in the grains during mid-developmental stages; whereas SSI gene was found endosperm specific (Li et al., 1999). Likewise, in rice, two isoforms of SSIII and SSIV have been reported (Dian et al., 2005), of which SSIII2 and SSIV1 genes are mainly expressed in the endosperm while the other two (SSIII1 and SSIV2) are expressed in the leaves (Dian et al., 2005). While SSIII and SSIV are found exclusively in the stroma, SSI and SSII are found distributed between the stroma and as granule associated proteins (Mu et al., 1994;

Denyer et al., 1995; Mu-Forster et al., 1996; Yu et al., 1998; Edwards et al., 1999; Li et al., 1999; Li and Corke, 1999; Roldan et al., 2007; Leterrier et al., 2008).

Sequence analysis shows that a highly conserved region of approximately 60 kDa is confined to the C-terminus of all starch synthases in higher plants and green algae.

However, in prokaryotic glycogen synthases, this region is distributed across the protein sequence (Fukukawa et al. 1990; Nichols et al. 2000). This highly conserved K–X–G–G–

L motif is thought to be involved in binding ADP-glucose in both prokaryotic glycogen synthase and in plant and algal SSs (Furukawa et al. 1990, 1993; Busi et al. 2008). The glucan primer preference of starch synthases is determined by the presence of lysine in

14 the K–X–G–G–L domain; whereas the glutamate and aspartate residues are vital for catalytic activity and substrate binding in maize SSs (Nichols et al. 2000; Gao et al.,

2004). The N-terminal extensions in starch synthases upstream of the catalytic domain have very little sequence similarity with each other and are described as flexible arms.

The N terminal region can vary greatly in length, ranging 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 characterization of N-terminal extensions in maize SSI, SSIIa and SSIIb has shown that these extensions can be involved in the regulation of binding to different substrates (e.g. glycogen and amylopectin) and that they can be removed from the sequence without affecting the enzyme kinetic properties significantly (Imparl-

Radosevich et al., 1999a).

Figure 1.4 Comparison of amino acid domains in different SS isoforms. The C- terminal catalytic domains represented in black contain the putative ADPG binding K-X-G-G-L motif. The N- terminal region represented by shaded bars are called flexible arms and are thought to be involved in binding to different substrates and also providing isoform specificity through controlled protein-

protein interactions. The genes coding the proteins are given in italics in brackets near respective protein and the numbers on the left denotes the number of amino acid residues. (Length of the sequences are not drawn to scale) (Derived from Tetlow, 2011)

1.3.1.2.1 Granule bound starch synthase (GBSS)

The two isoforms of GBSS, GBSSI and GBSSII, are restricted to the granule matrix of starch synthesizing tissues in plants. Granule bound starch synthases were initially thought to be involved specifically in the synthesis of amylose, but studies have shown that GBSSI may also be responsible for elongating extra-long glucan chains

(ELC) in amylopectin (Maddelein et al., 1994; van de Wal et al., 1998). In cereals,

GBSSI is encoded by the Waxy locus (Nelson and Rines, 1962; Shure et al., 1983) and is mostly found within the granule matrix. GBSSI is responsible for elongating amylose in storage tissues such as endosperm and tubers whereas GBSSII is found in tissues such as

16 pericarp, leaf, stem, and root (Vrinten and Nakamura, 2000) involved in transient starch synthesis. The waxy mutant with impaired GBSSI expression exhibited a lack of amylose production (Vrinten and Nakamura, 2000) but was able to form starch granules with semi-crystalline properties similar to the wild type suggesting that amylose is not required for insoluble granule synthesis (Denyer et al., 1999).

1.3.1.2.2 Starch synthase I (SSI)

Genetic and biochemical analyses reveal that SSI is responsible for the synthesis of the initial short glucan chains of up to 10 glucosyl units or less to form chains of degree of polymerization (DP) 8-12 (Commuri and Keeling, 2001; Nakamura, 2002;

Delvalle et al. 2005). Arabidopsis SSI mutants exhibited considerably different amylopectin structure with fewer short glucan chains and more outer chains (Delvalle et al., 2005) while the SSI deficient rice mutants exhibited an increased degree of polymerization in endosperm amylopectin (Fujita et al., 2006). Both observations are indicative of the role of SSI in synthesising short glucan chains. However, SSI mutants in transgenic potato plants exhibited no visible phenotypic changes (Kossmann et al., 1999) indicating that its function may be species specific. Kinetic studies with maize SSI have shown that SSI exhibited higher affinity for amylopectin (Kd= 0.2 mg/mL) than for glycogen (Kd= 1.0 mg/mL), amylose (Kd= 0.6 mg/mL) and starch (Kd= 0.49 mg/mL)

(Commuri and Keeling, 2001). It was also observed that when the outer chain lengths

(OCL) of glucan substrate (glycogen) were extended (to an average OCL of 21), using the phosphorylase enzyme, the affinity of SSI increased exponentially with a significant decrease in its catalytic activity (Commuri and Keeling, 2001). It was concluded that SSI has a very high affinity for the longer glucan chains of DP > 20, rendering it catalytically

17 incapable at longer chain lengths. Therefore it was proposed that during amylopectin synthesis, SSI extends shorter A and B1 chains to a certain chain length until they cannot be elongated further by this enzyme. Alternatively, SSI gets entrapped as a relatively inactive protein within the granule matrix and further glucan chain extension might be carried out by other SS enzymes which can extend the glucan chains further (Commuri and Keeling, 2001).

The relative contribution of SS isoforms towards total SS activity varies in different plant species. For example, in SSIIa deficient japonica rice (Nakamura et al.

2005) SSI accounts for 70% of the total SS activity (Fujita et al. 2006) whereas in potato

SSIII accounts for >70% of total SS activity (Abel et al. 1996).The double-recessive homozygous mutants from SSI null mutants with SSIII null mutants in japonica rice demonstrated the overlapping functions of these two SSs in the biosynthesis of starch in rice endosperm. While the heterozygous mutants produced fertile seeds, the double- recessive homozygous lines produced sterile seeds (Fujita et al. 2011). In developing wheat endosperm, expression and relative abundance of SSI in the amyloplast stroma varied with the stages of kernel development (Peng et al. 2001). The highest concentration of SSI in the soluble fraction was observed during 15-25 DPA (days post anthesis). However, the higher concentration of SSI in the granule matrix did not vary considerably with stages of kernel development (Peng et al. 2001). In maize, 85% of the total endosperm SSI was found strongly associated with starch granules (Mu- Forster et al., 1996).

1.3.1.2.3 Starch synthase II (SSII)

In monocots two SSII isoforms have been identified, SSIIa and SSIIb. The former predominates in cereal endosperms, while the latter is mostly confined to photosynthetic and vegetative tissues (Harn et al., 1998; Morell et al., 2003). In developing maize endosperm, SSIIa is found partitioned between the granule and stroma during the early stages of endosperm development. However, from mid to later stages of grain filling,

SSIIa existed predominantly in granule bound form (Li and Corke, 1999). Similarly in other plant species such as pea (Denyer and Smith, 1992) and Arabidopsis (Zhao, unpublished PhD thesis), SSIIa is found largely associated with the starch granule. SSIIa appears to elongate glucan chains produced by SSI leading to the production of medium length chains of DP 12-25 (Morell et al., 2003; Zhang et al., 2004); however its precise role in starch synthesis is still unknown (Zhang et al., 2004). In developing maize endosperm, higher accumulation of SSIIa mRNA was observed during the starch accumulation period (Harn et al. 1998). Mutants with impaired SSII exhibited reduced starch content, altered amylopectin chain length and arrangement and overall distorted granule morphology in both monocots and dicots (Craig et al., 1998; Morell et al., 2003;

Shimbata et al., 2005). SSIIa mutation in pea resulted in starch with altered granule morphology and amylopectin with oligosaccharides having reduced chain length as compared to the wild type (Craig et al., 1998). Similarly, a mutation eliminating soluble

SSII in Chlamydomonas reinhardtii (sta-3) also produced similar effects on granule morphology and amylopectin structure (Fontaine et al., 1993).

In the barley sex6 mutant lacking SSIIa activity, there was a significant reduction in amylopectin synthesis resulting in a shrunken endosperm phenotype with low starch

19 content (Morell et al., 2003). The mutant also had altered chain length distribution with a

20% decrease in the amount of medium length glucan chains (DP= 12-30) and a 15% increase in the amount of the shorter glucan chains and a reduced gelatinization temperature (Morell et al., 2003). Moreover the mutation resulted in pleotropic effects on other starch biosynthetic enzymes eliminating the binding of these proteins (SSI, SBEIIa and SBEIIb) to the starch granule without significantly affecting their expression levels in the soluble fraction (Morell et al. 2003). Similar alteration in the chain length distribution was observed in a sugary2 (su-2) mutant of maize lacking active SSIIa (Zhang et al.

2004; Liu et al., 2012b) and in the SSII mutant of Arabidopsis thaliana (Zhang et al.

2008). The allelic variant of maize su-2 expressing inactive SSIIa had reduced starch content (10%) but significantly higher amylose (double than wild type); a significant reduction in the proportion of glucan chains of DP 12-22 and a reduced gelatinization temperature (Liu et al., 2012b). Scanning Electron Microscopy analysis of starch granules showed that su-2 starches had altered granule morphology with a higher proportion of irregularly shaped granules (Liu et al., 2012b). Correspondingly, the su-2 mutant starch exhibited significantly reduced levels of crystallinity and disrupted glucan chain packing (Liu et al., 2012b). Previous studies have shown that in wheat and maize endosperms, SSIIa forms the core of an important functional enzyme complex with

SBEIIb and SSI in a phosphorylation dependent manner (Tetlow et al., 2008; Hennen-

Bierwagen et al., 2008). The study with the maize sugary2 (su-2) mutant thus demonstrated that the granule association of the trimeric protein complex containing SSI,

SSIIa and SBEIIb in maize endosperm depends on the glucan binding ability of SSIIa

(Liu et al., 2012b). It was postulated that the altered physicochemical properties of maize

20 su-2 starches could be due to the loss of glucan binding ability of the catalytically inactive SSIIa present in the allele studied, thereby preventing the other components of the trimeric protein complex (SSI and SBEIIb) to functionally associate with the starch granule (Liu et al., 2012b). All these studies strongly suggest the involvement of SSIIa in maintaining the well-organized crystalline structure of starch granule assembly. The importance of SSIIa in starch biosynthesis is discussed further in section 1.5 and in the following chapters (sections 2.1, 3.1 and 4.1).

1.3.1.2.4 Starch synthase III (SSIII)

Biochemical analysis of SSIII mutants suggest that it is mostly involved in amylopectin synthesis and is responsible for producing longer glucan chains of DP 25-35 or greater (Gao et al., 1998; Tomlinson and Denyer, 2003). Characterizing soluble starch synthases from maize endosperm, Cao and others (1999) found that SSIII (˃ 200 kDa in size) is coded by DUI gene and is expressed throughout the developmental stages exclusively in the stroma. However the size of this protein varies in various plant species, for example, in potato the molecular mass of SSIII is found as 139.2 kDa (Abel et al.,1996) whereas the isoforms found in rice endosperm and leaves are 138kDa and

201kDa respectively (Dian et al.,2005). Moreover, the mutation of SSIII leads to different effects dependent on the genetic background. While the SSIII mutation in potato caused altered amylopectin chain-length distribution (Abel et al., 1996), maize SSIII mutants exhibited a less significant phenotypic consequence that is only detected in Waxy mutants (Gao et al., 1998). The mutation in SSIII also influenced starch branching enzyme (Preiss and Boyer, 1979) function in maize causing a loss in SBEIIa activity specifically (Gao et al., 1998). In Arabidopsis, null mutants that lack SSIII activity

21 produced a starch excess phenotype in leaves indicating that SSIII can negatively regulate the biosynthesis of transient starch (Zhang et al., 2005). SSIII appears to play a vital role in the biosynthesis of transient starch while granule initiation involves the necessity of either SSIII or SSIV in Arabidopsis leaves (Szydlowski et al., 2009). The barley amo1

(high amylose Glacier, HAG) mutant is characterized by an increased amylose phenotype

(up to 45% amylose) (Banks et al., 1971). Genetic mapping strongly indicated that ssIIIa gene is tightly linked to the amo1 locus (Li et al., 2000; Li et al., 2011) suggesting that ssIIIa is a candidate gene underlying the amo1 mutation. Characterizing a barley population generated between the sex6 mutant and the amo1 mutant to evaluate the impact on amylose content (combining recessive mutations was expected to produce a higher amylose content with simultaneous decrease in starch content), it was observed that the amo1sex6 double mutant signiﬁcantly restored starch synthesis in the endosperm of sex6 mutants, through parallel increases in both amylose and amylopectin content (Li et al., 2011). Moreover the amylose content in the double mutant was not significantly higher than the amylose content observed for the sex6 mutation alone. It was also demonstrated that the amo1 mutation up-regulated the expression of SSI, SSIIa, SBEIIa, and SBEIIb in the granules (Li et al., 2011). It was therefore proposed that amo1 may be a negative regulator of other genes of starch biosynthesis in barley (Li et al., 2011).

Competition between different SSs for the substrate could partially explain the specific distribution of glucan chains within amylopectin (Zhang et al., 2008). A recent study in kidney bean revealed that the N-domain of SSIII has higher affinity towards amylose and amylopectin and functioned as a carbohydrate binding module (CBM) suggesting that SSIII can function both as a glucan elongator and a CBM (Senoura et al.,

2007). Investigating the function of starch binding domains (SBD) present on the N- terminal region of Arabidopsis thaliana Valdez et al. (2008) proposed that these domains have a regulatory role in the catalytic activity of SSIII, especially in terms of its starch binding ability. Analysis of a potato double mutant of SSII and SSIII (compared to individual mutations) with respect to the branched glucan chain-lengths starch granule morphology and the gelatinisation behaviour clearly suggested that different isoforms of starch synthase make distinct contributions to the synthesis of amylopectin, and that they act in a synergistic manner, rather than independently, during amylopectin synthesis

(Edwards et al., 2002).

1.3.1.2.5 Starch synthase IV (SSIV)

SSIV is present in the stroma of the plastids (Roldan et al., 2007; Leterrier et al.,

2008). The specific role of SSIV in starch biosynthesis is largely unknown. Of the two cloned isoforms of SSIII and SSIV in rice, SSIII -2 and SSIV-1 genes are expressed in the endosperm while the others (SSIII-1 and SSIV-2) are expressed in leaves (Dian et al.,

2005). In developing rice endosperm the expression of SSIV is higher at the later stages of grain filling (Dian et al., 2005). In wheat SSIV has been found to be preferentially expressed in leaves and has a unique N-terminus region containing two coiled-coil domains and a 14-3-3 protein recognition site (Leterrier et al., 2008). In their biological context, coiled –coil protein domains act as a versatile tool box of the cell carrying out diverse cellular functions such as transcriptional control of expression of proteins, organization of chromatin and mediating protein- protein interactions (Mason et al.,

2004; Rose et al., 2004); while 14-3-3 proteins interact with various signaling proteins like protein kinases and phosphatases (Comparot et al., 2003).

The exact function of SSIV being still unclear, it is speculated that it may serve as a primer in starch granule formation (Roldan et al., 2007; Szydlowski et al., 2009). SSIV mutants in Arabidopsis thaliana exhibited reduced (35-40%) leaf starch contents with respect to their wild type lines at the end of the illuminated period. Also, the total activity of the both cytosolic and plastidial forms of starch phosphorylase (SP) in the mutants increased by two fold without affecting the total SS activity, the reason of which is still unknown (Roldan et al., 2007). Most interestingly, the mutants had fewer starch granules per plastid but of larger size compared to the wild type, suggesting that SSIV might function in the control of the granule number (Roldan et al., 2007). In a recent study in

Arabidopsis thaliana, the ss3/ss4 double mutant failed to accumulate any measurable amounts of starch despite the dark or light conditions suggesting that the presence of either SSIII or SSIV is vital for the biosynthesis of transient starch in the leaves

(Szydlowski et al., 2009). However this speculation about SSIV function in starch granule initiation is not yet confirmed in any other plant species and the exact mechanism of starch granule initiation is still not known.

1.3.1.3 Starch Branching Enzymes (SBEs, EC 2.4.2.18)

Starch branching enzymes are responsible for generating the branching pattern and the subsequent structural arrangement of the amylopectin molecule. These enzymes form the  (16) linkages through cleavage of internal (14) glycosidic bonds and transfer of the reducing ends to the C6 hydroxyls creating branch points at (16) bonds. SBEs are associated to the α-amylase super-family of enzymes (Jespersen et al.,

1993). These enzymes are capable of producing α-(1- 6)-linkages on linear and branched glucan substrates. Subsequent to the cleavage of an α (1- 4)-linkage, SBEs can transfer

24 the sliced glucan to an acceptor chain which is either part of the original glucan chain, or part of a neighboring glucan chain (This is known as inter-chain transfer) (Jespersen et al., 1993; Tetlow, 2011). The multiple isoforms of SBE (SBEI, SBEIIa, and SBEIIb) are developmental or tissue specific in their expression and differ in the length of the glucan chain they transfer in vitro as well as their substrate specificity (Gao et al.,1997; Regina et al., 2005).

1.3.1.3.1 Starch Branching Enzyme I (SBEI)

SBEI exhibits a higher catalytic activity towards amylose and transfers longer chains compared to SBEII, which has a higher affinity towards amylopectin (Guan and

Preiss, 1993; Takeda et al., 1993; Rydberg et al., 2001). In developing maize endosperm,

SBEI is strongly expressed during the later stages of kernel development (22–43 DAA) while moderate expression of this enzyme is observed in the vegetative tissues as well

(Kim et al. 1998). In maize SBEI mutant (sbe1) the lack of SBEI was found to be compensated by other two SBE isoforms (SBEIIa and SBEIIb) resulting in the retention of total SBE activity as in the wild type lines (Blauth et al. 2002). Similarly, the amylopectin chain length distribution remained unchanged in this mutant as compared to the wild type suggesting that SBEI may not have a critical role in determining starch quantity or quality in leaves or endosperm (Blauth et al. 2002). However, this enzyme is highly conserved in plants and has been shown to interact with other starch biosynthetic enzymes (Tetlow et al. 2004a; Liu et al. 2009) suggesting its significance in regulating starch biosynthesis. In a recent study, the analysis of starch granules from the sbe1a mutant (deficient in SBEI) showed that these starches exhibited an altered branching pattern for amylopectin and amylose and were more resistant to digestion by pancreatic α-amylase (Xia et al., 2011). When kernels of sbe1a mutant were germinated,

25 they had shorter coleoptile length and higher residual starch content resulting from less efficient starch utilization during germination. The results suggested that SBEI is essential to synthesize starch granules for normal kernel development, promoting efficient hydrolysis and utilization of starch during seed germination (Xia et al., 2011).

1.3.1.3.2 Starch Branching Enzyme II (SBEII)

Although the two SBEII gene products (SBEIIa and SBEIIb) are closely related in monocots (Rahman et al., 2001), in wheat endosperm SBEIIa is expressed at higher levels than SBEIIb (Regina et al., 2005), whereas in maize SBEIIb is expressed approximately 50 fold higher than SBEIIa (Gao et al., 1998). Mutations in SBEII isoforms produced more pronounced phenotypic variations compared to SBEI mutations.

In maize, a single gene encodes SBEIIb which is predominantly expressed in endosperm and embryo during kernel development (Fisher et al., 1993). The mutation of this gene produces a high-amylose starch phenotype, known as the amylose extender (ae) characterized by less branched and longer glucan chains in amylopectin (Yu et al., 1998;

Nishi et al., 2001; Klucinec and Thompson, 2002).

In maize mutations eliminating SBEIIa significantly affected leaf starch without any significant changes in storage starches in the kernels (Blauth et al., 2001) suggesting that the phenotypic changes resulting from SBEIIa mutations are tissue specific. SBEIIb is the most abundant protein in the maize amyloplast stroma (Mu et al., 2001) and is regulated by protein phosphorylation (Tetlow et al., 2004a). In the wild type maize endosperm, SSI and SSIIa form the core of a phosphorylation dependent functional protein complex during amylopectin synthesis to which SBEIIb joins whereas in the amylose extender (ae) mutant in the absence of SBEIIb, SBEI compensates for SBEIIb

26 by forming a complex with SBEIIa and SP as well as with SSI and SSII (Liu et al.,

2009). In a recent study in barley, utilizing RNA-mediated silencing technology, a reduction (>80%) in the expression of both SBEIIa and SBEIIb was required to elevate the amylose content to >70% from ∼28% in the WT (Regina et al., 2010). When either of the SBE isoforms was reduced individually there was either no difference (in SBEIIb reduced line) or a minor increase (in SBEIIa reduced line) in amylose content. Analysis of the amylopectin chain length distribution indicated that the frequency of branches in amylopectin was significantly reduced only when both SBE IIa and SBE IIb were reduced ; whereas the branching frequency of amylose significantly increased when SBE

IIb alone was reduced (Regina et al., 2010). The study suggested that both SBEIIa and

SBEIIb play specific roles in determining the fine structure of amylose and amylopectin and that there is a high degree of functional overlap between these SBE isoforms such that the formation of α (1-6) linkages is determined by the relative expression levels of the two genes in the cereal endosperm (Regina et al., 2010).

1.3.1.4 Starch Debranching Enzymes (DBEs, EC 3.2.1.41 and EC 3.2.1.68)

Starch debranching enzymes play an important role in the development of crystalline amylopectin as other starch biosynthetic enzymes (James et al., 1995; Mouille et al., 1996; Zeeman et al., 1998). Based on their substrate specificity two groups of debranching enzymes occur in plants. Debranching enzymes that can hydrolyze the α (1-

6)-linkages in the fungal polymer of malto-triose units, pullulan and in limit-dextrins of amylopectin are known as pullulanases or limit-dextrinases, and those that cannot hydrolyze pullulan but can debranch amylopectin and its limit-dextrins are known as isoamylases (Nakamura, 1996; Zhu et al., 1998). Role of debranching enzymes in the

27 degradation of starch during germination has been previously reported (Manners, 1985).

Evidence from the study of cereal mutants, and Chlamydomonas reinhardtii, strongly indicate that these enzymes are equally important in starch biosynthesis. Sugary1 (su1) mutants from maize and rice, and the sta7 mutant of C. reinhardtii having reduced debranching enzyme (ISO1) activity (Pan and Nelson, 1984; James et al., 1995; Mouille et al., 1996; Nakamura,1996), accumulated a highly branched, water soluble, glucan polymer, phytoglycogen, in addition to or instead of starch (Mouille et al., 1996;

Nakamura, 1996; Dinges et al., 2001; Nakamura, 2002). Likewise, mutations that eliminate ISO and ISO2 caused significant reduction in granular starch content and increased accumulation of disordered water soluble phytoglycogen in potato (Bustos et al., 2004) and Arabidopsis (Delatte et al., 2005). Analysis of PUL mutants in maize and rice suggest that pullulanase could function both in the synthetic and degradative pathways of starch metabolism (Dinges et al., 2003; Fujita et al., 2009). The specific roles of isoamylase and pullulanase type DBEs in starch biosynthesis still being unclear, there are two models which have been suggested to explain their function. The first one, the “glucan trimming” model, proposes that DBEs trim the elongating glucan chain in order to facilitate amylopectin aggregation, allowing crystallization and development of the starch granule (Ball et al., 1996: Myers et al., 2000). The alternative “glucan clearing” model, suggests that DBEs clear away any soluble glucan not attached to the granule that would lead to phytoglycogen synthesis (Zeeman et al., 1998). Although the latter model would explain the increase of phytoglycogen in DBE mutants, it is possible these models are not mutually exclusive and they work in conjunction with one another to facilitate starch granule formation. Debranching enzymes, ISOI and ISO2 are shown to

28 form functional hetero- oligomeric protein complexes (Hussain et al., 2003). In maize two function isoamylase protein complexes were found, one of approximately 400 kDa contained both ISO1 and ISO2, and the other approximately 300 kDa complex contained

ISA1 but not ISA2 (Kubo et al., 2010). Observations from Arabidopsis leaves showed that isoamylase ISO3 is catalytically active on water soluble polysaccharides produced by the action of β- amylase and starch phosphorylase (Wattebled et al., 2005) suggesting that ISO3 could be involved in starch mobilization and degradation (Dinges et al., 2003).

1.3.1.5 Starch Phosphorylase (SP, EC 2.4.1.1)

The reversible transfer of glucosyl units from glucose-1-phosphate to the non- reducing end of α-1-4 linked glucan chains is catalyzed by starch phosphorylase. This reaction is driven in either direction by the relative concentrations of the soluble substrates (Schupp and Ziegler, 2004). There are two isoforms of SP, a cystolic form

(Pho2) and a plastidial form (Pho1) (Nakano and Fukui, 1986). The plastidial form of SP is the second most abundant protein in the maize amyloplast stroma after SBEIIb (Yu et al., 2001). As a glucan degradative enzyme, SP in the presence of orthophosphate (Pi) cleaves α-1-4-glucosyl bonds from the non-reducing end of α-glucan chain to produce glucose-1-phosphate (G-1-P) (Schupp and Ziegler, 2004). However the exact physiological function of SP in starch biosynthesis in higher plants has yet to be understood fully. Although the majority of studies propose a role in starch degradation, it has been demonstrated that Pho 1 contributes significantly to starch synthesis (Duwenig et al., 1997; Rathore et al., 2009). While Pho 1 possesses higher affinity towards low molecular weight linear malto-oligosaccharides (MOS) and amylose, the cystolic Pho2 prefers highly branched polyglucans like glycogen (Satoh et al., 2008). The

29 predominance of cytosolic forms (Pho2) of SP in germinating seeds suggests that they are involved in the utilization of α –glucans resulting from starch degradation (Schupp and

Ziegler, 2004). Studies with rice Pho1 mutants suggested that SP could function at two different phases of starch biosynthesis; at the starch initiation and starch elongation phases (Satoh et al., 2008). In developing wheat endosperm, Pho1 may play an important role in recycling glucosyl units from malto-oligosaccharides back into starch synthesis

(Tickle et al., 2009). The activity of starch phosphorylase is significantly associated with starch accumulation in potato tubers (Sonnewald et al., 1995), maize (Mu et al., 2001) and wheat (Schupp and Ziegler, 2004). Studies with a low-starch producing st1-1 mutant of Chlamydomonas reinhardtii defective in 3-phosphoglycerate (3-PGA) activation and orthophosphate inhibition of ADP glucose pyrophosphorylase (AGPase) clearly demonstrated the existence of the regulation of this phosphorylase enzyme by these metabolites and its significance in the biosynthesis of starch (Ball et al., 1991). This recessive Chlamydomonas reinhardtii mutant (st1-1) was exclusively defective for 3-

PGA activation and Pi inhibition of AGPase, consistent with a mutation in the structural gene of the multi-subunit enzyme (AGPase) or in a regulatory gene responsible for its sensitivity for 3-PGA (Ball et al., 1991).

Analysis of Arabidopsis mutants suggested that plastidial SP is not required for starch degradation in chloroplasts but might play an important role in developing stress tolerance in leaves by providing an alternate path for starch degradation (Zeeman et al.,

2004). Even though studies propose a degradative role of SP (Pho1) in starch metabolism

(Tickle et al., 2009), the evidence from Tetlow et al. (2004a) and Liu et al. (2009) clearly suggest its association in the formation of functional protein complexes with other starch

30 biosynthetic enzymes like SSI, SSII SBEIIb and SBEI in wheat and maize ae mutants in a phosphorylation dependent manner. Starch phosphorylase in maize amyloplast lysates exists in both tetrameric and dimeric states (Mu et al., 2001; Liu et al., 2009). In developing rice endosperm, plastidial phosphorylase (Pho1) accounts for approximately

96% of the total phosphorylase activity (Satoh et al., 2008). Oryza sativa Pho1 mutants deficient in Pho1 produced seeds with significant variation in the size and starch content that ranged from shrunken to pseudo normal. The mutant seeds had smaller starch granules with altered amylopectin structure. The mutant phenotype was also temperature dependent. Based on these results, Satoh and others (2008) proposed that Pho1 plays a vital role in starch biosynthesis in rice endosperm at low temperatures. Further, functional interactions of SP with SBE isoforms were observed in developing rice endosperm, suggesting that both SP and SBE isoforms associate physically and catalytically synergistic manner by each activating the mutual capacity of the other for chain elongation and branching during amylopectin biosynthesis (Nakamura et al., 2012). In a recent study with recombinant maize SP, it was found that multimeric forms of SP

(dimeric and tetrameric) interacted with other starch biosynthetic enzymes and these interactions were greatly regulated by protein phosphorylation (Subasinghe et al., 2013), suggesting a significant role for this enzyme in starch biosynthesis.

1.3.1.6 Disproportionating enzyme (D-enzyme, EC 2.4.1.25)

Disproportionating enzyme or D-enzyme catalyzes the hydrolysis of - (14) linkages of unbranched malto-oligosacharides (MOS) and subsequently transfers the glucan released at the non-reducing end to a non-reducing end of the acceptor molecule to form a new - (14) linkage. Arabidopsis D-enzyme mutants exhibited reduced

31 nocturnal starch degradation rates compared to the wild type suggesting an important role of D-enzyme in chloroplast starch degradation (Critchley et al. 2001). In conjunction with SP, D-enzymes contribute significantly towards starch biosynthesis in the phosphorolytic pathway (Takaha et al. 1998). It was proposed that the short-chain MOS liberated in the trimming reaction by DBEs are converted to longer-chain glucans by D- enzyme that serve as substrates for phosphorolysis by SP, liberating G-1-P for plastidial

AGPase to synthesize ADP-glucose (Takaha et al. 1998).

1.4 Regulation of starch biosynthetic enzymes

The architecture of the starch granule is highly conserved and organized across species (Jenkins et al., 1993) strongly suggesting that the activities of the specific enzymes involved in the synthesis of this organized, semi-crystalline, insoluble carbohydrate polymer are likely to be highly regulated and co-ordinated. Various regulatory mechanisms operate in controlling starch biosynthetic enzymes at multiple levels from their expression through their transcription, translation and post-translational stages (Preiss and Levi, 1980; Preiss, 1991; Morell et al., 1997; Scheible et al., 1997;

Nielson et al., 1998; Wu et al., 2002; Tiessen et al., 2003; Balmer et al., 2006; Smith et al., 2004). These multilevel regulatory mechanisms of starch biosynthetic enzymes allow the plant starch metabolic machinery to respond efficiently across a range of time scales to diverse physiological and environmental stimuli (Geigenberger, 2011).

1.4.1 Regulation at the transcriptional level

Transcriptional regulation of starch biosynthetic enzymes has been reported in potato (Geigenberger, 2003; Tiessen et al., 2003), Arabidopsis leaves (Solokov et al.,

1998; Smith et al., 2004) and cereal endosperms (Mangelsen et al., 2010). The most

32 researched enzyme in the starch biosynthetic pathway that has been subject to transcriptional regulation is plastidial AGPase. The expression of four genes encoding the large subunit and the single gene encoding the small subunit of AGPase in Arabidopsis is spatially and temporally controlled (reviewed in Geigenberger, 2011). Expression of the genes coding for the large subunit is restricted to certain tissues and under specific conditions allowing the production of the enzyme with varying degrees of sensitivity to potential allosteric regulators (Olive et al., 1989; Müller-Rober et al., 1990; Villand et al., 1992a &b; Weber et al., 1995; Duwenig et al., 1997). Sucrose stimulates the expression of the plastidial AGPase (Salanoubat and Belliard, 1989; Müller-Röber et al.,

1990; Sokolov et al.,1998; Tiessen et al.,2003) while nitrates and phosphates (Scheible et al., 1997; Nielson et al., 1998) suppress its expression thus regulating starch accumulation in accordance to the changing nutritional status and external environment

(Geigenberger, 2011). In potato leaves, during polymer (amylose) elongation is the transcript abundance and protein levels of GBSSI are found to be controlled by circadian rhythms and sucrose levels (Wang et al., 2001). Analysis of the starch synthesis transcriptome has revealed that the transcripts of many genes encoding enzymes that might be involved in the synthesis and degradation of starch as well as in the synthesis of

Suc (sucrose) from starch show strong diurnal fluctuations (Harmer et al., 2000; Smith et al., 2004). For example, the transcripts encoding two SSs, granule bound starch synthase

(GBS) and SSII (STS2,a gene coding for SSII class) increased substantially during transition from dark to light in Arabidopsis leaves (Smith et al., 2004). It was also observed that the transcripts encoding several starch synthesizing and putative starch degrading enzymes showed co-ordinated decrease in the dark followed by rapid

33 accumulation in the light (Smith et al., 2004). Suc-mediated regulation of starch synthesis in developing barley endosperm is found to involve a WRKY transcription factor,

SUSIBA2, which participates in source-sink interactions (Sun et al., 2003).Similarly transcriptional regulation of starch biosynthesis in rice endosperm is found to include the ethylene receptor ETRC (Wuriyanghan et al., 2009) and an AP2/EREBP family transcription factor (Fu and Xue, 2010). In a recent study, it was observed that SS4 gene expression and starch granule formation in Arabidopsis chloroplasts are strongly influenced by clock associated genes and transcription factors (AtIDD5 and COL)

(Ingkasuwan et al., 2012).

1.4.2 Allosteric regulation and redox modulation

At the post transcriptional level the starch biosynthetic enzymes have been shown to be regulated by various effector molecules which are often intermediate metabolites

(Ghosh and Preiss, 1966; Priess, 1991; Tetlow et al., 2004b). The most highly studied example in this category is the allosteric regulation of AGPase (Ghosh and Preiss, 1966) by glycerate-3-phosphate (3PGA) and Pi. In potato tubers, AGPase was activated by an increase in the 3PGA to Pi ratio allowing the rate of starch biosynthesis to attune to the alterations in the balance between photosynthesis and sucrose synthesis in leaves and the balance between sucrose synthesis and respiration in tubers (Stitt et al., 1987;

Geigenberger et al., 1998; Geigenberger, 2011). Moreover the relative sensitivity of

AGPase to these allosteric regulators varied depending on the type of tissue it is expressed and its subcellular localization. In contrast to this, the AGPases from barley and wheat endosperms were found insensitive to these allosteric effectors (Doan et al.,

1999; Tetlow et al., 2003; Geigenberger, 2011). A recent study by Martins et al. (2013) showed that trehalose -6-phosphate, a sugar signaling metabolite, plays an important role

34 in the feedback regulation of the breakdown of transient starch in Arabidopsis leaves potentially linking starch turnover to sucrose demand by growing sink tissues during night.

AGPase has been found to be regulated post translationally by redox modification involving the reversible formation of an intermolecular Cys (cysteine) bridge between

Cys82 of its two small subunits (Fu et al., 1998; Tiessen et al., 2002) (explained earlier in section 1.3.1.1). Several enzymes in the starch degradation pathway have also been found to be redox regulated implying the importance of the redox signals in the co-ordinated regulation of starch metabolism in plants (Kötting et al., 2010; Valerio et al., 2011;

Glaring et al., 2012). Performing a comprehensive analysis of the redox sensitivity of major starch biosynthetic enzymes in Arabidopsis leaf extracts, Glaring and others (2012) observed that the activities of several enzymes in the pathway including the isoamylase complex (AtISA1/ISA2), limit dextrinases (AtLDA), starch synthases (AtSS1& SS3) and starch branching enzymes (SBE2) were activated by reduction at physiologically relevant redox potentials.

1.4.3 Protein phosphorylation

1.4.3.1 Protein Phosphorylation- a universal post translational regulatory mechanism

In eukaryotes, protein phosphorylation and dephosphorylation represent the important regulatory mechanisms involved in switching cell signalling networks in response to changing external conditions (Hardie, 1996). This post translational modification of proteins and enzymes by the reversible covalent incorporation of

3- phosphate group (PO4 ) is vital in controlling almost all basic eukaryotic cellular processes including the control of cell metabolism (Hardie, 1996; Huber, 2007). In

35 general, regulatory phosphorylation / dephosphorylation causes important conformational changes to proteins, which result in inactivation, activation or changes in the allosteric properties of the target protein and is believed to modify over 50% of all proteins

(Kalume et al., 2003). During this process, the inorganic phosphate group is often donated to the target protein by ATP (adenosine triphosphate) catalysed by a protein kinase, while dephosphorylation by specific phosphatases involves simple hydrolysis generating orthophosphate (Pi). The broad spectrum of cellular activities including signal transduction, gene expression and metabolism occur through a large variety of dependent reactions and interestingly most of these target the OH groups associated with the serine or threonine residues of the target polypeptides and therefore the phosphorylation of serine, threonine and tyrosine are predominant in eukaryotic cells playing key regulatory roles (Moorhead et al., 2009).

Specific protein kinases (PKs) and phosphatases (PPs) are the key enzymes involved in the regulation of the phosphorylation status of proteins and it is found that these proteins constitute 2-4% of the genes in a typical eukaryotic genome (Manning et al., 2002; Hunter, 2004; Moorhead et al., 2009). The genome of the model plant

Arabidopsis thaliana encodes approximately (3.8%) 1050 different protein kinases out of the total ~ 27,400 protein coding genes (Gribskov et al., 2001; Wang et al., 2003; Martin et al., 2009). Based on the substrate specificity, protein kinases can be classified into five major groups. I) Histidine kinases (originally thought to be restricted in prokaryotes but found in higher plants). II) True histidine kinases (mammals and yeast). III) Serine- threonine specific kinases. IV) Tyrosine kinases which together for the “eukaryotic kinase super family”. V) Dual specificity kinases (target serine, threonine and tyrosine)

(Hardie, 1996). The five major subfamilies of eukaryotic protein kinases include; I) the

AGC family of cyclic nucleotide and lipid activated protein kinases that include cyclic-

AMP (PKA) and cyclic-GMP dependent (PKG) kinases and isoforms of protein kinase C family and ribosomal 56 kinase family. II) The CaMK family of Ca2+ dependent protein kinases and SNF 1 (sucrose non-fermenting) subgroup. III) The CMGC family of cyclin dependent protein kinases and relatives (Cyclin dependent PK, MAPK, GSK-3 and

Ceasin Kinase II type). IV) PTK family of protein tyrosine kinases found largely in multicellular animals (integral plasma membrane proteins involved in signal transduction pathways). V) Other protein kinase family (MAP kinase kinases, Raf kinases subgroup involved in ethylene response in plants) (Hardie, 1996).

The enzymes that catalyze dephosphorylation of serine, threonine and tyrosine residues form three groups based on the unique catalytic signatures/ domain sequences and substrate preferences (Moorhead et al., 2009). I) PPP (Phospho protein phosphatase) including PP1, PP2A, PP2B or PP3, PP4, PP5, PP6, and PP7 and PPM (metallo- dependent protein phosphatase/ PP2C) families that dephosphorylate phospho-serine and phospho-threonine. II) The PTP (protein tyrosine phosphatase) family defined by catalytic signature CX5R with some family enzymes recognized to dephosphorylate complex carbohydrates (reviewed in Moorhead et al., 2009). III) Aspartate based phosphatases with a catalytic aspartic acid signature (DXDXT/V) and includes the

FCP/SCP [TFIIF (transcription initiation factor IIF) - associating component of CTD (C- terminal domain) phosphatase/small CTD phosphatase] and HAD (halo acid dehalogenase) family enzymes.

Very little is known about the protein kinases and phosphatases in plant systems as compared to animal systems. Some well-characterized plant protein kinases include sucrose phosphate synthase kinase (SPS kinase), phospho enol pyruvate (PEP) carboxylase kinase (PEPC kinase), nitrate reductase kinase (NR kinase) and thylakoid bound protein kinases associated with chloroplast light harvesting complex (LHC)

(Huber et al., 1996; Huber and Huber 1996; Gadal et al., 1996; Buchanan et al., 2000;

Vainonen et al., 2008; Lemeille et al., 2009; Baginsky and Gruissem, 2009) and the phospho-protein phosphatase-2 type of phosphatase enzymes have been characterized and shown to be significantly involved in phosphorylation dependent regulation of the enzymes involved in plant carbon and nitrogen metabolism (Smith and Walker,1996).

Recent studies on large scale chloroplast phosphoproteome analysis in Arabidopsis have provided novel information about phosphorylation targets and have revealed more information about the chloroplast metabolic and regulatory functions that are potentially regulated by protein phosphorylation (Baginsky and Gruissem, 2009). After thirty years of research, kinases that are involved in the light dependent thylakoid protein phosphorylation are now revealed (Baginsky and Gruissem, 2009). An investigation of chloroplast protein kinases and phosphatases in Arabidopsis thaliana showed that the set of enzymes predicted to be involved in reversible phosphorylation of chloroplast proteins comprises of 6 chloroplast protein kinases (cpPKs) and 9 chloroplast protein phosphatases (cpPPs). However, the substrates and specific regulatory functions of these identified proteins still remain unknown (Schliebner et al., 2008). According to the database currently available, the major chloroplast protein kinases include Chloroplast kinase II (CKII) (Kanekatsu et al., 1998; Reiland et al., 2009), STN7 and its homolog

STN8 (Rochaix, 2007), the chloroplast sensor kinase (CSK) (Puthiyaveetil et al., 2008), thylakoid associated kinases (TAKs) (Snyder’s and Kohorn, 2001) and uncharacterized kinases (Schliebner et al., 2008). Arabidopsis genome-scale phospho proteomic survey conducted by Reiland and others (2009) listed 1617 chloroplast protein entries while a chloroplast proteome table published by Yu et al. (2008) comprised of 1808 proteins.

Comparing and integrating data from both these studies Baginsky and Gruissem (2009) developed a chloroplast proteome reference table consisting of 1156 chloroplast proteins.

Considering all the previously published reports verifying chloroplast protein kinases to date, Bayer and colleagues (2012) reported that from STN7, STN8 and cpCK2 represent the major protein kinases and that three additional protein kinases including unusual protein kinases like ABC1 protein kinases or PPK also carry out specialized functions in chloroplasts. Based on these reports it is estimated that a total set of 15 chloroplast protein kinases are found in different plant species to date (Bayer et al., 2012).

1.4.3.2 Protein phosphorylation and plant metabolism

Studies have shown that protein phosphorylation plays significant roles in plant metabolism and energy conversion (Huppe and Turpin, 1994; Huber et al., 1996; Huber and Huber, 1996; Buchanan et al., 2000). The effects of protein phosphorylation in regulating the C/N balance in plants through the regulation of the activities of SPS

(sucrose phosphate synthase) and NR (nitrate reductase), along with PEP carboxylase, and in energy conversion reactions during photosynthesis are well documented (Huppe and Turpin, 1994; Huber et al., 1996).

Sucrose phosphate synthase which catalyzes the synthesis of sucrose from fructose-1, 6 bisphosphate was one of the first plant enzymes shown to be regulated by

39 protein phosphorylation (Huber and Huber, 1996). In the dark, the enzyme is phosphorylated resulting in its inactivation (Huber et al., 1996). SPS is apparently phosphorylated on multiple seryl- residues, however Ser-158 is phosphorylated by SPS kinase (either CDPK or SNF 1 –Related Protein Kinase, SnRK1) and is found to be the major regulatory site (McMichael et al., 1993; Huber et al., 1996). The activation of SPS during the dark to light transition is modulated by a type 2A protein phosphatase (Siegl et al., 1990). Similarly the activity of nitrate reductase (NR), a major enzyme involved in plant nitrogen metabolism is regulated by reversible seryl phosphorylation (Huber et al.,

1996). Light and oxygen are the external triggers that modulate NR activity, while sugars and/or phosphates are the internal triggers that regulate the specific PKs and PPs involved in the reversible NR phosphorylation (Kaiser and Huber, 2001). NR phosphorylation is catalyzed by Ca dependent PKs and SNF1 Related Protein Kinases (Kaiser and Huber,

2001) and the dephosphorylation is catalyzed by type 2A PPs (Mackintosh, 1992) like

SPS. In C4 plants and CAM plants, PEP carboxylase (PEPC) plays an important role in primary CO2 fixation and is diurnally regulated by reversible phosphorylation (Terada et al., 1990; Jiao and Chollet, 1992; Ueno et al., 2000). The specific phosphorylation site targeted by PEPC-PK was confirmed as Ser 15 in maize (Ueno et al., 1997) and Ser 8 in sorghum (Wang et al., 1992).

Protein phosphorylation also serves as a major regulatory mechanism in adjusting the light absorption properties of light harvesting complexes (LCH), and the energy distribution between photosystem I (PSI) and II (PSII), to the prevailing light conditions through the activation of thylakoid bound chloroplast protein kinases. The efficiency of photosynthetic electron transport in chloroplasts is largely controlled by protein

40 phosphorylation (Reiland et al., 2009; 2011). It is also well known that chloroplast protein phosphorylation is not restricted to the modulation of photosynthetic functions but also regulates other metabolic processes as well (Baginsky and Gruissem, 2009).

1.4.3.3 Carbohydrate metabolism is regulated by protein phosphorylation – from bacterial glycogen to plant starch In many living organisms, glycogen and starch represent the major storage carbohydrates. Bacterial and animal cells synthesise glycogen from glucose while plants store glucose as highly organized semi-crystalline insoluble polymer, starch (Bollen et al., 1998; Roach, 2002). Although glycogen has twice as many branch points as amylopectin, the branch point distribution does not result in a well-organized cluster formation and hence it is soluble (Blennow et al., 2002; Kötting et al., 2009). However, both these carbohydrate polymers contain significant amounts of covalently linked phosphate groups (Blennow et al., 2002) and their synthesis and degradation are highly regulated by the phosphorylation of specific enzymes in their metabolic pathways. While eukaryotes synthesize glycogen from UDP glucose, most bacteria make their storage polysaccharide glycogen, through an ADP-glucose-based pathway (Cohen, 1999; Ball and Morell, 2003; Jope and Johnson, 2004). Unlike in the bacterial system, the plant starch metabolic pathway consists of multiple isoforms of enzymes catalyzing each step in the pathway with each form having a specific, distinct and conserved role in starch metabolism (Ball and Morell, 2003).

The first major rate –controlling step of the glycogen synthesis pathway in bacteria is the synthesis of ADP glucose by ADP glucose pyrophosphorylase. But unlike in plants, bacterial glycogen synthase cannot prime the transfer of activated glucose to the

41 growing glucan chain and also the post- translational modification and regulation of this enzyme has not so far been reported in bacterial system (Ball and Morell, 2003).

Glycogen metabolism requires the concerted action of several enzymes including glycogenin, glycogen synthase, branching enzyme, debranching enzyme and glycogen phosphorylase (Bollen et al., 1998). Even though, the anabolic and catabolic pathways of glycogen metabolism (Fig. 1.5) are identical in all tissues, the enzymes involved are uniquely adapted to the specific role of glycogen in different cell types (Bollen et al.,

1998). The phospho-enol pyruvate (PEP):carbohydrate phospho-transferase system (PTS) is unique in bacteria, catalyzing the transport and phosphorylation of numerous monosaccharides, disaccharides, amino sugars, polyols, and other sugar derivatives and thereby plays a significant role in carbohydrate uptake and metabolism (Deutscher et al.,

2006).

Animals synthesize glycogen from UDP glucose through a simple but strictly ordered pathway (Fig.1.5). During glycogen synthesis, Glycogen synthase (GS), a member of Family 3 retaining glycosyl transferases catalyzes the transfer of the glucosyl unit of UDP-glucose to the non-reducing end of the growing glycogen polymer by forming a 1-4 glycosidic linkage (Campbell et al., 1997; Roach, 2002). In mammals, there are two isoforms of GS, one expressed in muscle and many other tissues, and the other in liver having 70% sequence identity and similar properties (Browner et al., 1989;

Nuttall et al., 1994). Glycogen synthase is regulated by protein phosphorylation, allosteric activation and enzymatic translocation (Cohen 1999; Jope and Johnson, 2004).

The enzyme is inactivated upon phosphorylation by glycogen synthase kinase (GSK3) on up to nine residues, resulting in its decreased sensitivity to allosteric activators (Lawrence

42 et al., 1977). Two phosphorylation sites on the C terminus appear to have most influence on the activity of GS. It was found that glycogen synthase kinase 3 (GSK3) phosphorylates these sites in a hierarchical fashion after casein kinase II phosphorylates site 5 (Coghlan et al., 2000; Roach, 2002). GSK3 itself is inactivated by phosphorylation of its N-terminus (Sutherland et al., 1993). In the presence of insulin, the GSK 3 is inactivated, resulting in the activation of protein phosphatase I which dephosphorylates and activates the enzyme. Further, the binding of glucose 6- phosphate causes unfolding of the enzyme and overcomes the inhibitory effect of phosphorylation. Glucose-6- phosphate binding also favours dephosphorylation of glycogen synthase through conformatory changes (Lawrence et al., 1977; Lawrence and Roach, 1997). Glycogen synthase has multiple phosphorylation sites and it has been found that three serine residues at the C- terminal could be dephosphorylated upon stimulation by insulin, favouring glycogen synthesis at high blood sugar levels (Jope and Johnson, 2004).

Evidences suggest that the phosphatase responsible for dephosphorylating glycogen synthase is type 1 family of glycogen associated phosphatases PP1Gs (Bollen, 2001;

Roach, 2002). These phosphatases have specific glycogen targeting subunits that contain a C-terminal hydrophobic sequence that anchors the protein to membranes (Tang et al.,

1991; Roach, 2002). Under low sugar conditions stimulated by glucagon, glycogen breaks down to glucose 6- phosphate catalyzed by glycogen phosphorylase which again is modulated by protein phosphorylation. In addition, a protein primer called glycogenin which is attached to the glycogen molecule is found to be essential for the initiation of glycogen synthesis (Smythe and Cohen, 1991) since the glycosyl tranferase activity of this protein catalyzes the transfer of glucose residues from UDP glucose to the α 1-4

43 glucan chain until it becomes long enough (8-10 residues) for the action of glycogen synthase (Cao et al., 1993).

The major enzymes involved in glycogen synthesis (glycogen synthase (GS)) and degradation (glycogen phosphorylase/phosphorylase (GP)) can be enzymatically interconverted between two forms with distinct kinetic and allosteric properties through a complex of cascade of reactions called cyclic cascade (Voet and Voet, 2004). The inter- conversions of the active and inactive forms of these enzymes involve enzyme catalyzed covalent modifications like phosphorylation and demodification reactions. In a bicyclic cascade the enzymes that modulate the target enzyme (e.g. GSK modifying GS) are themselves under the allosteric control of some effector molecules (e.g. under high glucose conditions, insulin activates protein phosphatases that dephosphorylate and inactivate GSK thus activating GS). Thus, a small change in the concentration of the effector of the modifying enzyme causes a large change in the concentration of the active modified target enzyme resulting in signal amplification. Bicyclic signaling cascades

(Fig. 1.6) control the activities of GS and GP.

Figure 1.5 Glycogen metabolism- steps involved in the synthesis and degradation of glycogen derived from Bollen et al. (1998). – Glucose; - glucose-1-phosphate; in the glycogen molecules shown at right the broken lines represent the bulk of the glycogen structure

Figure 1.6 Schematic diagram showing the bi-cyclic cascades involving major enzymatic modifications in the control of glycogen metabolism in mammalian muscle tissue (derived from Voet and Voet, 2004).

Protein phosphorylation also plays an important role in sugar signalling and in the control of carbon flow into starch biosynthetic reactions. Sucrose synthase (SuSy) is a key enzyme that degrades photosynthetic sucrose into glucose and fructose, generating substrates for starch biosynthesis. The activity of this enzyme is modulated by phosphorylation, and binding of 14-3-3 proteins to the major phosphorylation site in the enzyme resulted in decreased activity (Alexander and Morris, 2006). The SnF1 related protein kinases are involved in sucrose- induced expression of sucrose synthase in plants

(Purcell et al., 1998). It was found that SnF1 related protein kinase and hexokinase are involved in sugar signalling by modulating AGPase by post translational redox activation sucrose/sugar levels and addition of these sugars enhanced the redox activation (Tiessen et al., 2003) (see the section on AGPase).

1.4.4 Phosphorylation dependent protein –protein interactions in starch biosynthesis

Biosynthesis of starch is a result of highly co-ordinated actions of various starch biosynthetic enzymes whose activities are controlled by various regulatory mechanisms operating at different levels of enzyme expression (transcription, translation and post- translation) (Preiss and Levi, 1980; Preiss, 1991; Morell et al., 1997; Wu et al., 2002;

Tiessen et al., 2003; Balmer et al., 2006). Analysis of functional mutants in the starch biosynthetic pathway demonstrates pleiotropic effects, further indicating that such regulatory mechanisms involving functional multi-protein complexes operate in the control and co-ordination of starch biosynthetic enzymes in cereal endosperms (Nishi et al., 2001; Colleoni et al., 2003; Dinges et al., 2001 & 2003; Tetlow et al., 2004a; Tetlow et al., 2008; Hennen-Biewagen et al., 2008; 2009; Liu et al., 2009). For example, studies have shown that in the endosperm of the rice ae (amylose extender) mutant (lacking

SBEIIb), the catalytic activity of soluble SSI was considerably reduced (Nishi et al.,

2001). Similarly, the loss of SSIIa in the barley sex6 mutant resulted in a 20% decrease in the amount of amylopectin compared to the wild type, and also resulted in the complete loss of SSI, SBEIIa and SBEIIb binding to the starch granule matrix even though none of these enzymes showed decreased affinity towards amylopectin. This is consistent with the hypothesis that these proteins exist in a complex within the granule (Morell et al.,

2003; Umemoto and Aoki, 2005). Even though the specific function of these multi- enzyme complexes is not clearly understood, it is possible that these protein associations could improve the efficiency of the starch biosynthetic enzyme machinery. This might involve co-ordinating individual enzyme activities via substrate channelling, regulating and altering the enzyme kinetics via conformational alterations, and it could also be possible that such high molecular weight enzyme complexes protect the growing glucan polymer from the action of degrading enzymes as well. Interestingly, studies have shown that the formation of some of these functional starch biosynthetic enzyme complexes are dependent on post translational regulatory mechanisms like phosphorylation (Tetlow et al., 2004 a and b; Tetlow et al., 2008; Hennen–Bierwagen et al., 2009) indicating that phosphorylation of one or more proteins associated with these complexes makes them physically and functionally stable.

Investigating the role of phosphorylation as a mechanism of regulation of starch biosynthesis in developing wheat endosperm, Tetlow et al.(2004a) identified that forms of starch branching enzymes, SBEI, SBEIIa and SBEIIb (in amyloplasts) and SBEI and

SBEIIa (in chloroplasts) were phosphorylated. Incubation of intact wheat plastids with -

32P-ATP followed by phospho amino acid analysis showed that they were all

48 phosphorylated on serine residues (Tetlow et al., 2004a). Interestingly, granule bound

SSIIa and another unidentified SS isoform were also found to be phosphorylated. The activity of SBEIIb in amyloplasts and SBEIIa (both in chloroplasts and amyloplasts) was enhanced by phosphorylation whereas dephosphorylation using alkaline phosphatase reduced their catalytic activity. However phosphorylation did not have any effect on SBE

I activity (Tetlow et al., 2004a). It was also observed that along with the branching enzymes (SBEI and SBEIIb), starch phosphorylase (SP) was also co-immunoprecipitated in a phosphorylation dependent manner, indicating the possible association of these enzymes in a complex (Tetlow et al., 2004a). In recent work in wheat endosperm, using immunoprecipitation techniques and cross linking strategies, Tetlow et al., (2008) detected physical interactions between SS’s and SBE’s and identified two distinct complexes that are phosphorylation-dependent. It was also observed that this high molecular weight protein complex had higher affinity towards glucan substrates than monomers (Tetlow et al., 2008) which indicates that the phosphorylation dependent protein- protein interaction might improve the functional efficiency of these biosynthetic enzymes. Exploring the role of phosphorylation in the association of starch biosynthetic enzymes to the starch granule, Grimaud et al. (2008) observed that granule-associated starch biosynthetic enzymes GBSS, SBEIIb and SP are regulated by phosphorylation. It was also observed that the granule association of one protein was affected by the absence of the other, suggesting the importance of individual enzymes in the stability of these functional protein complexes. In maize amyloplasts, Hennen-Bierwagen et al., (2008) identified two functional protein complexes- one containing SSII, SBEIIa, and SBEIIb and another containing SSII, SSIII, SBEIIa, and SBEIIb using gel permeation

49 chromatography. Even though SSI was not identified as involved in these complexes, evidence from immunoprecipitation and affinity purification experiments showed that

SSI and SSII interact with each other and therefore there is high probability of SSI also being present in the SS complexes. Subsequently, it was observed that SSIII associated with remaining portion of the protein complex (SSIIa, SBEIIa and SBEIIb) in a phosphorylation dependent manner, suggesting that phosphorylation of one or more proteins in the complex was required for its stability (Hennen-Bierwagen et al., 2009).

Interestingly, an important plastidial protein, pyruvate orthophosphate dikinase (PPDK)

(involved in the phosphorylation of PEP during CO2 fixation in C4 plants which it turn regulates AGPase), AGPase and a sucrose synthase isoform (SUS-SHI which directly function in the production of ADP-glucose for starch biosynthesis) also co- immunoprecipitated along with the starch biosynthetic protein complex (Hennen-

Bierwagen et al., 2009). Based on this result, it was proposed that these proteins, PPDK and SUS-SHI, which are not directly involved in the starch biosynthetic pathway might act as regulators and influence the activity of the proposed hetero protein complex

(SSIIa-SBEIIa-SBEIIb) (Hennen-Bierwagen et al., 2009). The study clearly demonstrated how protein complexes function in global regulation of carbon partitioning between various metabolic pathways in a developing endosperm (Hennen-Bierwagen et al., 2009).

In a study with a maize ae mutant (lacking SBEIIb), Liu et al. (2009) demonstrated that in the amyloplasts from developing mutant endosperm, a functional protein complex containing SSI, SSII, SBEI, SBEIIa and SP was formed instead of the normal SSI-SSII-SBEIIb complex observed in wild type amyloplasts, suggesting that the

50 loss of SBEIIb in the mutant can be compensated by SBEI and SBEIIa. It was also observed that ATP enhanced, and alkaline phosphatase decreased, these protein interactions in wild type amyloplasts, while neither ATP nor APase had any effect on the stability of the protein complexes in the ae mutant. However, radiolabelling experiments demonstrated that SP and SBEI were phosphorylated within the mutant protein complex

(Liu et al., 2009). Therefore it was proposed that during starch biosynthesis, SSI and

SSIIa form the core of a phosphorylation –dependent functional protein complex which in wild type endosperm recruits SBEIIb, while in ae mutant SBEI is recruited with

SBEIIa and SP in the absence of SBEIIb. Such differences in stromal protein complexes were reflected in the complement of the starch biosynthetic enzymes detected in the starch granules in each genotype, suggesting that such specific phosphorylation dependent protein assemblies in the amyloplasts are responsible for modulating the unique architecture of starch in each genotype (Liu et al., 2009).

14-3-3 proteins, a highly conserved group of eukaryotic regulatory proteins involved in protein interactions through specific phosphorylated target sites (Roberts,

2003) are also proposed to be involved in starch metabolism. The regulation of enzyme activities through protein- protein interactions in some cases requires phosphorylation followed by the association of these regulatory proteins to form complexes (Roberts,

2003). A significant involvement of 14-3-3 proteins in the metabolism of leaf starch was demonstrated by Sehnke et al., (2001) by showing that a repression of the granule- associated 14-3-3 proteins by antisense technology resulted in leaf starch accumulation in

Arabidopsis. They speculated that this regulatory effect on leaf starch content is possibly through a reduction in the inhibition of starch synthase III binding to the 14-3-3 protein

(Sehnke et al., 2001). 14-3-3 proteins in both plant and animal systems recognize a well- conserved binding motif, Arg-X-X-pSer/pThr-X-Pro, where X is any amino acid and Ser or Thr is phosphorylated (Sehnke et al., 2000). The reason why the starch synthase III

(SSIII) family was suggested as the target for the 14-3-3 proteins was that, among the plastid localized starch metabolic enzymes, uniquely all the members of this family contain a conserved 14-3-3 phospho serine-threonine binding motif (RYGSIP) ( a hexa- peptide motif very similar to the 14-3-3 binding side of NR) that makes them putative targets for 14-3-3 binding and complex formation after phosphorylation (Sehnke et al.,

2001). The 14-3-3 proteins present in non-photosynthetic tissues in barley have been found to be potentially involved in the regulation of carbohydrate metabolism (starch biosynthesis and modification and sucrose biosynthesis), cell signalling and also in plant defence mechanisms (Alexander and Morris, 2006). Since dimerised 14-3-3 proteins have been shown to bind to two phosphorylated protein targets at once (Sehnke et al.,

2000) it is possible that 14-3-3 proteins could act as a scaffold protein, holding plastidic starch biosynthetic enzymes together in a phosphorylation-dependant complex and thus regulate their activity (Alexander and Morris, 2006). In developing barley endosperm,

14-3-3 proteins were found interacting with starch biosynthetic enzymes SSI, SSII,

SBEIIa and GBSS in a phosphorylation dependent manner suggesting their possible regulatory role in starch biosynthesis (Alexander and Morris, 2006). However, a direct role of 14-3-3 proteins in starch biosynthesis remains to be determined (Alexander and

Morris, 2006; Tetlow, 2011).

1.4.5 Regulation of starch turnover by protein phosphorylation

Recent studies revealing the association of specific protein phosphatases (Smith et al., 2005; Kerk et al., 2006; Nittlyla et al., 2006; Kötting et al., 2009) and protein kinases

(e.g. GSK in alfalfa) (Kempa et al., 2007) with the starch granule and their suggested roles in starch degradation strongly suggest that they could be involved in the regulation of starch metabolism through reversible phosphorylation at the site of synthesis and degradation (Tetlow, 2006).The significance of the association of covalently linked phosphate groups to starch and glycogen has recently become apparent (Blennow et al.,

2002). Phosphorylation of amylopectin seems to play a crucial role in the degradation of transient leaf starch (Blennow et al., 2002; Zeeman et al., 2004). Two starch phosphorylating enzymes, glucan water dikinase (GWD) and phospho glucan water dikinase (PWD) are known to be responsible for this glucan phosphorylation making the starch granule accessible to degradative enzymes (Ritte et al., 2006). During in vivo starch synthesis and degradation, GWD transfers the β-phosphate of ATP to either the C6 or C3 position of glucosyl residues, (but prefers the C6 position) within amylopectin

(Delatte et al., 2005; Ritte et al., 2006) whereas PWD, a predicted chloroplastic enzyme exclusively transfers the β-phosphate of ATP to the C3 position (Kötting et al., 2005;

Ritte et al., 2006). Mass spectrometric quantification of phosphorylated glycosyl residues has shown that 70-80% of phosphate residues are found attached to the C6 position and

20-30% in C3 position of potato tuber starch (Haebel et al., 2008). Using crystalline malto dextrins, Hejazi et al. (2008) demonstrated that phosphorylation of insoluble glucans facilitates their solubilisation.

A chloroplastic glucan binding phosphatase encoded at the Starch Excess (SEX4;

At3g52180) locus was reported to be required for normal starch degradation in

Arabidopsis (Nittlyla et al., 2006). The SEX4 protein contains a dual-specificity phosphatase domain and a carbohydrate binding module and members of the dual- specificity phosphatase family have been shown to act on diverse substrates including phosphorylated protein kinases involved in signalling pathways (Pulido and Hooft van

Huijisduijnen, 2008). Therefore it was proposed that the SEX4 protein might dephosphorylate a kinase involved in phosphorylation of starch granules (Nittlyla et al.,

2006). Laforin, a phosphatase required for normal glycogen metabolism in vertebrates and a closely related protein to SEX4, is able to dephosphorylate solubilized amylopectin or glycogen in vitro, demonstrating that it is in fact a glucan phosphatase, (Lafora and

Glück, 1911 ; Worby et al., 2006 ; Gentry et al., 2007). It was hence suggested that laforin and SEX4 are functionally equivalent (Gentry et al., 2007). Recently it was demonstrated that SEX4 like laforin, functions as a phospho-glucan phosphatase required for starch degradation in Arabidopsis thaliana (Kötting et al., 2009). In Arabidopsis,

SEX4 is found to hydrolyze both C6- and C3-phosphate esters introduced by related starch dikinases (GWD and PGWD) and thereby affect the phase transition of α-glucans

(Hejazi et al.,2010).

Glycogen synthase kinase 3 (GSK3) is an important regulator of glycogen synthesis in animals inactivating glycogen synthase by reversible phosphorylation.

Interestingly, this serine/threonine protein kinase has recently been found linked to plant carbohydrate metabolism. In alfalfa a novel plastid localised GSK 3 like kinase, MsK4

(Medicago sativa GSK-3-like kinase) has been found closely associated with starch granules indicating that this enzyme could be an important modulator of carbon metabolism under environmental stress (Kempa et al., 2007). It was observed that under

54 salt stress conditions, the over-expression of MsK4 resulted in increased starch accumulation in these plants (Kempa et al., 2007). This pinpoints the presence of functionally similar kinase proteins in both plant and animal systems.

1.5 Importance of starch synthase IIa (SSIIa) in starch biosynthesis

The role of protein phosphorylation in the regulation of the catalytic activity of starch biosynthetic enzymes in cereal endosperms lies in facilitating the formation of functional enzyme complexes as discussed in the previous sections. Starch synthase IIa

(SSIIa) is an important SS isoform that synthesizes a critical glucan intermediate required for the biosynthesis of amylopectin, and contributes significantly to the total SS activity in cereal endosperms. In cereal endosperms such as wheat and maize, SSIIa forms the core of a well-defined trimeric functional enzyme complex with SSI and SBEIIb which plays some role in the synthesis of the crystalline structure of amylopectin (Tetlow et al.,

2008; Hennen-Bierwagen et al., 2008; Liu et al., 2009; 2012b). It was observed that in wheat endosperm, SSIIa can be phosphorylated (Tetlow et al., 2004a) and a high molecular weight functional protein complex consisting of SS isoforms (SSI, SSIIa) and

SBEs (SBEIIa and SBEIIb), was found to be dissociated upon dephosphorylation, suggesting that any /all of these starch biosynthetic enzymes could be phosphorylated to form a protein complex (Tetlow et al., 2008). Further, in maize, SS isoforms SSIIb, SSIIa and SBEIIa co-immunoprecipitated with SSIII in a phosphorylation-dependent manner

(Hennen-Bierwagen et al., 2009). This indirectly suggests the possibility that SSIIa could also be phosphorylated since more frequent interactions between SSIIa and SSI , SBEIIb and SBEIIa were observed in in vivo protein-protein interaction tests in yeast

(Saccharomyces cerevisiae) nuclei, immunoprecipitation, and affinity purification experiments in a previous study (Hennen-Bierwagen et al., 2008).

The functional importance of individual enzymes in the starch biosynthetic pathway has been demonstrated through the detailed characterization of their purified forms in combination with the corresponding mutant analysis in selected plant species

(Tetlow, 2011). However the interpretation of the results from studies of mutants is challenging due to the increasing evidence of the occurrence of functional protein – protein interactions in the starch biosynthetic pathway (Tetlow et al., 2008; Hennen-

Bierwagen et al., 2008; Liu et al., 2009; 2012 a &b). The phenotype resulting from a single enzyme mutation could be a product of not only its specific function but also its interactions with other enzymes in the pathway (Liu et al., 2012b). The loss of SSIIa activity in corn endosperm results in significant modifications in the structure and morphology of starch granules, including major alterations in the chain length distribution (CLD) and in the amylose-amylopectin ratio (Kramer and Whistler, 1949;

Pfahler et al., 1957; Campbell et al., 1994; Perera et al., 2001). Unlike other SS mutations, SSII mutation in cereals caused alterations in the characteristic amylopectin crystallinity of the storage starch granules (Morell et al., 2003; Fujita et al., 2006; Ryoo et al., 2007) indicating its important function in determining amylopectin structure.

In maize, the loss of SSIIa activity in the endosperm results in one of the best characterized starch phenotypes called sugary -2 (su2) (Zhang et al., 2004). Biochemical analysis of an allelic variant of maize su2 mutant that expresses a catalytically inactive form of SSIIa with two single amino acid substitutions, namely, Asp146→Val and

Gly522→Arg revealed that the same trimeric protein complex consisting of SSI, SSIIa and SBEIIb found in the wild type amyloplasts exists in this mutant (Liu et al., 2012b).

However, the activities of SSI and SBEIIb associated with the su2 protein complex were

56 significantly reduced probably due to the presence of a catalytically inactive and possibly misfolded SSIIa (Liu et al., 2012b). This was due to the fact that SSIIa formed the core of the protein complex interacting with SSI and SBEIIb which didn’t interact with each other directly. Furthermore, the mutation at Gly522 is thought to be significant for glucan binding as it is found in the glycosyl transferase domain of all forms of SSII (Liu et al.,

2012b). As a result, in the mutant, the loss of glucan binding ability of SSIIa led to the inability of SSI and SBEIIb to bind to the granule and reduced their activities in the complex which in turn altered the morphology, crystallinity and other physicochemical properties of the starch granules (Liu et al., 2012b). These observations clearly demonstrate the importance of SSIIa-mediated functional protein complexes in the structural organization and biosynthesis of amylopectin (Liu et al., 2012b).

Recent studies have suggested that maize SSIIa could be phosphorylated along with SSI and SBE forms in maize, and that protein phosphorylation alters the mobility of the SSIIa in the presence of glucan polymers when separated by native gel electrophoresis (Polack, 2009). The phosphorylation of recombinant starch synthases did not occur in the absence of amyloplast extracts confirming that one or more kinases associated with these organelles is required for phosphorylation. However the specific role and effects of protein phosphorylation on the regulation of starch synthase IIa

(SSIIa) are largely unknown. Investigation of how protein phosphorylation regulates this protein and its potential to form functional protein complexes with other enzymes in the pathway will provide a better understanding of how these enzymes are being regulated in the starch biosynthetic process. The present study is therefore an investigation of the

57 effect of protein phosphorylation in starch synthesising amyloplasts of developing maize

(Zea mays L.) endosperm, focusing specifically on the regulation of SSIIa.

1.6 Hypothesis and Objectives of the study

Hypothesis:

The project hypothesizes that protein phosphorylation influences starch biosynthesis in (Zea mays L.) maize endosperm by regulating the activity of SSIIa and its association with other starch biosynthetic enzymes.

Objectives: the objectives of the study are

(a) To investigate whether SSIIa in the maize amyloplast lysates is

phosphorylated.

(b) To understand the effect of phosphorylation on the catalytic activity of maize

SSIIa and its affinity towards different glucans.

and dephosphorylation

(d) To study the effect of protein phosphorylation on functional protein

interactions of maize SSIIa with other starch biosynthetic enzymes.

The results presented in the following chapters discuss the biochemical investigation of protein phosphorylation of SSIIa in partially purified maize amyloplasts and in partially purified recombinant SSIIa from E coli lysates and explores how protein phosphorylation affects the catalytic activity, affinity of SSIIa towards glucan substrates and its interaction with other starch biosynthetic enzymes in the amyloplast lysates.

CHAPTER 2

CHAPTER 2: INVESTIGATION OF PROTEIN PHOSPHORYLATION OF STARCH SYNTHASE IIA IN MAIZE ENDOSPERM

2.1 Introduction

Starch synthases (SS, EC 2.4.1.21) are an important group of glycosyl transferases that catalyze the transfer of the glucosyl moiety of ADP-glucose to the non- reducing end of an - (14)-linked glucan primer generating the two major glucan polymer components of starch viz., amylose and amylopectin. Of the five major isoforms of soluble starch synthases, SSII specifically catalyses the synthesis of intermediate glucan chains of DP12-25 by elongating the shorter chains produced by SSI (Fontaine et al.,1993, Imparl-Radosevich et al., 2003; Zhang et al.,2004). Two isoforms of SSII ( two classes of genes) are found in monocots -SSIIa and SSIIb, while only a single form of

SSII has been reported in dicots studied to date (Morell, 2003). SSIIa predominates over

SSIIb in cereal endosperm (Morell et al., 2003) and is partitioned between the stromal region and the granular matrix in early endosperm development (Li et al., 1999).

Expression of SSIIb is mostly confined to the photosynthetic tissues and its specific role in leaf starch biosynthesis is not clear (Harn et al., 1998; Morell et al., 2003).

The significant role of SSIIa in synthesizing intermediate glucan chains and maintaining the crystalline structure of amylopectin has been well demonstrated through mutant studies in algae, monocots and dicots (Fontaine et al., 1993; Craig et al., 1998;

Morell et al., 2003; Zhang et al., 2004; Shimbata et al., 2005). The importance of SSIIa in the biosynthesis of starch has been discussed previously (see section 1.5). Loss of SSII

60 activity in the starch synthesising tissues of several plant species results in significantly reduced amounts of starch and major alterations in chain length distribution, amylose: amylopectin ratio and in the structure and morphology of starch granules (Kramer and

Whistler, 1949; Campbell et al., 1994; Craig et al., 1998; Yamamori et al., 2000; Perera et al., 2001; Umemoto et al., 2002; Morell et al., 2003). Moreover SSIIa mutations have been shown to have pleotropic effects on other starch biosynthetic enzymes specifically

SSI, SBEIIa and SBEIIb (Yamamori et al., 2000; Morell et al., 2003).

Previous studies have shown that starch biosynthetic enzymes including SSI (Mu et al., 1994; 2001), SSIIa (Zhang et al., 2004) and SBEIIb (Mu-Foster et al., 1996) exist as both soluble proteins in the amyloplast stroma and as granule- associated proteins even though it is unclear whether the stromal form, the granule associated form or both forms are physiologically important for starch biosynthesis (Grimaud et al., 2008). In the barley sex6 mutant, loss of SSIIa activity abolished the binding of SSI, SBEIIa and SBEIIb to the starch granule, even though these proteins were normally expressed in the soluble fraction (Morell et al., 2003). Utilizing genetic, immunological and proteomic approaches to comprehensively explore the proteome and phosphoproteome of maize starch granules,

Grimaud et al. (2008) identified the granule association of SSIII, SBEI, SBEIIa and SP along with previously identified proteins GBSS, SSI, SSIIa and SBEIIb. Mutations affecting the expression of starch biosynthetic proteins like SSIIa have been shown to have pleiotropic effects in eliminating specific internal granule –associated proteins

(Grimaud et al., 2008). Analysis of these mutants revealed that they displayed altered association of more than one protein to the starch granule, thereby affecting even proteins other than the product of the mutant gene (Grimaud et al., 2008). Studies with

61 developing wheat and maize endosperms have demonstrated that SSIIa is a critical component in the major functional protein complexes containing SSI and SBEs (Tetlow et al., 2004a; Tetlow et al., 2008; Hennen –Biewagen et al., 2008; 2009; Liu et al., 2009).

Similarly the maize su2- mutant (lacking SSIIa) exhibited decreased association of SSI,

SBEIIb and SSIII with the starch granule and increased levels of SSI in the amyloplast stroma, resulting from the loss of SSIIa in the mutant (Grimaud et al., 2008). It was previously proposed that either SSIIa could directly bind to other starch biosynthetic proteins and thus regulate their granule association or that the loss of SSIIa could alter the starch granule structure in a way that the general or specific protein binding to the granule is reduced (Morell et al., 2003; Grimaud et al., 2008).

Portraying the crucial role of maize SSIIa in trafficking SSI and SBEIIb into the granular matrix, in a study with an allelic variant of the maize sugary-2 mutant possessing a catalytically inactive SSIIa, Liu et al. (2012b) demonstrated that these enzymes viz., SSI, SSIIa and SBEIIb are partitioned into the granular matrix as a result of their physical interactions, and that SSIIa forms the central core of these protein complexes and facilitates this trimeric protein complex (SSI-SSIIa-SBEIIb) to become granule bound (Fig.2.1). Interestingly, the activities of SSs (SSI and SSIIa) and SBE in the su-2 trimeric complex were significantly reduced as compared to the wild type, due to the presence of catalytically inactive SSIIa in the mutant trimeric protein complex (Liu et al., 2012b). The altered physicochemical properties of these su-2 starch granules therefore arise from non-functional hetero-protein complexes, containing inactive SSIIa, reinforcing their importance in the structural organization of amylopectin (Liu et al.,

2012b). Further, the SS activity of this trimeric complex was enhanced by ATP in both

62 wild type and su-2 amyloplasts suggesting the possibility of the post- translational modification of SSIIa and other participating proteins of this hetero-complex by protein phosphorylation (Liu et al., 2012b).

Whilst the mechanisms underlying the physical associations of these enzymes to form functional heteromeric enzyme complexes remain largely unresolved, their formation appears to be dependent on the phosphorylation status of the individual enzymes participating in the protein complex (Tetlow et al., 2004a; Liu et al., 2009). The first indication of post- translational regulation of starch biosynthetic enzymes, including

SSIIa, by protein phosphorylation was reported in wheat endosperm (Tetlow et al.,

2004a). In developing wheat endosperm, Tetlow et al., (2004a) identified that granule bound SSIIa was phosphorylated, along with starch branching enzymes SBEIIa and

SBEIIb, when incubated with -32P-ATP. It was also found that protein phosphorylation had a significant effect on the catalytic activity of starch branching enzyme isoforms

(Tetlow et al., 2004a). Grimaud et al. (2008) observed that granule-association of starch biosynthetic enzymes GBSS, SBEIIb and SP in maize endosperm is regulated by protein phosphorylation. Analysis of the starch granule phosphor proteome using phospho- protein specific dye ProQ Diamond (Steinberg et al., 2003) revealed that granule bound

GBSS, SBEIIb and SP were phosphorylated (Grimaud et al., 2008). They also suggested that, the fact that granule-bound SSIIa did not stain with ProQ Diamond in their study did not rule out the possibility of it being phosphorylated, since ProQ Diamond staining is comparatively less sensitive compared to radioactive labelling. It was also observed that phosphorylation of GBSS seemed to be affected by mutations in other starch biosynthetic enzymes, specifically SSIIa as indicated by the intensity of ProQ Diamond staining. The

GBSS phosphorylation signal upon ProQ Diamond staining was stronger in the mutant lines lacking SSIIa and weaker and more diffuse in the lines lacking SBEIIb (Grimaud et al., 2008). These observations reinforce the physiological significance of protein phosphorylation-dependent, protein-protein interactions between starch biosynthetic enzymes in the granular matrix facilitating the synthesis of crystalline amylopectin.

A B

Figure 2.1: Proposed model (Liu et al., 2012b) demonstrating the critical role of SSIIa in the granule deposition of the major amylopectin synthesizing enzymes SSI, SSIIa and SBEIIb in maize amyloplasts. (A) In wild type amyloplasts, the trimeric protein complex consisting of SSI, SSIIa and SBEIIb is directed towards the granule matrix and becomes granule bound. This protein complex is hypothesised to synthesize the amylopectin clusters. The central core of this protein complex, SSIIa directs itself and the other interacting proteins, SSI and SBEIIb, into the granule matrix through a glucan binding domain whose exact location is still unidentified. (B) In sugary 2 amyloplasts possessing a catalytically inactive SSIIa with a glucan binding domain lacking affinity towards amylopectin, the proteins in the trimeric complex are not trafficked towards the starch granule and therefore unable to synthesize the amylopectin clusters.

Phosphorylation dependent functional hetero-protein complexes containing SSIIa and other starch biosynthetic enzymes have been identified in wheat and maize amyloplasts (Tetlow et al., 2008; Hennen-Bierwagen et al., 2008 & 2009; Liu et al.,

2009). Phosphorylation of both stromal and granule associated wheat SSIIa was observed when the intact amyloplasts were phosphorylated in vitro with γ-[32-P] ATP (Tetlow et al., 2004a; 2008). The indirect evidence that SSIIa in maize endosperm could also be phosphorylated comes from the fact that it forms a major component of a high molecular weight complex with SBEIIb, SBEIIa and SSIII in a phosphorylation-dependent manner

(Hennen-Bierwagen et al., 2009). A study by Liu et al. (2009) using the maize amylose extender (ae) mutant lacking SBEIIb activity revealed that during amylopectin synthesis,

SSI and SSIIa form the central core of a functional protein complex with SBEIIb in a normal wild type maize endosperm and that the formation of this trimeric protein complex is also phosphorylation-dependent.

There is no direct evidence so far as to whether maize SSIIa is regulated by protein phosphorylation during starch biosynthesis. Further, the effect of such a post- translational modification on catalytic activity and kinetic properties has not been determined. The objective of this chapter is to investigate whether SSIIa in the developing maize endosperm is subject to post-translational protein phosphorylation.

Biochemical investigation of the phosphorylation of SSIIa, partially purified from maize amyloplast extracts, and also using recombinant maize SSIIa expressed in Escherichia coli, is described in the following sections.

2.2 Materials and Methods

2.2.1 Materials

2.2.1.1 Plant materials

The amyloplast extract samples for the various experiments were obtained from wild type field grown maize (Zea mays L). The cobs were collected at about 22 days after anthesis (DAA) from field grown wild type maize plants and were stored at 4°C until used for extracting amyloplasts. In order to prepare whole cell extracts later, kernels were collected from wild type maize plants and quick frozen in liquid nitrogen and stored at -

80°C for future use.

2.2.1.2 Chemicals

All the chemicals used in the experiments were obtained from Sigma Aldrich,

Canada unless otherwise specified.

2.2.2 Methods

2.2.2.1 Isolation of amyloplasts from maize endosperms

The synthesis of storage starch takes place in the amyloplasts of the developing endosperm. Hence amyloplast lysates served as the source of native starch biosynthetic proteins (including the putative protein kinases) and the protein complexes that were investigated in this study. Amyloplasts were extracted from kernels harvested at 22 DAA from wild-type maize plants. The extracted amyloplasts were purified to remove any unwanted cell debris and contaminating proteins from the maize whole cell lysates.

Purification of amyloplasts was performed as described by Liu et al. (2009).

Approximately 100g of endosperms were separated from the developing maize kernels using a small 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′-ethanesulphonic acid (HEPES)/KOH, pH 7.5, containing 0.8 M sorbitol, 1 mM KCl, 2 mM MgCl2, and 1 mM Na2-EDTA) in a petri dish kept on ice until a finely chopped uniform creamy solution was obtained. This creamy solution of whole cell extract was then filtered through four layers of Mira-cloth (Cal Biochem) soaked in the same extraction buffer.

The filtrate (5-10ml) was then carefully layered onto 15 mL of 3% (w/v) Histodenz

(Nicodenze, dissolved the in amyloplast extraction buffer) and centrifuged at 100 g at

4°C for 20 min. The supernatant was carefully discarded and the precipitated pellet with intact amyloplasts was ruptured by mixing and vortexing 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 plant protease inhibitor cocktail (Plant

Protease Arrest™[100X] , G Biosciences) (10μl per 1 mL buffer). The amyloplast extract mixture was transferred into micro-centrifuge tubes and centrifuged at 13,000rpm, at 4°C for 5 min to precipitate starch. The supernatant was quick frozen in liquid nitrogen and stored in -80°C for further use. These preserved amyloplast lysates were ultra-centrifuged

(Beckman Coulter Optima-Max–XP ultracentrifuge (at 25 psi)) at 100,000 rpm for 15 min prior to use to remove the plastidial membranes.

2.2.2.2 Preparation of maize whole cell extracts

Maize whole cell extracts were prepared following the procedure described by

Tetlow et al. (2003) for wheat. About 10 g of endosperm tissue was quick- frozen in

68 liquid nitrogen and immediately ground into a fine powder in a chilled mortar and pestle kept on ice. This frozen powdered endosperm mixture was then mixed with ice-cold rupturing buffer (composition same as the rupturing buffer used in amyloplast purification) and a plant protease inhibitor cocktail (Plant Protease Arrest™ [100X], G

Biosciences) (10μl per 1 mL buffer). After mixing, the mixture was allowed to stand on ice for 5 min followed by centrifugation at 13,000rpm for 5 min at 4°C. The supernatant was collected and ultra-centrifuged at 100,000 g for 15 min in a Beckman Coulter

Optima-Max–XP ultracentrifuge (at 25 psi) to remove plastidial membranes and other particulate materials.

2.2.2.3 Isolation of starch granule bound proteins

Granule bound proteins were isolated following the method described by Tetlow et al. (2004a). After extracting the amyloplasts and separating the soluble protein fractions by centrifugation (see section 2.2.2.1), the remaining pellets (approximately 1g) were re-suspended 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 at 4°C. Washing was repeated 8 times.

The pellet was then washed three times with 1 mL acetone followed by three washes of

1mL 2% (w/v) SDS and centrifuged at 3000g for 2 min at 4°C. Starch granule bound proteins were extracted by boiling the washed starch in 1x SDS buffer (62.5 mM Tris-

HCl, pH 6.8, 2% (w/v) SDS and 5mM DTT) for 5 min. at 90°C. The boiled sample was cooled and centrifuged at 13,000rpm for 10 min and supernatant separated by SDS-

PAGE.

For dephosphorylation experiments, the supernatant was collected and subjected to overnight dialysis into a buffer containing 62.5 mM Tris-HCl, pH 6.8, 5mM DTT and

10mM MgCl2 using GE Dialysis Kit (GE- HealthCare – Life Sciences). These samples were then used for dephosphorylation experiments by treating them with 40U of soluble alkaline phosphatase (APase) following the protocol given in section 2.2.5.1. After APase treatment, the samples were boiled 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 90°C. The boiled samples were cooled and centrifuged at 13, 000 rpm for 5 min and supernatant was separated by SDS-PAGE.

2.2.3 Proteomic analysis

2.2.3.1 Quantification of proteins

Protein concentration in the samples was determined according to the method developed by Bradford using the Bio-Rad protein assay kit following the manufacturer’s instructions using bovine serum albumin (BSA) as the standard (Bradford, 1976).

2.2.3.2 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

SDS-PAGE was performed following the method developed by Laemmli (1970) using a Mini-Protean III Vertical Electrophoresis System (Bio-Rad) according to the manufacturer’s instructions. Protein samples were prepared by boiling for 5 minutes in

SDS 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) at 90°C and were separated by SDS-PAGE on 10% polyacrylamide gels prepared by using a Bio- Rad Model 485

Gradient Former. The compositions of the stacking gel and the separation gel are shown

70 in Table 2.1. The gel was run in SDS running buffer (0.25M Tris, 192 mM Glycine, 0.4%

SDS) at 120V for 1.5hr.

Table 2.1: Composition of the stacking and resolving gels used for SDS-PAGE

Stock Solution Composition of SDS PAGE gel (10ml)

Stacking gel (ml) Resolving gel (ml) (5% acrylamide) (10% acrylamide)

ProtoGel® Acrylamide 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 (APS) 0.112 0.1 Distilled water 7.0 3.8 Tetramethylethylenediamine (TEMED) 0.008 0.01

2.2.3.3 Coomassie blue staining

After electrophoresis, the 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 overnight and destained for 2-3hrs in 42% [v/v] methanol/ 18% [v/v] acetic acid, till the protein bands become clearly visible. The gels were then washed in distilled water and observed.

2.2.4 Immunological techniques

2.2.4.1 Preparation of peptides and antisera

Polyclonal rabbit antisera targeted to maize SSIIa, SSI, SBEI, SBEIIa, SBEIIb, SP and SSIV were raised against synthetic peptides prepared commercially (Anaspec) The specific peptide sequences used to generate the various antibodies are given in Table 2.2.

Table 2.2: The synthetic peptide sequences derived from the amino acid sequences of various starch biosynthetic enzyme isoforms in maize. The sequence location and the Gene bank accession numbers are given below.

Enzyme Peptide sequence Location in the Gene bank full length sequence accession no:

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 SP YSYDELMGSLEGNEGYGRADYFLV 917–943 AAS33176

2.2.4.2 Purification of polyclonal maize antibodies

Maize polyclonal antibodies were purified from crude rabbit antisera using prepared peptide affinity columns following the method described by Liu et al. (2009).

To make a 1 mL peptide affinity 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). Two mL sulpho-link resin slurry (Pierce) was washed with 1 mL Tris-HCl, pH 8.5 for 6 times. The dissolved peptide was then added to 1 mL washed resin in a Falcon tube and incubated on a rotor for 15min. at room temperature and for additional 30 min. without rotating. The peptide-resin mixture was then added to a Bio-Rad Poly-Prep® Chromatographic column

(9cm high, 2mL bed volume (0.8 x 4cm) 10ml reservoir volume) and 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 mixed with 3 mL of

PBS /0.01% [w/v] sodium azide were applied to the column and mixed on a rotator overnight at 4°C. The next day, 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, and 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 content measured. The column was neutralized by adding 10 mL of 10 mM Tris-

HCl pH 7.8/0.05% [w/v] sodium azide and stored at 4°C.

2.2.4.3 Immunoblot analysis

Following gel electrophoresis proteins in the polyacrylamide gels were transferred to nitrocellulose membranes (Bio-Rad) using a Mini Trans-Blot® Electrophoretic

Transfer Cell (Bio-Rad) following the manufacturer’s instructions. The transfer buffer used for the western blot transfer contained 1X running buffer (see section 2.2.3.3), 20% methanol (Fischer-Scientific) and 70% distilled water. After transfer, the membrane was blocked with 1.5% bovine serum albumen (BSA) in 1X TBS buffer [0.2 M Tris Base,

1.5M NaCl; pH 7.2-7.4 with HCl] and incubated overnight in diluted antibodies using the methods described by (Harlow and Lane, 1999). The anti-maize antibodies used in the immunoblot analyses were diluted in 1.5% BSA in 1X TBS buffer as follows 1:800 for

SSIIa and SSI, 1:1000 for SBEI and SBEIIb and 1:500 for SP, SSIII 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 solution (BCIP/NBT) (Invitrogen).

2.2.4.4 Immunopurification of SSIIa from maize amyloplast lysates

SSIIa from wild type maize amyloplasts was immunopurified using peptide- specific anti maize SSIIa antibodies following the method described by Liu et al.

(2012b). Purified maize SSIIa antibodies were added (to a final concentration of 30 µg

/mL) to 1ml of maize amyloplast lysates (1-1.5mg/mL) and incubated for an hour on a rotator at room temperature. SSIIa was immunoprecipitated by adding 80-100 μL of 50%

(w/v) Protein A-Sepharose slurry. The Protein A-Sepharose slurry was made by adding the phosphate buffer saline PBS, (137 mM NaCl, 10 mM Na2HPO4, 2.7 mM KCl, and 1.8 mM KH2PO4, pH7.4) to the Protein A-Sepharose beads and incubated for 1hr at room temperature. Protein A-Sepharose- antibody /protein complex was then centrifuged at

100 g for 2 min at 4°C in a refrigerated micro centrifuge. The supernatant was collected and denatured by boiling with the SDS loading buffer to use as an indicator of the immunoprecipitation efficiency. The remaining pellet was washed five times (1.3mL each) with PBS containing 1M NaCl followed by three washes with 10 mM (4-(2- hydroxyethyl)-1-piperazineethanesulfonic acid ) HEPES/NaOH, pH 7.5 (at 100 g, 2 min centrifugation). The immunoprecipitated pellet containing Protein A-Sepharose- antibody/protein complex was boiled in 2X SDS loading buffer for 8 min. The protein bound to the sepharose beads as well as the supernatant were analysed by SDS-PAGE and immunoblotted with peptide specific antibodies for SSIIa and other SSs and SBEs to check the efficiency of immunopurification.

2.2.5 Detection of phosphorylation of SSIIa in maize endosperm

2.2.5.1 Phosphorylation and dephosphorylation of SSIIa in maize amyloplast stroma

To detect in vitro phosphorylation of native maize SSIIa, 1mL of amyloplast lysate/crude extract (22 DAA) (protein content 1-1.5mg/mL) was incubated with 1 mM

ATP (for 40 min at room temperature) and a phosphatase inhibitor cocktail Phosphatase

Arrest TM (G Biosciences; contents given in section 3.3.6) (10 μL/ 1mL lysate) to the latter in order to minimize dephosphorylation of protein by native phosphatases present in the amyloplast lysates. Addition of ATP is presumed essential for the activity of protein kinases present in the endosperm in order to catalyze protein phosphorylation. For in vitro dephosphorylation, amyloplast lysates were treated with either 30-40 units of soluble alkaline phosphatase (recombinant APase from E. coli. in 3.2 M ammonium sulphate containing 1 mM MgCl2 and 0.1 mM ZnSO4 from Megazyme) or 100 units of insoluble alkaline phosphatase conjugated to agarose beads (APase, insoluble form suspension in (NH4)SO4). All samples, including the control, included 10mM Mg Cl2,

5mM DTT and plant protease inhibitor cocktail (Plant Protease Arrest™ [100X], G

Biosciences) (10μL per 1 mL lysates). Amyloplast lysates that were not treated with ATP and/or APase served as controls. Rupturing buffer was added to balance the final volumes of each assay (500 µL to 2 mL). All treatments were conducted in triplicate.

2.2.5.2 Phos-tagTM phosphate affinity acrylamide gel electrophoresis

In vitro phosphorylation of native SSIIa in maize amyloplasts was detected by using affinity-based Mn2+-Phos-tagTM (Wako chemicals Ltd.) poly-acrylamide electrophoresis according to the method described by Kinoshita et al. (2006) with some modifications. 50 µM of Phos-Tag affinity ligand (Phos-tagTM AAL) was incorporated into a 10% SDS-PAGE gel. The di-nuclear metal complex (Mn2+) - Phos-tag, acts as a selective phosphate-binding tag molecule and retards the movement of the phosphorylated forms of proteins in an SDS-PAG compared to the mobility of dephosphorylated forms (Kinoshita et al., 2006). Stromal SSIIa from amyloplast lysates pre-treated with ATP (1mM) or insoluble alkaline phosphatase (APase) (100U) and the granule bound SSIIa were separated by 10% SDS-PAGE containing 50µM Phos-tag ligand, followed by immuno-blot analysis using SSIIa specific antibodies. Several trials were conducted to optimize the conditions of Phos-tag gel electrophoresis and improve separation of the phosphorylated and dephosphorylated forms of the protein. Trials included trying various ligand concentrations (25-100µM), duration of electrophoresis (6-

24h) and electric current (0.01 & 0.04A). As a result of optimization, Phos-tag gels were run at 0.04A for 6-7hrs at room temperature followed by immunoblot at 30V for 2.5h.

Following electrophoresis, Mn2+ was removed from the gel by washing it with transfer

76 buffer (20% of methanol in 25 mM Tris and 192 mM glycine running buffer) containing

1 mmol/L EDTA for 10 min, followed by further washing in transfer buffer without

EDTA for 10 min, before western blotting. All experiments were replicated at least three times. To compare and analyze the mobility shift of the phosphorylated form of the protein, the same samples were run on a standard 10% SDS-PAGE lacking Phos-tag, (as a control) and immuno-probed with anti-SSIIa specific antibodies.

The compositions of the required stock solutions and the Phos-tag gel prepared (Table

2.3) are given below.

Stock Solutions

1. 5.0 mmol/L Phos-tagTM AAL-107 (Wako chemicals Ltd.) solution- 10mg of Phos- tagTM AAL-107 was mixed with 0.10 mL (3% v/v) MeOH and 3.2 mL distilled water in a rotator overnight in dark at 4°C. This oily product was aliquoted in 0.5mL in micro- centrifuge tubes (Eppendorf) stored in dark at 4°C until use.

2. 10 mmol/L MnCl2 solution - 0.10 g MnCl2 4(H2O) (FW: 198) was dissolved in in 50 mL of distilled water and stored at 4°C until use.

Table 2.3: The composition of Phos-Tag TM phosphate affinity acrylamide gel

Composition of Phos-Tag TM SDS PAGE gel Stock Solution Stacking gel (for 3ml) Resolving gel ( for 7ml) (5% acrylamide) (10% acrylamide)

(50µM Phos-Tag TM

Acrylamide AAL-107)

30% (w/v) Acrylamide solution 0.50 mL 2.55mL 1.5 mol/L Tris/HCl solution, pH 8.8 - 1.95mL 0.5M Tris (pH 6.8) 0.38 mL - 5 mmol/L Phos-tag AAL solution - 50µL

10 mmol/L MnCl2 solution - 50µL 10% (w/v) SDS solution 0.03mL 75µL 10% (w/v) APS 0.03mL 0.1mL Distilled water 2.1mL 2.85mL TEMED 2µL 7.5µL

2.2.5.3 Pro-Q diamond phospho-protein gel staining

In vitro phosphorylation of native SSIIa in maize amyloplasts was also detected by utilizing a method for selectively staining phospho-proteins in SDS-PAGE gels. The

Pro-Q® Diamond phosphoprotein gel staining (Invitrogen) is a simple staining system that allows direct, in-gel detection of phosphate groups attached to tyrosine, serine, or threonine residues of phosphorylated proteins (Steinberg et al.,2003). SSIIa was immunopurified (section 2.2.4.4) from maize amyloplast stroma pre-treated with ATP

(1mM) or alkaline phosphatase (APase) (100U) and was separated by 10% SDS-PAGE.

Pro-Q® Diamond phosphoprotein gel staining was conducted following the standard protocol for staining mini-gels given in the manufacturer’s manual with slight modifications. The molecular weight of the protein in the gel was compared using the

Peppermint Stick™ phosphoprotein molecular weight standards (Invitrogen) provided by the manufacturer, along with the stain. After gel electrophoresis, the gel was fixed in

100mL of 50% methanol and 10 % acetic acid overnight and washed with three changes of 100mL deionized water for 10 min per wash with gentle agitation. The gel was then stained with Pro-Q® Diamond phosphoprotein gel stain (diluted two-fold with distilled water) for 1hr in dark with gentle agitation. For destaining the gel was immersed in 80–

100 mL of Pro-Q® Diamond phospho-protein destain solution with gentle agitation for 30 minutes at room temperature, protected from light. This step was repeated twice. Finally, the gel was washed twice with ultrapure water at room temperature, 5 minutes per wash, and visualized on a Typhoon 9200 trans-illuminator (Amersham Biosciences) using excitation at 532–560 nm. To identify the SSIIa protein, the protein samples were also

79 run on SDS-PAGE gels and Coomassie stained and immunoblotted with anti-SSIIa antibodies.

2.2.5.4. In vitro phosphorylation of maize amyloplasts using γ- [32-P] ATP

An additional approach to investigate the in vitro phosphorylation of SSIIa in maize amyloplast stroma involved radiolabelling of protein with the terminal phosphate group of ATP. Amyloplast lysate (22 DAA) (protein content 1-1.5mg/mL) was incubated with 1 mM [γ-32P] ATP (specific activity of 10Ci/mmol; 2mCi/mL; 250µCi per bottle) on a rotator for an hour at room temperature in the presence of 10mM Mg Cl2, 5mM DTT and plant protease inhibitor cocktail (Plant Protease Arrest™ [100X], G Biosciences)

(10μL per 1 mL lysates). SSIIa was then immunopurified using anti-SSIIa specific antibodies bound to Protein-A sepharose beads following the procedure described in the section 2.2.4.4. Non-specific proteins bound to the Protein-A-Sepharose beads/SSIIa antibody complex were removed by washing the centrifuged pellet five times, each with

1.5 mL PBS containing 1M NaCl, followed by three washes with 10 mM HEPES/NaOH, pH 7.5 buffer (at 100 g, 1 min centrifugation). The immunoprecipitated pellet containing

SSIIa was boiled in 2X SDS loading buffer for 8 min and separated by SDS-PAGE.

2.2.5.5 Autoradiography

The immunopurified SSIIa from maize amyloplasts lysates after radiolabelling with [γ-32P] ATP was separated by SDS-PAGE. After electrophoresis, the gel was immunoblotted and analyzed using SSIIa specific antibodies. The immunoblot was exposed to X-ray film for two weeks at -80°C. Phosphorylation of SSIIa was detected by

80 auto radiographic analysis of the X-ray film after aligning it with the developed immunoblot, which was probed with anti-SSIIa specific antibodies.

2.2.5.6 Expression and purification of recombinant maize SSIIa in Escherichia coli

Recombinant plasmid, containing maize full length SSIIa cDNA cloned into the pET-29b expression vector (Novagen) was kindly provided by Dr. Amina Makhmudova,

University of Guelph. The pET system has a 15 amino acid S-tag at the N-terminus

(utilized for immobilization on S-protein agarose beads for purification steps and interaction reactions), a thrombin digestion site (to remove the overexpressed protein from the tag) and a strong T7 promoter along with all necessary transcriptional and translational control sequences (Fig.2.2) The recombinant plasmid was transformed into

E coli strain BL21-CodonPlus (DE3)-RP (Stratagene). An individual colony picked up from an overnight culture was inoculated into 5mL of LB broth (Luria Bertani) with

50µM kanamycin and grown at 37°C overnight. The overnight cultures were sub-cultured in 2L of LB media without the antibiotic until the cell density reached 0.4-0.5 at D600.

Expression of the recombinant protein was induced by IPTG (isopropyl β-D-1- thiogalactopyranoside) to a final concentration of 1 mM. The cultures were further grown for 6h by shaking at 250rpm at 16°C. E. coli cells were then collected by centrifugation

(at 13,000 rpm at for 20 min.) and lysed by incubating with Bug Buster Protein

Extraction Reagent (Novagen) in the presence of plant protease inhibitors (Plant Protease

Arrest™ [100X], G Biosciences) (10μl per 1 mL buffer) for 20 min at room temperature on a rotator. After centrifuging this lysed mixture at 13,000 rpm for 20 min at 4°C, the soluble fraction containing recombinant SSIIa (supernatant) was collected and the total protein content was estimated. The expression level of the protein was tested by running

81 both the induced and un-induced (control) soluble fractions from E coli cell lysates (the resulting E.coli cellular components after disrupting the cell membranes with lysozyme) on an SDS-PAG followed by Coomassie staining and immunoblotting with anti S-tag specific antibodies. The activity of the recombinant protein was assessed using 14C- labelled ADP-glucose as substrate (section 3.2.7.4).

Figure 2.2 Sequence structure of Novagen pET29b expression vector containing a 15 amino acid S-tag on the N-terminus with a thrombin digestion site, and a T7 promoter (derived from www. snapgene.com). Restriction enzyme digestion sites are shown in bold. Abbreviations shown in the large arrows are; lacI- lac promoter, ori- origin of replication, KanR- kanamycin resistance, rop- repressor of primer sequence. 82

2.2.5.7 Immobilization of recombinant maize SSIIa on S-protein agarose beads and pull- down assay

The S-tagged recombinant SSIIa protein was immobilized to S-protein agarose beads (Novagen) following the procedure described by Liu et al. (2009) with some modifications. Approximately 500µL of the recombinant SSIIa soluble fraction (total protein concentration of 3mg/mL) was incubated with 300µL of S-protein agarose bead slurry on a rotator at 4°C overnight. The sample was centrifuged at 500g for 5 min in a micro centrifuge at 4°C, and the supernatant discarded. The slurry containing recombinant protein bound to S agarose beads was transferred to a 10 mL Bio-Rad Poly

Prep® chromatography column and thoroughly washed with 200mL of buffer (20 mM

Tris-HCl pH 7.5, 150 mM NaCl, 0.1% (v/v) Triton X-100 and 5mM DTT). The washed

S-agarose bead- protein pellet was then transferred back to a micro-centrifuge tube and centrifuged at 4°C for 5 min at 500g. After removing the supernatant, the pellet was boiled in 100 μL of 20mM Tris-HCl pH 7.5 and 2X 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 7 min at 95°C. A sample without recombinant SSIIa protein lysate served as a control. The recombinant SSIIa in the boiled samples was analyzed by SDS-PAGE and immunoblotting with anti -SSIIa and anti-S-tag specific antibodies.

2.2.5.8 In vitro phosphorylation of S- tag immobilized recombinant SSIIa using γ- [32-P] ATP and autoradiography

The S-tagged recombinant SSIIa protein was immobilized to S-protein agarose beads (Novagen) as described above. One mL of recombinant SSIIa soluble fraction was

83 incubated with 300µL of S-protein agarose bead slurry on a rotator at 4°C overnight. The sample was centrifuged at 500g for 5 min in a micro centrifuge at 4°C and the supernatant discarded. The protein -agarose bead pellet was washed at least five times with washing buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% (v/v) Triton X-100 and 5mM DTT) , 1-1.5mL each time to avoid non-specific binding of contaminating proteins. The immobilized recombinant SSIIa pellet was mixed with 300µL of maize amyloplast extract (protein concentration of 1-1.5mg/mL) prepared in rupturing buffer

(section 2.2.2.1), pH 7.8, as a source of protein kinases and incubated with 1 mM [γ-32P]

ATP (specific activity of 10Ci/mmol) in the presence of 10mM Mg Cl2, 5mM DTT, plant protease inhibitor cocktail (Plant Protease Arrest™ [100X], G Biosciences) (10μl per 1 mL lysates) and 25mM of tetra-sodium pyrophosphate ( to prevent dephosphorylation by native phosphatases in the amyloplast lysate) on a rotator for one hour at room temperature. Following incubation with ATP, the reaction suspension was transferred to a

10 mL Bio-Rad Poly Prep® chromatography column and the S-agarose bead – protein pellet was washed with washing buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1%

(v/v) Triton X-100 and 5mM DTT) as explained in the previous section and boiled in 100

μL of 20mM Tris-HCl pH 7.5 and 2X SDS loading buffer (0.31M Tris-HCl pH 6.75,

25% (v/v) 2-mercaptoethanol, 10% (w/v) SDS, 50% (v/v) glycerol, 0.005% (w/v)

Bromophenol Blue) for 7 min at 95°C. The recombinant SSIIa in the boiled, protein-bead sample was separated by SDS-PAGE and identified by immunoblotting with anti- S-tag antibodies. The immunoblot was exposed to X-ray film for two weeks at -80°C.

Phosphorylation of recombinant SSIIa was detected by the auto radiographic analysis of the X-ray film after aligning it with the developed immunoblot, which was probed with

84 anti-S-tag antibodies. A control sample without maize amyloplast extract (rupturing buffer instead) was also run to test whether the phosphorylation of the recombinant protein was dependent on native protein kinases from the amyloplast extract.

2.3 Results

2.3.1. Detection of starch synthase IIa in the amyloplast stroma and in the starch granule

To detect the subcellular localization of SSIIa in the maize endosperm, proteins from wild-type maize plants (22 DAA) were extracted from amyloplast stroma, from starch granules separated from amyloplasts and within whole cell extracts of the endosperm. SSIIa was clearly detected in the amyloplast stroma, starch granule and in whole cell lysates (Fig. 2.3). In the immunoblot probed with anti-SSIIa antibody, SSIIa protein was detected at an approximate molecular weight of 86 kDa (Fig. 2.3.). SSIIa was found distributed between the amyloplast stroma and in the granular matrix.

Figure 2.3: The presence of SSIIa in maize seed whole cell extract, amyloplast stroma and in the starch granule. Western blot was probed with anti SSIIa antibodies. Equal amounts of proteins (30µg) were run on SDS-PAGE. 85

2.3.2 Optimization of Phos-tag TM phospho- protein affinity gel electrophoresis

In order to determine the concentration of Phos-tag affinity ligand to be incorporated into the SDS poly-acrylamide gel, and the running conditions required to optimize the separation of phosphorylated and dephosphorylated forms of SSIIa, several trials were run under varying ligand concentrations and running conditions. Increasing the concentration of the Phos-tag TM ligand beyond a certain concentration (75 µM) resulted in a distorted curvature of the protein marker bands (100 µM) making it difficult to relate the molecular weight of the protein with the protein marker, e.g. see the MW marker in Figure 2.4 C. The reason for the distortion of the band is unclear. A Phos-tag

TM ligand concentration of 50µM (final) produced optimal separation of the ATP and

APase treated forms of SSIIa with minimum “curvature” of the protein bands migrating through the gels (Fig 2.4). Leaving a lane between the protein marker lane and the sample lane also helped to reduce this curvature of the protein bands.

Differentially migrating forms of SSIIa separated well when the gel was run on constant current of 0.04A for 6-7 hrs, and by changing the running buffer at least once during electrophoresis. Running gels at lower current (as low as 0.01A) for longer periods (12 -24hrs) did not improve separation (Fig. 2.5). Following electrophoresis, the gel was soaked in a general transfer buffer (section 2.2.4.3) containing 1 mmol/L EDTA for 10 min and then in a general transfer buffer without EDTA for 10 min with gentle agitation. This was necessary to eliminate manganese ions from the gel that might interfere with the electro blotting. These steps enhanced the transfer efficiency of the protein onto the nitrocellulose membrane. Results from the optimization experiments

86 showed that electrophoresing the ATP- and APase-treated forms of SSIIa on SDS gels containing 50µM Phos-tag TM phospho protein affinity ligand at 0.04A for 6-7hrs resulted in better separation of two distinct, migrating forms of SSIIa (see Fig 2.6A).

Figure 2.4: Optimization of Phos-tag TM phospho protein affinity gel electrophoresis. SSIIa from wild type maize amyloplast lysates pretreated with 1mM ATP or 40U (soluble) or 100U (insoluble) APase for 40min at room temperature was separated in denaturing SDS gels containing (A) 25µM (B) 50µM and (C) 100µM Phos-tag TM phospho protein affinity ligand. To test the reversibility of the effects of ATP and APase on SSIIa mobility, stromal proteins were also incubated first with 100U insoluble APase for 40 min and then incubated with 1mM ATP for 40 min for re-phosphorylation (APase, ATP). After incubation, the insoluble APase present in the samples was removed by centrifugation. Following Phos-tag gel electrophoresis at 0.04A for 6-7hrs, SSIIa was identified by probing the western blots with anti-SSIIa specific antibodies. The APase protein present in the samples treated with soluble APase is indicated by a red arrow.

25µM 50µM A B

Figure 2.5: Mobility of SSIIa in Phos-tag acrylamide gel electrophoresis. SSIIa from wild type maize amyloplast lysates pretreated with 1mM ATP or 40U (soluble) or 100U (insoluble) APase for 40 min was separated in denaturing SDS TM gels containing 50µM final concentration of Phos-tag phospho protein affinity ligand at 0.04A for 6hrs (left) and at 0.01A for 20hrs (right). To test the reversibility of the effects of ATP and APase on SSIIa mobility, stromal proteins were also incubated first with 100U insoluble APase for 40 min and then incubated with 1mM ATP for 40 min for re-phosphorylation (last lane in the blots). After incubation, the insoluble APase present in the samples was removed by centrifugation. Following Phos-tag gel electrophoresis, SSIIa was identified by probing the western blots with anti-SSIIa specific antibodies.

2.3.3 Detection of in vitro phosphorylation of SSIIa in the amyloplast stroma using Phos-tagTM phospho- protein affinity gel electrophoresis

The phosphorylation status of maize SSIIa in the amyloplast stroma was determined by measuring the differential mobility of SSIIa protein following incubation with ATP or APase and SDS-PAGE on gels containing the Mn2+–Phos-tagTM phospho protein affinity ligand (Fig. 2.6). Results from the Mn2+-Phos-tagTM phospho protein affinity gel electrophoresis showed that SSIIa in the maize amyloplast stroma incubated with ATP was retarded relative to the untreated and APase-treated controls (Fig. 2.6A) suggesting that these SS isoforms could be phosphorylated in the wild-type maize amyloplast stroma. When stromal SSIIa was first treated with immobilized APase followed by incubation with ATP, the protein was again retarded as compared to the

APase-only treated samples (last lane in Fig. 2.6 A). Following SDS-PAGE lacking the affinity ligand, the untreated, ATP and APase treatments had no effect on the migration of SSIIa (Fig. 2.6 B).

Figure 2.6: Detection of in vitro phosphorylation of SSIIa in amyloplast stroma. SSIIa from wild type maize amyloplast lysates pretreated with 1mM ATP or 100U (insoluble) APase for 40 min at room temperature, was separated in (A) denaturing SDS gels

containing 50µM Phos-tag TM phospho protein affinity ligand and (B) in SDS gels lacking the ligand. To test the reversibility of the effects of ATP and APase on SSIIa mobility, stromal proteins were also incubated first with 100U insoluble APase for 40 min and then incubated with 1mM ATP for 40 min for re-phosphorylation (last lane in the blots). After incubation, the insoluble APase present in the samples was removed by centrifugation. Following Phos-tag gel electrophoresis at 0.04A for 6-7hrs, the differential mobility of the ATP- and APase- treated forms of SSIIa were identified by -P probing the western blots with anti-SSIIa specific antibodies.

2.3.4 Comparison of the phosphorylation status of maize SSIIa in amyloplast stroma and starch granule

The phospho-protein affinity based Mn2+–Phos-tagTM acrylamide gel

electrophoresis was used to detect and compare the phosphorylation status of native

maize SSIIa in the amyloplast stroma and in the granule matrix. The mobility of

untreated granule bound SSIIa was retarded compared to the untreated stromal SSIIa,

suggesting that SSIIa in the granule matrix is phosphorylated (Fig 2.7). Upon treatment

with 40U of soluble APase, the mobility of the solubilized, granule bound SSIIa migrated

to the same position as protein in the untreated stromal sample (Fig. 2.7) (note that the

90 non-specific bands picked up by the anti-SSIIa antibody in the APase (soluble) sample lane is due to APase- ammonium sulphate buffer in the reagent and not interfering proteins).

Figure 2.7: Phosphorylation state of SSIIa in amyloplast stroma and starch granules. Amyloplast stromal proteins and the granule bound proteins were isolated separately. SSIIa from amyloplast stroma was treated with 1 mM ATP or 40U of soluble APase

for 40 min. After extraction, the granule bound proteins were dialyzed overnight at 4°C into a buffer - Tris HCl, pH 6.5, with 5mM DTT and 10mM MgCl2 and then incubated with 40U of soluble APase for 40 min at room temperature. Protein samples (20-30µg of protein was loaded onto each lane) were separated by Phos-tag phospho protein affinity gel electrophoresis at 0.04A for 6-7hrs. The differential mobility of the ATP and APase treated forms of stromal SSIIa and the untreated and APase treated granule bound SSIIa was detected by probing the western blots with anti-SSIIa specific antibodies.

2.3.5 Detection of phosphorylation of maize SSIIa using Pro-Q diamond gel staining

Immunopurification of SSIIa from amyloplast lysate using peptide specific anti-

SSIIa antibodies provided a convenient method to purify the protein from other starch biosynthetic enzymes in the amyloplast stroma. The thorough washing of the protein – sepharose bead complex with high salt 1X PBS buffer (see 2.2.4.4) avoided the binding of other interacting starch biosynthetic enzymes from the beads. The SSIIa – sepharose bead protein complex was separated by SDS-PAGE and immunoblotted with antibodies specific to other proteins including SSI, SSIII, SSIV, SP and SBEs to confirm their absence on the beads (see Fig.3.15 in Chapter 3). In vitro phosphorylation of immunopurified SSIIa from maize amyloplast stroma (amyloplast stroma was pretreated with 1mM ATP and SSIIa was immunopurified) was detected by fluorescent staining of

SDS-PAGE gels with Pro-Q Diamond phospho-protein gel stain. Phosphorylated proteins appear intensively black in comparison with the background. ATP- treated immunopurified SSIIa appeared as a dark black protein band as compared to the untreated and APase treated controls when the SDS-PAGE gel was stained with Pro-Q

Diamond stain (Fig. 2.8 A). SSIIa was treated with ATP in the presence of amyloplast lysates, as the source of endogenous protein kinase(s). This result strongly indicated that

SSIIa in the stroma could be phosphorylated by protein kinases within the amyloplast.

Immunopurified SSIIa was also identified by Coomassie staining (Fig. 2.8 C) and immunoblotting with anti-SSIIa specific antibodies (Fig. 2.8 B). Figure 2.8 C suggests that the immuno affinity separation of SSIIa from amyloplast stroma resulted in a highly purified protein preparation (SSIIa) with very little contaminating protein.

Figure 2.8: In vitro phosphorylation of immunopurified SSIIa from wild type maize amyloplast stroma detected using Pro-Q Diamond phospho-protein gel staining. SSIIa was immunopurified from amyloplast lysates pretreated with 1mM ATP or 100U APase using anti- SSIIa antibodies. After incubation with ATP or APase (for 40min) followed by anti- SSIIa antibodies (for 1h), Protein-A -Sepharose beads were added into the mixture and incubated for one hour. The beads were then washed with 1X PBS with 1M NaCl as explained in section 2.2.4.4 to remove contaminating proteins, followed by SDS-PAGE. (A) Following electrophoresis the gel was stained with Pro-Q® Diamond stain and visualized on a Typhoon 9200 trans-illuminator (Amersham Biosciences) as described in section 2.2.5.3 (B) SSIIa was identified by immunoblotting with anti –SSIIa antibodies and (C) also by Coomassie staining of protein.

2.3.6 Detection of phosphorylation of maize SSIIa by γ- [32-P] ATP labeling and autoradiography

In vitro phosphorylation of SSIIa in the wild type maize amyloplast stroma was further examined by radiolabelling proteins with γ- [32-P] ATP followed by auto radiographic analysis. ATP-treated, immunopurified SSIIa from amyloplast lysates exhibited strong signals of 32P labeling when the immunoblots were exposed to auto- radiographic analysis (Fig. 2.9).

Figure 2.9: Auto radiography of immunopurified SSIIa from wild type maize amyloplast stroma following radiolabelling with γ- [32-P] ATP. Stromal proteins were incubated with 1mM γ- [32-P] ATP for 40 min at room temperature. SSIIa was immunopurified using anti-SSIIa specific antibodies followed by washing the Protein-A-Sepharose beads with 1X PBS with 1M NaCl (section 2.2.4.4). The protein -bead complex was separated by SDS-PAGE and SSIIa was identified by immunoblotting with anti-SSIIa specific antibodies. The autoradiograph was developed from the same immunoblot by incubating on an X-ray film in the dark

for two weeks at -80°C. Arrow indicates the SSIIa protein in both the immunoblot and autoradiograph in comparison with the standard protein marker.

2.3.7 The effect of protein kinase inhibitors on phosphorylation of SSIIa

To test the hypothesis that protein kinases are involved in the phosphorylation of

SSIIa, amyloplast lysates were incubated with known protein kinase inhibitors, FSBA (5- fluorosulfonylbenzoyl-5΄adenosine) (10mM) and K252a (staurosporine) (100µM). In order to optimize the concentration of ATP to be used to visualize any possible effect of protein kinase inhibitors, a range of ATP concentrations was used (sections 3.3.2 and

3.3.5 in the following chapter). 25μM ATP proved optimal in inducing a significant alteration in SSIIa mobility which was inhibited by FSBA and K252a. Therefore, amyloplast lysates pretreated with FSBA and K252a were then incubated with 25μM

ATP. The phosphorylation state of SSIIa in the amyloplast stroma was detected by Phos- tag gel electrophoresis followed by immuno-detection with anti-SSIIa antibodies. The

ATP-dependent mobility retardation of SSIIa in the Phos-tag gel was prevented by FSBA and K252a (Fig. 2.10) suggesting inhibition of the protein kinases that facilitate the in vitro phosphorylation of SSIIa in the amyloplast stroma. Even though the ATP treated and protein kinase inhibitors pre-treated- ATP treated forms of SSIIa migrated differently in the phos-tag gel, all protein bands including the protein marker were distorted as described previously (2.3.2).

Figure 2.10: Phosphorylation state of stromal SSIIa in maize amyloplast lysates is inhibited by protein kinase inhibitors. Stromal proteins from maize amyloplasts were incubated with known protein kinase inhibitors, FSBA (5-fluorosulfonylbenzoyl- 5´adenosine) (10mM) and K252a (staurosporine) (100µM) for 20 min at room temperature prior to the incubation with ATP (25ìM) for 30-40 min in the presence of 10mM Mg Cl2, 5mM DTT and plant protease inhibitor cocktail (Plant Protease Arrest™ [100X], G Biosciences) (10μl per 1 mL lysates). Proteins were separated by Phos-tag gel electrophoresis and SSIIa was identified by immunoblotting with anti- SSIIa specific antibodies. 95

2.3.8 Phosphorylation of recombinant maize SSIIa

2.3.8.1 Expression of recombinant SSIIa in E coli cell lysates

Recombinant plasmid incorporating the maize SSIIa cDNA, cloned to the pET-

29b (Novagen) expression vector was transformed into E coli strain BL21-CodonPlus

(DE3)-RP (Stratagene). Expression of the recombinant protein was induced by 1mM

IPTG as described in the section 2.2.5.6. The expression level of the recombinant SSIIa was qualitatively assessed by separating the proteins from the E coli cell lysates by SDS-

PAGE and comparing equal amounts of soluble proteins obtained from the induced and uninduced cell lysates by Coomassie staining, (Fig. 2.11A) as well as by immuno- detection with anti-S-tag specific antibodies (Fig. 2.11B). The IPTG-induced E coli cell lysates containing the recombinant plasmid showed higher level of SSIIa expression when compared to the uninduced E coli cell lysates. The immunoblot probed with anti-S tag antibodies confirmed the higher expression was due to the expressed S- tagged recombinant SSIIa protein (Fig 2.11). The catalytic activity of the recombinant SSIIa expressed in E coli cells was measured with ADP-(U-14C) glucose, using 0.3% maize amylopectin as the glucan primer (section 3.2.6). The soluble cell lysates from the induced cell cultures exhibited significantly higher activity (13.6 nmol ADP-(U-14C) glucose/hr/µg of protein) as compared to the cell lysates from the uninduced controls (2 nmol ADP-(U-14C) glucose/hr/µg of protein).

Figure 2.11: Expression of recombinant maize SSIIa in E coli. Expression of recombinant SSIIa in BL21-CodonPlus (DE3)-RP E coli cells was induced by 1mM IPTG. Following induction, the cultures were further grown and E. coli cells were collected by centrifugation as described in section 2.2.5.7. The harvested cells were lysed using Bug Buster Protein Extraction Reagent (Novagen) in the presence of plant protease inhibitors (2.2.5.7) and the lysed mixture was centrifuged as described in section 2.2.5.7 to yield soluble cell lysate (supernatant containing recombinant SSIIa) and inclusion bodies (the resulting precipitate after centrifugation which is an accumulation of overexpressed protein into insoluble aggregates). Soluble cell lysate containing recombinant SSIIa was collected and the total protein content was estimated. The expression level of the protein was tested by running both the induced and un-induced (control) soluble fractions (15-20µg) on an SDS-PAG and (A) the proteins were visualized by Coomassie staining. (B) Western blot of the same SDS-PAGE gel was probed with anti-S-tag specific antibodies. The induced SSIIa protein is shown by the arrows.

2.3.8.2 Immobilization of recombinant maize SSIIa on S-protein Agarose beads

The S-tagged recombinant SSIIa in soluble E coli cell lysates was immobilized to

S-protein agarose beads as described in section 2.2.5.7. The agarose beads were washed with 200mL of buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% (v/v) Triton X-100 and 5mM DTT) before electrophoresis. Recombinant SSIIa was detected with anti-S-tag

(Fig. 2.12A) and anti-SSIIa antibodies (Fig. 2.12B). Recombinant SSIIa was successfully bound to the S-protein agarose beads showing strong immuno-reactive bands (Fig 2.12).

Recombinant SSIIa protein (approx. 86 kDa + 1.7 kDa of the S-tag) carrying the 15 amino-acid, S-tag peptide (Lys-Glu-Thr-Ala-Ala-Ala-Lys-Phe-Glu-Arg-Gln-His-Met-

Asp-Ser; MW= 1.75 kDa), migrated less than amyloplast SSIIa (approx. 86 kDa). To check the specificity of protein binding to the S-protein agarose beads, a control lacking recombinant SSIIa was also electrophoresed and immunoblotted. No S-tagged protein bands were observed in the control lacking the recombinant protein (Fig. 2.12 A).

Figure 2.12: Immobilization of recombinant maize SSIIa to S- protein agarose beads. Approximately 1-1.5mg of S-tagged recombinant maize SSIIa was immobilized to S- protein agarose beads. Following overnight incubation, the sample was centrifuged at 500g for 5 min at 4°C and the supernatant was discarded. The bead- protein pellet was thoroughly washed to remove any non- specific binding of proteins from the soluble E coli cell lysate (section 2.2.5.8). The washed pellets (15-20µg protein loaded in each lane) were subjected to SDS- PAGE. Immobilized recombinant SSIIa was detected by immuno-probing the western blots with (A) anti-S-tag specific and (B) anti-SSIIa specific antibodies.

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2.3.8.3 In vitro phosphorylation of recombinant maize SSIIa using γ- [32-P] ATP labeling and autoradiography The S-tagged recombinant- SSIIa from soluble E coli cell lysates was immobilized to S-protein agarose beads. The protein-bead slurry was then incubated in

300-400µg of soluble protein from maize amyloplast lysates, as a source of protein kinase(s) with 1mM γ- [32-P] ATP for an hour as described in 2.2.5.8. Following incubation, 15-20 µg of protein was subject to SDS-PAGE followed by western blotting.

Immobilized recombinant SSIIa was detected by anti- S-tag specific antibody in samples incubated with amyloplast lysates and rupturing buffer (control) (Fig. 2.13 A).

Radiolabelling of recombinant SSIIa was detected following autoradiography of X-ray film (Fig. 2.13 A&B). Signals of 32P were absent in the samples lacking amyloplast lysates (lane 2) indicating that in vitro phosphorylation of recombinant SSIIa occurs only in the presence of amyloplast lysates.

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Figure 2.13: In vitro phosphorylation of recombinant maize SSIIa. S-tag immobilized recombinant SSIIa protein was incubated with 1mM γ- [32-P] ATP for 40 min at room temperature in the presence of 450-500 µg of soluble amyloplast protein. After incubation, the recombinant protein –bead complex was thoroughly washed as described in section 2.2.5.8 to remove non-specifically bound proteins. (A) The S-tag immobilized recombinant SSIIa was separated by SDS-PAGE and was identified by immunoblotting with anti S-tag specific antibodies. (B) Autoradiograph was developed from the same immunoblot following exposure to X-ray film, in the dark, for two weeks at -80°C. Arrows indicate SSIIa protein in both immunoblot and autoradiograph relative to standard protein markers.

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2.4 Discussion The primary focus of this chapter was to investigate the hypothesis that maize

(Zea mays L.) SSIIa is subject to post translational regulation by protein phosphorylation.

Various biochemical and proteomic strategies including immunopurification of SSIIa from amyloplast stroma, SDS-PAGE followed by Pro-Q® Diamond phospho-protein gel staining, Phos-tagTM phosphate affinity acrylamide gel electrophoresis, γ- [32-P] ATP labeling and autoradiography of native and recombinant maize SSIIa and SS zymogram analysis were used. Initially, the subcellular localization of SSIIa in a wild type maize endosperm was examined. In order to investigate whether SSIIa could be phosphorylated by endogenous protein kinase(s) and to elucidate the effects of phosphorylation on this enzyme, extraction of pure and detectable amounts of this protein from the plant tissue was essential. Earlier studies have experienced difficulty in purifying these SS isoforms from endosperm tissues because of their low abundance, instability and association with other starch biosynthetic enzymes (Imparl-Radosevich et al., 1999a &b). It was reported that in maize endosperm, major grain filling and starch accumulation occurs during the growth period from 9-24 DAA (Tsai and Nelson, 1969;Yu et al., 2001) and all the major starch biosynthetic enzymes in the amyloplasts are expressed and active during this period (Hennen-Bierwagen et al.,2008; Liu et al.,2009). Kernels were examined at 22

DAA and the results indicated that SSIIa is partitioned between the amyloplast stroma and the starch granular matrix (Fig. 2.3). Like other starch biosynthetic enzymes, SSI and

SBEIIb, SSIIa is proportioned between the amyloplast stromal region and nascent starch granule in developing cereal endosperm (Li et al., 1999). In some species such as pea

(Denyer and Smith, 1992), Arabidopsis (Zhao, unpublished) and developing wheat

103 endosperm (Li et al., 1999), SSIIa predominantly exists as a granule bound protein.

Studies with wheat and maize endosperms have also shown that the granule association of SSI and SBEIIb is mediated by SSIIa and that SSIIa forms the core of a major trimeric protein complex within the starch granule (Tetlow et al., 2008; Hennen-Bierwagen et al.,

2008; Liu et al., 2012b).

The observation that SSIIa forms a major component of a functional protein complex with other starch biosynthetic enzymes, the formation of which is regulated by protein phosphorylation (Tetlow et al., 2004 a &b; Tetlow et al., 2008; Hennen-

Bierwagen et al., 2008&2009; Liu et al., 2009; Liu et al., 2012a) indirectly suggests the possibility that SSIIa could also be phosphorylated. In a previous study in maize, Polack

(2009) provided preliminary evidence that recombinant maize SSIIa could be phosphorylated.

In order to phosphorylate and dephosphorylate SSIIa, maize amyloplast lysates or immunopurified SSIIa (with amyloplast lysates used as a source of protein kinases) were incubated with ATP or alkaline phosphatase (APase). Several techniques including phospho-protein affinity gel electrophoresis, phospho protein gel staining, immuno- detection and auto radiography were employed to investigate the phosphorylation of

SSIIa in wild type maize amyloplasts, and discussed below.

Phos-tag™ Acrylamide gel electrophoresis allows a simple and convenient means to detect phosphorylated and non-phosphorylated forms of proteins as different bands using Phos-tag™ SDS-PAGE (Kinoshita et al., 2006). During electrophoresis, the phosphorylated form of the protein migrates with its phosphate group bound to the

104 divalent metal ion (Mn2+) in Phos-tag™. As a result, the migration speed of the phosphorylated form is retarded compared to its non-phosphorylated counterpart, allowing the two forms to be separated (Fig.2.14).

Dephosphorylated protein

Figure 2.14: Principle and application of Phos-tag TM acrylamide gel electrophoresis. http://www.wako-chem.co.jp/english/labchem/product/life/phos-tag-aal- guidebook/index.htm

Following incubation of maize amyloplast lysates with ATP, stromal SSIIa was significantly retarded compared to the untreated and APase treated samples (Fig. 2.6A) consistent with in vitro phosphorylation of maize SSIIa in the amyloplast stroma.

However, stromal SSIIa in the untreated amyloplast lysates migrated similarly to the

APase-treated control, suggesting that SSIIa exist in a dephosphorylated form in the amyloplast stroma under the extraction conditions. Whether this reflects the native phosphorylation state of the protein in a developing endosperm is unclear, as the turnover of phosphate on the protein is likely to alter during amyloplast extraction. To test the

105 reversibility of protein phosphorylation, stromal proteins were first dephosphorylated with APase followed by re-phosphorylation with ATP. Interestingly, the ATP-induced mobility retardation was restored in the rephosphorylated SSIIa sample (last lane in Fig.

2.6A). This strongly suggested that the observed retardation of the SSIIa protein band in the Phos-tag gel is the consequence of a phosphorylation-induced modification of SSIIa and that the process is reversible. The absence of the phosphorylation-dependent, mobility shift during normal SDS-PAGE in the absence of the Phos-tag ligand further indicated that the altered mobility of phosphorylated forms of SSIIa in the Phos-tag gel was due to the incorporation of phosphate group(s) on the protein (Fig. 2.6 B).

Maize SSIIa bound to the starch granule migrated differently in the Phos-tag gel as compared to the untreated stromal SSIIa (Fig 2.7) suggesting that starch granule-bound

SSIIa exists in the phosphorylated form. Tetlow et al. (2004a) also found that granule bound SSIIa in wheat endosperm is phosphorylated but did not investigate this phenomenon in detail.

Recently using Mn2+- Phos-tag TM phospho-protein affinity gel electrophoresis,

Liu et al.(2012a) confirmed the phosphorylation status of SBEs (SBEIIb and SBEI) in the stromal and granule bound fractions in wild type and ae 1.2 (amylose extender) mutant of maize possessing an inactive SBEIIb. The phosphorylated and dephosphorylated forms of stromal SBE (SBEIIb and SBEI) exhibited differential mobility in the Phos- tag gel. The mobility of the phosphorylated forms was markedly retarded compared to the dephosphorylated forms in both genotypes, indicating that they are phosphorylated (Liu et al., 2012a). In a Phos-tag gel, the phosphorylated proteins show differential retardation in their electrophoretic mobility corresponding to the number of phosphorylated residues;

106 for e.g., dephosphorylated forms with no phosphate residues show the greatest mobility and increased numbers of phosphorylated residues cause greater retardation (Fig. 2.14).

In those studies multiple bands of SBEIIb were observed indicative of more than one phosphorylation site (Liu et al., 2012a). By contrast, data from the present study indicate that SSIIa might be subject to phosphorylation only on one site, given the patterns of electrophoretic migration observed.

Recently Liu et al. (2012b), immunopurified SSIIa successfully from maize amyloplast lysates using peptide specific anti-SSIIa antibody. This method was used here to obtain highly purified SSIIa from stroma, and to investigate the implications of protein phosphorylation on the functional regulation of maize SSIIa from endosperm tissue. The absence of other starch biosynthetic enzymes on the Protein-A-Sepharose beads indicated that SSIIa was significantly immunopurified from those enzymes present in the amyloplast stroma under the conditions employed (e.g. see Fig. 3.15 in the following chapter). In vitro phosphorylation of immunopurified SSIIa from maize amyloplast lysates was further confirmed by Pro-Q® diamond phospho-protein gel staining and by auto radiographic analysis following radiolabelling by γ- [32-P] ATP. The increased intensity of SSIIa staining with Pro-Q diamond (Fig.2.8A) reinforces the hypothesis that stromal maize SSIIa can be phosphorylated by endogenous protein kinase(s) present in the amyloplasts.

Radiolabelling of immunopurified stromal SSIIa following incubation with γ- [32-

P] ATP and maize amyloplast lysates also argues for direct phosphorylation of SSIIa

(Fig. 2.9). The observation that phosphorylation of recombinant SSIIa was dependent on

107 the presence of maize amyloplast lysates is indicative of a dependence on protein kinase(s) within the organelle (Fig. 2.12).

To test the hypothesis that putative protein kinases from the amyloplasts facilitate protein phosphorylation of SSIIa, stromal SSIIa was pretreated with protein kinase inhibitors FSBA and K252a, prior to incubation with ATP to inhibit potential native protein kinases that could possibly be involved in the process. FSBA (5- fluorosulfonylbenzoyl-5΄adenosine) and K252a (Staurosporine) are broad spectrum potent kinase inhibitors (Adams and Parker, 1992; Ohto and Nakamura, 1995). FSBA covalently modifies a conserved lysine present in the ATP binding site of most of the protein kinases and competitively inhibit them (Adams and Parker, 1992). K252a is an efficient serine/threonine protein kinase inhibitor that inhibits potent protein kinase C and cyclic dependent protein kinases (Tapley et al., 1992). Interestingly, FSBA and K252a prevented the phosphorylation-dependent, mobility shift of ATP-treated SSIIa following

Phos-tag electrophoresis (Fig.2.9). This result further strengthens the argument for the involvement of endogenous protein kinases in the amyloplast, in the phosphorylation of stromal SSIIa.

The results discussed in this chapter strongly indicate that maize SSIIa can be phosphorylated by one or more protein kinase(s) present in the amyloplasts of developing maize endosperm. Importantly, the results also suggest that maize SSIIa bound to the starch granule exists in the phosphorylated state. Analysis of wheat starch granule associated phospho-proteins following in vitro phosphorylation using γ-32P-ATP demonstrated that the most strongly phosphorylated phosphoprotein within the starch granules was SSIIa (Tetlow et al., 2004a). Previous studies in wheat and maize

108 endosperms have clearly shown the importance of protein phosphorylation in regulating the physical associations of major starch biosynthetic enzymes (SSs and SBEs) including

SSIIa to form functional multi-enzyme complexes (Tetlow et al.,2004a; Tetlow et al.,2008; Hennen-Bierwagen et al., 2008 & 2009; Liu et al.,2009). Reversible protein phosphorylation can influence the activity, subcellular localization and stability of the target proteins that interact each other, to form functional multi- enzymes complexes

(Stone and Walker, 1995; Cohen, 2000; Schliebner et al., 2008; Bayer et al., 2012).

Hence the formation of phosphorylation-dependent complexes of starch biosynthetic enzymes through protein-protein interactions could possibly enhance the efficiency of the starch biosynthetic machinery by coordinating and regulating individual enzyme activities via altering enzyme kinetics and conformation of the participating enzymes (

Tetlow et al.,2004a; Tetlow et al.,2008; Hennen-Bierwagen et al., 2008 & 2009; Liu et al.,2009).

Phospho-amino acid analysis of the phosphorylated polypeptides (putative

SBEs) in both amyloplasts and chloroplasts of wheat revealed that in all cases, phosphorylation occurred on one or more Ser residues (Tetlow et al., 2004a). In vitro phosphorylation of recombinant maize SSI and phospho- amino acid analysis have shown that recombinant maize SSI is phosphorylated on one or more serine residues (Barker and

Romanova, Pers. Comm.). Bio-informatic prediction of putative phosphorylation sites of maize SSIIa indicates that the possible phosphorylation sites include 8 serine (Ser), 4 threonine (Thr) and 7 tyrosine (Tyr) residues (Fig. 2.15). Considering the above observations, it might be proposed that maize SSIIa could be phosphorylated on one or more serine residues. Retardation of phosphorylated SSIIa on Phos-tag phospho- protein

109 affinity gels might imply a single phosphorylation site but further detailed investigation on the number of phosphorylation sites on maize SSIIa was not conducted in this study.

NetPhos 2.0 Server - Prediction Results

Phosphorylation sites predicted: Ser: 8 Thr: 4 Tyr: 7

Serine predictions

Name Pos Context Score Pred ______v______Sequence 15 PPERSGDAA 0.570 *S* Sequence 30 RNAVSKRRD 0.995 *S* Sequence 44 GRYGSATGN 0.398 . Sequence 56 TGAASCQNA 0.632 *S* Sequence 69 VEIKSIVAA 0.490 . Sequence 77 APPTSIVKF 0.290 . Sequence 92 MILPSGDIA 0.064 . Sequence 110 PLHESPAVD 0.072 . Sequence 117 VDGDSNGIA 0.135 . Sequence 152 AKDDSRVGA 0.966 *S* Sequence 161 DDAGSFEHY 0.717 *S* Sequence 170 GDNDSGPLA 0.012 . Sequence 189 AAECSPWCK 0.006 . Sequence 277 IYGGSRQEI 0.035 . Sequence 342 QYTRSVLVI 0.006 . Sequence 401 VVTVSRGYL 0.055 . Sequence 423 DIIRSNDWK 0.004 . Sequence 450 VHLRSDGYT 0.099 . Sequence 457 YTNYSLETL 0.761 *S* Sequence 548 WVGFSVPMA 0.031 . Sequence 566 LVMPSRFEP 0.312 . Sequence 637 KYGESWKSL 0.969 *S* Sequence 640 ESWKSLQAR 0.018 . Sequence 647 ARGMSQDLS 0.094 . Sequence 651 SQDLSWDHA 0.887 *S* ______^______

Threonine predictions

Name Pos Context Score Pred ______v______

110

Sequence 46 YGSATGNTA 0.053 . Sequence 49 ATGNTARTG 0.351 . Sequence 52 NTARTGAAS 0.070 . Sequence 76 AAPPTSIVK 0.117 . Sequence 99 IAPETVLPA 0.426 . Sequence 124 IAPPTVEPL 0.378 . Sequence 133 VQEATWDFK 0.065 . Sequence 194 PWCKTGGLG 0.060 . Sequence 320 NDWHTALLP 0.007 . Sequence 340 LMQYTRSVL 0.076 . Sequence 399 DRVVTVSRG 0.721 *T* Sequence 410 WELKTVEGG 0.179 . Sequence 454 SDGYTNYSL 0.026 . Sequence 460 YSLETLDAG 0.224 . Sequence 521 VMLGTGRAD 0.716 *T* Sequence 556 AHRITAGAD 0.126 . Sequence 583 MAYGTVPVV 0.280 . Sequence 596 GLRDTVAPF 0.815 *T* Sequence 611 GLGWTFDRA 0.059 . Sequence 630 HCLDTYRKY 0.778 *T* ______^______

Tyrosine predictions

Name Pos Context Score Pred ______v______Sequence 42 PVGRYGSAT 0.199 . Sequence 86 PAPGYRMIL 0.486 . Sequence 139 DFKKYIGFD 0.250 . Sequence 165 SFEHYGDND 0.974 *Y* Sequence 222 VVPRYGDYV 0.172 . Sequence 225 RYGDYVEAF 0.670 *Y* Sequence 236 GIRKYYKAA 0.019 . Sequence 237 IRKYYKAAG 0.056 . Sequence 248 LEVNYFHAF 0.029 . Sequence 274 QDDIYGGSR 0.792 *Y* Sequence 306 GGVCYGDGN 0.146 . Sequence 326 LLPVYLKAY 0.026 . Sequence 330 YLKAYYRDH 0.014 . Sequence 331 LKAYYRDHG 0.419 . Sequence 339 GLMQYTRSV 0.012 . Sequence 362 DEFPYMDLP 0.983 *Y* Sequence 369 LPEHYLQHF 0.088 . Sequence 376 HFELYDPVG 0.668 *Y* Sequence 404 VSRGYLWEL 0.025 . Sequence 453 RSDGYTNYS 0.976 *Y*

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Sequence 456 GYTNYSLET 0.016 . Sequence 577 LNQLYAMAY 0.235 . Sequence 581 YAMAYGTVP 0.176 . Sequence 631 CLDTYRKYG 0.408 . Sequence 634 TYRKYGESW 0.031 . Sequence 659 AAELYEDVL 0.971 *Y* Sequence 668 VKAKYQW-- 0.259 . ______^______

Figure 2.15: Identification of potential phosphorylation sites within maize SSIIa protein. Potential serine, threonine and tyrosine phosphorylation sites were identified and scored by using NetPhos 2.0 server. The putative phosphorylation sites scoring above the 0.500 threshold are shown in bold print and included 8 serine, 4 threonine and 7 tyrosine residues.

112

Liu et al. (2012b) demonstrated that the presence of functionally active SSIIa was essential for the trimeric protein complex (SSI-SSIIa-SBEIIb) to become granule bound, to retain normal SS and SBE activities within the complex, and to maintain the morphology and crystallinity of starch granules. Notably, the SS activity of this heteromeric protein complex (combined activity of SSI and SSIIa) was considerably enhanced by ATP (Liu et al., 2012b). Together, all the experimental evidence suggests the post translational modification of SSIIa by protein phosphorylation. Investigations on the effect of protein phosphorylation on the regulation of maize SSIIa with respect to its affinity towards glucan substrates, and catalytic activity, are considered in Chapter 3.

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CHAPTER 3

114

CHAPTER 3: THE EFFECT OF PROTEIN PHOSPHORYLATION ON REGULATION OF MAIZE SSIIA

3.1 Introduction

The role of protein phosphorylation in coordinating the activities of potential amylopectin synthesizing enzymes (isoforms of SSs and SBEs) required for the starch granule assembly has been demonstrated in previous studies (Tetlow et al., 2004a;

Tetlow et al., 2008; Hennen –Biewagen et al., 2008; 2009; Liu et al., 2009; reviewed in

Kötting et al., 2010). The results presented in the previous chapter have clearly revealed that SSIIa in maize amyloplast stroma could be phosphorylated by endogenous protein kinases present in the amyloplasts. Protein phosphorylation has been previously shown to regulate starch branching enzyme activity (Tetlow et al., 2004a), the physical association of enzymes involved in starch biosynthesis (Tetlow et al., 2008; Hennen –Biewagen et al., 2008; 2009; Liu et al., 2009) and their association with starch granules (Grimaud.et al., 2008; Liu et al., 2009). Phosphorylation-dependent, functional heterocomplexes incorporating SSIIa have been found in the endosperms of wheat and maize during starch biosynthesis (Tetlow et al., 2008; Hennen –Biewagen et al., 2008; 2009; Liu et al.,

2009). Formation of a 260 kDa protein complex between SSI, SSIIa and SBEIIb in maize endosperm (with SSIIa being the core of the complex) required protein phosphorylation and, in vitro, dephosphorylation dissociated the complex (Liu et al., 2009; 2012a). The importance of SSIIa in this trimeric complex in governing the binding of SSI and SBEIIb to the starch granule to facilitate amylopectin synthesis has been clearly demonstrated by

Liu and coworkers (2012b).

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Biochemical characterization of various isoforms of maize SSs expressed in

Escherichia coli revealed that recombinant maize SSIIa exhibited a slightly higher Vmax with amylopectin (corn) as the primer (29.2±2.3 µmol/min/mg) compared to glycogen

(rabbit liver) (24.0±0.6 µmol/min/mg) (Imparl-Radosevich et al., 1999a). Site-directed mutagenesis of the conserved Arg 214 in recombinant maize SSIIa resulted in a significantly higher Km for amylopectin suggesting that this amino acid is critical for the apparent affinity of SSIIa for its glucan primer (Imparl-Radosevich et al., 1999b). Like other SSs, the highly conserved KXGGL motif present in the C-terminal catalytic domain of SSIIa is found to regulate ADPG binding, while the N-terminal domain is thought to be involved in regulating its binding to different glucan substrates (Furukawa et al. 1990,

1993; ; Imparl-Radosevich et al., 1999a ; Busi et al., 2008). It was also observed that the specific activity of recombinant maize SSIIa was at least 2-3fold lower than that of SSIIb

(Imparl-Radosevich et al., 1999a). A summary of the kinetic properties of recombinant form of maize SSIIa is given in the Table 3.1.

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Table 3.1 Kinetics of recombinant maize SSIIa expressed in Escherichia coli

Km Vmax

(µmol/min/mg)

Primer Rabbit liver Glycogen 0.14±0.02 (mg/ml) 24.0± 0.6 Corn Amylopectin 0.16±0.06 (mg/ml) 29.2 ± 2.3

ADP-Glucose

Rabbit liver Glycogen 0.16±0.01 (mM) 19.5± 0.1 Corn Amylopectin 0.11±0.01 (mM) 30.6 ± 1.2

(Derived from Imparl-Radosevich et al., 1999a)

Recently Liu et al. (2012b) purified soluble SSIIa from plant tissue (maize amyloplast stroma) by immunopurification with peptide-specific maize anti-SSIIa antibodies. The authors observed that the specific activity of immunopurified SSIIa for glycogen (rabbit liver) (9.9±0.1 nmol/h/µg) was twofold higher than that for amylopectin

(corn) (4.3±0.1 nmol/h/µg).

The post translational regulation of glycosyl transferases (like starch synthases) by protein phosphorylation has been widely explored in animal systems. In mammals, during glycogen synthesis, the transfer of the glucosyl unit of UDP-glucose to the non- reducing end of the growing glycogen polymer by forming a 1-4 glycosidic linkage is

117 catalyzed by glycogen synthase (GS), a member of family 3 retaining glycosyl transferases (animal counterpart for starch synthases) (Campbell et al., 1997; Roach,

2002). Mammalian GS enzymes are regulated allosterically by binding ligands, such as glucose-6-phosphate, and by protein phosphorylation (Roach, 2002). Under high glucose conditions, glucose 6-phosphate enhances the activity of GS through conformational changes caused by dephosphorylation (Stalmans et al., 1987; Cadefau et al., 1997). At high blood sugar levels, insulin inactivates glycogen synthase kinase 3 (GSK3) resulting in the activation of protein phosphatase I, which dephosphorylates the three serine residues at the C- terminal of GS and activates the enzyme (Jope and Johnson, 2004).

A great deal of experimental proof highlights the significance of protein phosphorylation in regulating functional protein–protein interactions between major biosynthetic enzymes during the synthesis of storage starch in cereal endosperms

(reviewed in Tetlow et al., 2011). In vitro phosphorylation of intact wheat amyloplasts with γ- [32-P] ATP resulted in the phosphorylation of both stromal and granule associated

SSIIa (Tetlow et al., 2004a; 2008). It was also observed that dephosphorylation with

APase reduced the SBE activity by threefold in chloroplasts and by four fold in amyloplasts (Tetlow et al., 2004a). The reduction in the activities of different SBE isoforms was clearly observed by zymogram analysis (Fig. 3.1).

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Figure 3.1 Effects of dephosphorylation on SBE activity from amyloplast stroma and chloroplasts of wheat (taken from Tetlow et al., 2004a). (A) SBE activity

visualized by with I2/KI after separation of amyloplast and chloroplast protein lysates pretreated with either ATP (1mM) or APase (10units) in non-denaturing polyacrylamide gels. (B) Western blots of SBE zymograms: amyloplast (lanes 1, 3, 5) and chloroplast (lanes 2, 4, 6) stromal proteins developed with antibodies against SBEI (lanes 1 and 2), SBEIIa (lanes 3 and 4), and SBEIIb (lanes 5 and 6).

Studying the interdependence of key maize starch biosynthetic enzymes (SSI,

SSIIa and SBEs) in developing maize endosperm, Hennen –Biewagen et al., (2009) observed that these enzymes were capable of associating with each other directly or indirectly and that their interactions were largely regulated by the phosphorylation of one or more of the participating enzymes. In the two major protein complexes identified by gel permeation chromatography (670 kDa and 300 kDa) SSIIa appeared to be a major component interacting with SSI, SSIII and SBEIIa and SBEIIb (Hennen –Biewagen et al., 2009). In a recent study, Liu et al. (2012b) observed that in wild type maize endosperm, the total SS activity associated with the functional protein complex

119 containing SSI, SSIIa and SBEIIb was increased by 43% and the SBE activity was increased by 15% upon treatment with ATP. The total SS activity associated with this trimeric (immunopurified) complex, which represented the combination of both SSI and

SSIIa (5.6 nmol/h/µg of the protein) was comparable with the activity of SSIIa alone

(4.3nmol/h/µg of the protein) in the immunopurified SSIIa from amyloplasts (Liu et al.,

2012b). Therefore a significant proportion of the total SS activity measured in these experiments is likely contributed by SSIIa and, interestingly, this catalytic activity was enhanced by prior treatment with ATP. These results strongly suggest a possible role of protein phosphorylation in regulating the catalytic activity and function of SSIIa in these functional heteromeric protein complexes during the biosynthesis of amylopectin.

The effect of post translational protein phosphorylation on the catalytic activity and kinetics of SSIIa in wild type maize amyloplast stroma has yet to be characterized.

The experiments presented in the previous chapter provided compelling evidence for the post-translational modification of maize SSIIa by protein phosphorylation. Chapter 3 investigates the effect of phosphorylation and dephosphorylation on the catalytic activity and the kinetics of maize endosperm SSIIa. The information gained by this investigation should throw more light on the role of reversible protein phosphorylation in regulating the biosynthesis of starch in maize endosperm.

3.2 Materials and Methods

3.2.1 Zymogram analysis of SS activity To visually detect the activity of maize SSIIa in the amyloplast stroma under phosphorylating and dephosphorylating conditions, starch synthase zymogram analysis was conducted as described by Liu et al. (2009) based on the published work by Buléon

120 et al. (1997). After incubation with ATP (1mM) or APase (100U), the amyloplast lysate

(section 2.2.5.1) was electrophoresed in non-denaturing gels containing 0.3% of corn amylopectin, rabbit liver glycogen and corn starch. Zymograms consisted of native 5%

(w/v) polyacrylamide gels containing 10mg of the α-amylase inhibitor Acarbose

(Prandase, Bayer), and 0.3% (w/v) of glucan substrate (Table 3.2). Protein samples were mixed with the native loading buffer (62.5mM Tris HCl pH6.7, 10% glycerol, 0.005%

(w/v) bromophenol blue) at a ratio of 10:1 and electrophoresed in running buffer (25 mM

Tris- HCl pH 8.3,190 mM glycine) at 100 V, 4°C, until the loading dye ran off the bottom of the gel. The gel was then incubated in the SS zymogram incubation buffer at

30°C for 48 to 72 hours (50mM glycylglycine, pH 9.0, 100mM sodium citrate, 20mM

DTT, 5mM MgCl2, 0.5mg/ml BSA, 4mM ADP-Glc). After incubation, the zymograms were developed in Lugol’s solution (0.2% [w/v] Lugol’s iodine and 2% [w/v] potassium iodide) and immediately analyzed. Simultaneously, an identical gel was immunoblotted onto nitrocellulose membrane and SSIIa protein detected using anti- SSIIa antibody following the procedure given in section 2.2.4.3.

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Table 3.2: Composition of non –denaturing starch synthase zymogram gels containing different glucan substrates

Stock solution Resolving gel (10ml)

(5% acrylamide; no SDS)

Distilled water 3.7 (mL) 1.5% glucan stock (Corn amylopectin, Corn starch 2.0 (mL) (0.3%) and rabbit liver glycogen) 30%Acrylamide 1.8 (mL) 1.5M Tris, pH 8.8 2.5 (mL) 10% APS 40 (μL) Acarbose 80 (μL) TEMED 10 (μL)

3.2.2 Effect of ATP concentration and duration of incubation on the mobility of maize SSIIa during non-denaturing PAGE

In order to estimate the optimal concentration of ATP required to observe its effect on the mobility shift of native SSIIa, maize amyloplast lysates were incubated with a range of ATP concentrations (from 0-1mM) for 30 -40 min under room temperature.

For the optimization of incubation time, the amyloplast lysates were treated with 1mM

ATP for a range of time periods (0- 40 min) and the reaction was stopped after each time period by adding 20mM EDTA. All samples, including the control, included 10mM

MgCl2, 5mM DTT and plant protease inhibitor cocktail (Plant Protease Arrest™ [100X],

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G Biosciences) (10μl per 1 mL lysates). Untreated amyloplast lysates were used as controls. Rupturing buffer was added to balance the final volumes of each assay.

Following ATP treatment, the proteins in the lysate were subjected to non-denaturing gel electrophoresis followed by immunoblotting with anti-SSIIa specific antibody as described in section 2.2.4.3.

3.2.3 Substrate affinity electrophoresis Varied concentrations of glucan substrates (rabbit liver glycogen and corn amylopectin) were incorporated into the non-denaturing polyacrylamide gel polymerization mixture following the procedure described by Tetlow et al. (2008).

Proteins in the maize amyloplast lysates pretreated with ATP (1mM) or APase (100U)

(see section 2.2.5.1) were separated on non-denaturing native 5% (w/v) polyacrylamide gels containing 10mg of the α-amylase inhibitor Acarbose (Prandase, Bayer), and different concentrations of corn amylopectin or rabbit liver glycogen as a glucan chain primer. The migration distance of native maize SSIIa was measured after immunoblotting and probing with anti-SSIIa specific antibodies as described previously (section 2.2.4.3).

Dissociation constants (Kd) of the enzyme-substrate complex were calculated following the methods described by Commuri and Keeling (2001). Following affinity electrophoresis and immunoblotting, the relative migration (Rm) of SSIIa in native gels in the presence of different concentrations of glycogen and amylopectin was measured.

Relative migration (Rm) of SSIIa was determined by reference to migration of SSIIa protein in the absence of the glucan substrate used. The reciprocal of the values of relative migration (1/Rm) of SSIIa were linearly related to the concentration [S] of the glucan used in the gels (Takeo and Nakamura, 1972; Matsumoto et al., 1990). Kd values

123 were determined (x- intercept of the plotted graph) as mg/ml. The experiment was repeated three to five times with different biological replicates. Students’ T- tests were used to compare Kd and apparent affinity constants under different treatment conditions.

3.2.4 Preparation of radio-labelled ADP-[U-14C] glucose as substrate for starch synthase

In order to quantitatively estimate the catalytic activity of maize SSIIa, radiolabelled ADP-[U-14C] glucose was required as the glucan substrate to conduct 14C- labelled ADP-glucose substrate SS assay. The need to synthesize ADP-[U-14C] glucose from [U-14C] glucose-1-phosphate (PerkinElmer) using purified E. coli AGPase resulted from the unavailability of a cheaper source of this reagent commercially.

3.2.4.1 Expression and purification of E coli recombinant AGPase (adenosine 5- diphosphate glucose pyrophosphorylase)

The expression of recombinant AGPase in E coli cells and the purification of the recombinant protein were conducted following Liu et al. (2012). The E. coli-derived

AGPase gene (glgc) built in the pETM-11vector with a His6 tag fused at its N-terminus was kindly provided by Dr. John Lunn, Max Planck Institute of Molecular Plant

Physiology, Golm, Germany. The recombinant plasmid was transformed into E. coli

Arctic Express cells. A single colony picked from an overnight plate was inoculated into

5ml of LB (Luria–Bertani) broth with 50µM Kanamycin and 20µM Gentamycin and grown at 37°C overnight. The overnight cultures were sub cultured in 2L of LB media, without the antibiotics, and grown at 37°C until the cell density reached 0.4-0.5 at D600.

Expression of the recombinant protein was induced by IPTG (isopropyl β-D-1- thiogalactopyranoside) to a final concentration of 1 mM. The cultures were further grown for 24h by shaking at 250rpm at 10°C. E. coli cells were then collected by centrifugation

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(at 13,000 rpm at for 20 min.) and lysed using Bug Buster Protein Extraction Reagent

(Novagen) in the presence of plant protease inhibitors (Plant Protease Arrest™ [100X], G

Biosciences) (10μl per 1 mL buffer) and the soluble fraction containing recombinant

AGPase was collected. Recombinant AGPase in the soluble cell lysate was then purified using the Invitrogen Ni-NTA (Ni2+ -nitrilotriacetate) Purification System following the manufacturer’s protocol. The expression level of the protein and its purity was examined by running the induced soluble fraction and eluates from the His-tag purification column, on SDS-PAGE followed by Coumassie staining. The uninduced soluble lysate served as the control. The protein content was determined following the method by Bradford

(Bradford, 1976). The recombinant protein was stored at -20°C in 40% (v/v) glycerol and the catalytic activity checked every 2-3 months.

The specific activity of the recombinant AGPase was determined following the method described by Tetlow et al. (2003). The pyrophosphorolytic activity of AGPase was measured by continuously monitoring the formation of NADH at 340 nm at 25 °C using Beckman-Coulter DU 800 UV-Visible spectrophotometer. The standard assay mixture included 10 mM MgCl2, 2 mM DTT, 5 mM NaF, 0.5 mM NAD+, 1 mM ADP glucose, 100 mM glucose 1,6-bisphosphate, 1 mM tetra-sodium pyrophosphate (PPi) and

2 units each of phosphoglucomutase (from rabbit muscle) and glucose 6-phosphate dehydrogenase (from Leuconostoc mesenteroides). The reaction was initiated by the addition of PPi. The specific activity was calculated from the molar extinction co- efficient for (6.22) NADH using Beer- Lambert’s equation.

3.2.4.2 Synthesis of ADP- [U14C] Glucose using recombinant AGPase ADP-[U-14C] glucose was synthesized from [U-14C] glucose -1-phosphate

(PerkinElmer) using purified E. coli AGPase following the method used by Liu et al.

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(2012). The 200µl reaction mixture contained 10 mM Tris/HCl, pH 7.5, 1 mM MgCl2, 10 units of inorganic pyrophosphatase (from E. coli, MCLAB IPE-200), 100 units of recombinant E. coli AGPase, .02 μmol of [U-14C] glucose -1-phosphate (∼4 μCi) and

0.025 μmol of ATP.The reaction mixture was incubated at 30°C for 5 h. The reaction was stopped by heating at 95°C for 5 min followed by centrifugation at 14000 g for 10 min.

To test the efficiency of conversion, following the ADP-[U-14C] glucose synthesis reaction, the ADP-[U-14C] glucose present in the supernatant was collected and spotted on a TLC sheet and separated using Ethyl acetate: Isopropanol: Water, 65:25:10 v/v/v as the solvent phase. When the solvent phase reached 2-3 cm from the top of the sheet, the

TLC was removed and the solvent front marked. The TLC sheet was then sprayed with

Naphthoresorcinol (Dihydroxy-naphthalene)-H3PO4 mixture and the separated samples were visualized by developing the sprayed TLC sheet at 100°C in a hot air oven for few minutes until the spots developed. An autoradiograph was also developed by incubating the TLC sheet on an X-ray film in the dark for 10 days at -80°C. Unreacted AGPase from the remaining stopped reaction mixture was removed by precipitation with ice-cold acetone, and the supernatant was dried down in a speed vac. The dried product was resuspended in 200 mM Bicine/KOH, pH 8.5, and stored at −20°C for use. The radioactivity of the synthesized ADP-[U-14C] glucose was measured using the Beckman LS

6500 Liquid Scintillation counter; radioactivity was determined in µCi/µmol and compared with the radioactivity of commercial ADP-[U-14C] glucose (Perkin Elmer).

3.2.5 Determining the effect of protein phosphorylation on the catalytic activity of SSIIa in maize amyloplast stroma

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SSIIa from maize amyloplast stroma pretreated with ATP (1mM) or APase

(100U) was immunopurified using anti -SSIIa specific antibodies following the protocol described in the section 2.2.4.4. The catalytic activity of immunopurified SSIIa pretreated with ATP or APase was measured by using the ADP-[U-14C] glucose assay following the procedure described by Liu et al. (2012). Immunopurification of SSIIa protein bound to the Protein -A-Sepharose was analyzed by SDS-PAGE and immunoblotting with antibodies specific to various maize SSs to determine whether there was any non-specific binding. Following immunopurification, the amount of SSIIa protein on Protein –A-Sepharose beads was quantified. Each 200µl of SS assay mixture contained 100mM Bicine-KOH, pH 8.5, 50 mM potassium acetate, 200 mM sodium citrate and 10mM Na2-EDTA and 3mg/ml corn amylopectin as the glucan precursor.

Following the addition of 20µg of the immunopurified SSIIa, reactions were initiated by the incorporation of 14C from ADP-[U-14C] glucose (5kBq per assay) (specific activity

1kBq/µl) synthesized from-[U-14C] Glucose-1-phosphate (Perkin Elmer) and incubated at

25°C for 20 min. Reactions were terminated by heating the assay mixture at 95°C for 5 min and the remaining 14C- labelled glucan was precipitated by methanol/KCl (75%v/v methanol,1%w/v KCl). The 14C- labelled products were released into the supernatant by vortexing the precipitate with distilled water. The catalytic activity was measured by counting the 14C- labelled products using the Beckman LS 6500 Liquid Scintillation counter and expressed as nmoles of ADP-[U-14C] glucose incorporated /hr/µg of the protein. The experiments were conducted in 3-5 biological replicates and the data were statistically analyzed.

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3.3 Results

3.3.1 Effect of ATP and APase on the activity of native SSIIa as visualized in the SS activity zymogram The activity of maize SSIIa in the amyloplast stroma pretreated with ATP (1mM) or APase (100U) was visualized by zymogram analysis in the presence of 0.3% of either corn amylopectin, rabbit liver glycogen or corn starch as glucan primers (Fig. 3.2). In all three glucan containing non-denaturing gels, ATP-treated SSIIa migrated further compared to the untreated and APase treated forms (Fig. 3.2 (1-3B)). Some of the activity bands in the zymogram ( labelled ‘b’, ’c’, ’d’ and ‘e’) corresponded to the SSIIa protein bands in the immunoblots in zymograms containing corn amylopectin and corn starch

(Fig 3.2 (1 and 3)) suggesting that those SS activities could be due to SSIIa protein in the amyloplast lysate. Several bands of SS activity were visible on the zymograms containing rabbit liver glycogen (Fig. 3.2 (2A)). Two distinct activity bands (‘h’ and ‘i’ shown by blue arrows) appeared towards the bottom of the gel (all three treatments). Likewise, two activity bands, ‘f’ (1.4cm) and ‘g’ (1.5cm) appeared in the untreated and APase treated samples. Interestingly, only a single prominent activity band ‘g’ was apparent in the same position in the ATP-treated samples (Fig. 3.2 (2A)). However, the results showed that none of the activity bands on the zymogram gel containing rabbit liver glycogen aligned with the SSIIa protein bands in the immunoblot (Fig. 3.2 (2A and B)), indicating that those activity bands could be due to other SSs present in the amyloplast lysate.

SS zymogram gels containing amylopectin and starch exhibited similar numbers and electrophoretic migration patterns of SS activity bands (Fig. 3.2 (1&3 A)). Activity

128 bands are labelled alphabetically (‘a’ to ‘e’) for convenience. A band of SS activity, labelled ‘a’, appeared at the top margin of the gel (migrating 0.2 cm under conditions employed) in all three treatments (Fig. 3.2 1&3A; Fig. 3.3 A&B). However this band did not correspond to any SSIIa protein bands identified by anti-SSIIa antibody in the immunoblots (Fig. 3.2 1&3B). Interestingly, band ‘a’ was identified by anti-SSI antibody in the immunoblot for SSI (Fig. 3.3).

ATP treatment caused a substantial modification in the electrophoretic mobility of

SSIIa (Fig. 3.2 (1-3B). In gels containing amylopectin and starch, the SSIIa protein band

(activity band ‘b’) migrated significantly further (band ‘c’) when samples were pretreated with 1mM ATP prior to electrophoresis, compared to the untreated and APase treated samples (Fig. 3.2 1&3 A&B). Interestingly, in the ATP-treated samples, two distinct and prominent activity bands ‘c’ and ‘d’ appeared in zymograms containing amylopectin and starch (Fig. 3.2 1A & 3 A). SS activity bands ‘b’ and ‘c’ in the amylopectin and starch zymogram gels were detected by anti-SSIIa antibody (Fig. 3.2 (1 and 3). Another band of SS activity (Fig. 3.2 (1A & 3A))‘d’ showed faint interaction with anti-SSIIa antibody in the western blots of amylopectin-containing gels (Fig. 3.2

(1)A&B). Following treatment with ATP, another activity band, ‘e’, appeared in the amylopectin containing zymogram, and showed some interaction with anti-SSIIa antibody in the corresponding immunoblot (Fig. 3.2 (1) A&B). For comparison, amyloplast lysates from a sugary-2 maize mutant lacking SSIIa, (unknown genetic background) were also electrophoresed side by side with the wild type amyloplast lysates in non-denaturing gels containing amylopectin. The zymogram showed that activity band

‘b’ seen in the wild type amyloplast sample, was not detected in the sugary -2 mutant

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(lacking SSIIa activity) (Fig. 3.3), corresponding to the loss of detectable SSIIa protein

(Fig. 3.3).

g f & g (1.4 &1.5 cm)

h (2.3cm) i (2.7 cm)

Figure 3.2: Effect of ATP on SS activity and mobility of maize SSIIa in non- denaturing gels in the presence of different glucan primers. After incubation with ATP (1mM), or APase (100U), maize amyloplast lysates were electrophoresed in non-denaturing gels containing (1) 0.3% corn amylopectin (2) 0.3% rabbit liver glycogen or (3) 0.3% corn starch. (A) Starch synthase activity was visualized using 4mM ADP –glucose as substrate followed by staining with KI-I2. (B) Replicate zymograms were immunoblotted and SSIIa detected with anti-SSIIa antibody. The activity bands are labelled alphabetically, with the corresponding SSIIa protein bands indicated by arrows.

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Figure 3.3: Effect of ATP on SS activity and mobility of maize SSIIa and SSI in non-denaturing PAGE. After incubation with ATP (1mM) or APase (100U) wild type (WT) maize amyloplast lysates and untreated sugary 2 amyloplast lysates (lacking SSIIa protein) were electrophoresed in non-denaturing gels containing 0.3% corn amylopectin. Starch synthase activity was visualized using 4mM ADP –glucose as substrate followed by staining with KI-I2 (left). Zymograms were immune-blotted and SSIIa and SSI detected by anti-SSIIa and anti- SSI antibodies (right). The activity bands are labelled alphabetically, with the corresponding SSIIa and SSI protein bands indicated by arrows.

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3.3.2 Effect of ATP concentration on the mobility of native maize SSIIa

The ATP- induced, mobility-shift of maize SSIIa was analyzed in non-denaturing gels containing 0.3% amylopectin after pre-incubating amyloplast lysates with various concentrations of ATP (0-1mM). A shift in mobility of the protein was observed using concentrations of ATP as low as 5µM (Fig. 3.4). The mobility of a significant portion of

SSIIa was altered when the amyloplast lysates were treated with ≥ 20µM ATP. As the concentration of ATP increased from 25µM to 1mM, the majority of SSIIa protein migrated to the lower position (Fig. 3.4). The results suggested that ATP concentrations as low as 5µM could induce an electrophoretic mobility shift of maize SSIIa and that 0.5-

1mM ATP is optimal to detect the effect of phosphorylation on the mobility of maize

SSIIa in non-denaturing gels.

Figure 3.4: Effect of ATP concentration on the mobility of native, maize SSIIa following non-denaturing PAGE. After incubation with various concentrations of ATP from 0 to 1mM, amyloplast lysates were electrophoresed in non-denaturing polyacrylamide gel containing 0.3% corn amylopectin. The mobility of SSIIa protein was detected by probing the immunoblots with maize anti-SSIIa specific antibody. SSIIa protein bands are indicated by arrows.

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In order to optimize the incubation- time, maize amyloplast lysates were incubated with 1mM ATP at different time intervals ranging from 1- 40 min. The results indicate that 1mM ATP could result in a complete shift in SSIIa mobility within 5min

(Fig. 3.5). For all other experiments discussed in this thesis, samples containing SSIIa were incubated with 1mM ATP (unless otherwise specified) for 30-40 minutes providing ample time for post-translational modification to occur.

Figure 3.5: Time-dependence of ATP-induced, mobility-shift of maize SSIIa. After incubating with 1mM ATP for different time intervals, stromal SSIIa in the amyloplast lysate was electrophoresed in non-denaturing polyacrylamide gels containing 0.3% corn amylopectin. 20mM EDTA was added to the reaction mixture to ensure that each reaction was stopped at the time shown. The mobility of SSIIa protein was detected by probing the immunoblots with maize anti-SSIIa specific antibody. The mobility of SSIIa protein is indicated by arrows.

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3.3.3 Effect of substrate and ATP on the electrophoretic mobility of maize SSIIa in non-denaturing polyacrylamide gels

The effect of ATP on the electrophoretic mobility of SSIIa from maize amyloplasts was observed in the presence and absence of glucan substrate in non- denaturing gels (Fig. 3.6 (A & B). Migration of SSIIa was reduced by the inclusion of amylopectin into the gel in all treatments (Fig 3.6B). Interestingly, in the absence of glucan, the SSIIa antibody detected two protein bands (bands ‘a’ and ‘b’) both in the untreated and APase-treated samples (Fig. 3.6 A). In the presence of amylopectin only a single prominent band (‘b’) was detected, the mobility of which changed (band ‘c’), following pre-treatment with ATP (Fig. 3.6B).

The mobility of SSIIa from maize amyloplast stroma in non-denaturing gels was analyzed after incubating with 1mM ATP or 100U APase, (APase conjugated to agarose beads was used, in order to remove it from the sample, after incubation, by centrifugation

(section 2.2.5.1)). The proteins in the lysate were electrophoresed in non-denaturing gels, with and without 0.3% amylopectin. The mobility of SSIIa was detected in immunoblots probed with anti-SSIIa specific antibody (Fig.3.6). As mentioned in the previous sections,

ATP significantly affected the mobility of native SSIIa. ATP- treated SSIIa migrated notably further in the gels as compared to the untreated and the APase treated samples

(Fig. 3.6 (A & B).

In order to test whether this mobility shift was due to phosphorylation of SSIIa and whether this effect could be reversed, an untreated sample of amyloplast lysate was pretreated with 100U APase to dephosphorylate proteins prior to incubation with 1mM

ATP (lane 4 in Fig. 3.6 (A & B). The agarose-immobilized APase was removed from the

134 reaction mixture by centrifugation prior to incubation with ATP. Interestingly the mobility of the rephosphorylated SSIIa was restored to the same position as the sample treated only with ATP.

Figure 3.6: Effect of glucan substrate on electrophoretic mobility of phosphorylated and dephosphorylated forms of maize SSIIa. After incubation with ATP (1mM) or insoluble APase (100U), stromal SSIIa in the amyloplast lysate was electrophoresed in non-denaturing polyacrylamide gels (A) without and (B) with 0.3% corn amylopectin. The mobility of SSIIa protein was detected by probing the immunoblots with maize anti-SSIIa specific antibody. The mobility of SSIIa protein is indicated by arrows. The protein bands are labelled alphabetically and are described in detail in the text.

3.3.4 Specificity of nucleotide phosphate-induced mobility shift of stromal maize SSIIa in non-denaturing gels

In order to confirm whether the mobility shift of native maize SSIIa in non- denaturing gels was specific to ATP, the effects of other mono, di and triphosphates on the mobility of native SSIIa were investigated (Fig. 3.7). While ATP could induce a detectable mobility-shift of SSIIa at a concentration as low as 2µM, AMP, ADP, GMP

135 and GDP failed to alter the protein’s mobility up to a concentration of 1mM (Fig. 3.7).

However, the nucleotide triphosphates GTP, UTP and CTP also caused a shift in SSIIa mobility at the highest concentrations used (0.5mM and1mM) (Fig. 3.7). The commercial preparations of these triphosphates were reportedly ≥95% pure. Therefore it is possible that they might contain as much as approximately 0.25% of ATP at even 5% of other triphosphates which could perhaps be responsible for the mobility-shift of SSIIa in non- denaturing gels at higher concentrations of these triphosphates. Thus a 1mM solution of these triphosphates (GTP, CTP and UTP) might contain 25-50µM of ATP which can cause a significant shift in SSIIa mobility. However, the ATP content of these commercial preparations was not determined directly.

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Figure 3.7: Effect of mono-, di- and tri-nucleotide phosphates on the mobility of maize SSIIa in non-denaturing PAGE. After incubating maize amyloplast lysates with varying concentrations of different nucleotide phosphates for 30-40 min, stromal SSIIa in the amyloplast lysates was electrophoresed in non-denaturing polyacrylamide gel containing 0.3% corn amylopectin. The mobility of SSIIa protein was detected by probing the immunoblots with maize anti-SSIIa specific antibody. SSIIa protein is indicated by arrows.

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3.3.5 Effect of protein kinase inhibitors on ATP- induced polyacrylamide gel mobility shift of maize SSIIa To determine whether the ATP induced mobility shift of native SSIIa was due to post-translational protein phosphorylation and whether protein kinases are involved in the process, maize amyloplast lysates were pretreated with known protein kinase inhibitors (5-fluorosulfonylbenzoyl-5΄adenosine) (10mM) and K252a (staurosporine)

(100µM) prior to incubation with ATP followed by electrophoresis on non-denaturing gels containing corn amylopectin.

In order to optimize the concentration of ATP to be used in order to visualize any possible effect of protein kinase inhibitors, a range of ATP concentrations was used

(Fig. 3.8). At higher concentrations of ATP (100µM and 1mM) the mobility shift of

SSIIa was unimpaired by the concentrations of FSBA (10mM) and K252a (100µM) used

(Ohto and Nakamura, 1995; Adams and Parker, 1992). At lower concentration of ATP

(20µM) a portion of the SSIIa protein remained in its initial location (Fig. 3.8). 25µM

ATP proved optimal in inducing a significant alteration in SSIIa mobility which was susceptible to inhibition by FSBA and K252a at the concentrations used (Fig 3.8 and Fig.

3.9).

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Figure 3.8: Effect of protein kinase inhibitors and ATP concentration on the electrophoretic mobility of maize SSIIa. Stromal proteins in the maize amyloplast were incubated with known protein kinase inhibitors, FSBA (5- fluorosulfonylbenzoyl-5΄adenosine) (10mM) and K252a (staurosporine) (100µM) for 20 min at room temperature prior to the incubation with various concentrations of ATP (40 min.). Proteins were separated by non- denaturing gel electrophoresis and SSIIa identified by immunoblotting.

Figure 3.9: Effect of protein kinase inhibitors on the ATP- induced, mobility shift of maize SSIIa. Stromal proteins in the maize amyloplast were incubated with known protein kinase inhibitors, FSBA (5-fluorosulfonylbenzoyl-5΄adenosine) (10mM) and K252a (staurosporine) (100µM) prior to incubation of amyloplast lysates with ATP (25µM). Proteins were separated by non- denaturing gel electrophoresis and SSIIa identified by immunoblotting.

139

3.3.6 Effect of phosphatase inhibitors on the mobility of maize SSIIa in non- denaturing gels

The phosphatase inhibitor cocktail, Phosphatase Arrest™ (G Biosciences) is a broad-spectrum phosphatase inhibitor cocktail consisting of known phosphatase inhibitors including sodium fluoride, sodium vanadate and sodium pyrophosphate which targets serine/threonine- specific, tyrosine- specific and dual- specificity phosphatases. In order to determine the effect of the individual components of the phosphatase inhibitor cocktail on the mobility of maize SSIIa on non-denaturing gels, maize amyloplast lysates were treated with 25mM of either sodium fluoride, sodium vanadate or sodium pyrophosphate, prior to electrophoresis on non-denaturing gels containing corn amylopectin. In the samples treated with phosphatase inhibitor cocktail (10µl/ml of the lysate), and in the absence of ATP, a major portion of SSIIa protein migrated further through the gel, comparable to the ATP-treated (25µM) sample (Fig. 3.10). Among the three phosphatase inhibitors, tetra-sodium pyrophosphate most notably induced the phosphorylation-dependent mobility shift of SSIIa on amylopectin gels, i.e. SSIIa protein in the tetra-sodium pyrophosphate treated amyloplast sample behaved like the ATP- treated (25µM) and phosphatase inhibitor cocktail treated forms, and migrated further in to the gel (Fig. 3.10). Sodium fluoride and sodium metavanadate had no effect on the migration of SSIIa (Fig. 3.10). The results indicate that sodium pyrophosphate is the key phosphatase inhibitor in the cocktail mixture. Given that no ATP had been added to the samples containing phosphatase inhibitor, this raises the question of what is responsible for this mobility shift, and will be considered further during the Discussion.

140

Figure 3.10: Effect of phosphatase inhibitors on the electrophoretic mobility of SSIIa from maize amyloplast lysates. Maize amyloplast lysates were incubated with 25mM of each phosphatase inhibitor. An untreated and ATP (25µM) treated sample were electrophoresed for comparison. Proteins were separated by non-

denaturing gel electrophoresis and SSIIa identified by immunoblotting.

3.3.7 Glucan binding properties of maize SSIIa under conditions of phosphorylation and dephosphorylation

Affinity gel electrophoresis was conducted to quantify the dissociation constant

(Kd) of maize stromal SSIIa pre-treated with ATP (1mM) or APase (100U) (Fig.3.11 and

Fig. 3.12) by measuring its relative migration (Rm) on non-denaturing gels in the presence of different concentrations of α-(1-4) –linked glucans (amylopectin and glycogen). The migration of SSIIa on glucan containing gels was visualized by immunoblotting and probing with anti-SSIIa specific antibody. Relative migration (Rm) of SSIIa was

141 determined in reference to the distance travelled in the absence of the substrate in each experimental condition. The reciprocal of Rm values of the ATP- and APase- treated samples of SSIIa were plotted against the concentration of glucan substrate in the gels and the dissociation constant (Kd) was determined (Fig 3.12 and Table 3.3).

Irrespective of the presence of glucan substrate in the gels, the ATP-treated form of SSIIa migrated further on native gels compared to the untreated and the APase treated forms (Fig. 3.11 A and B). As the concentration of corn amylopectin increased in the gels, the migration of both forms of SSIIa was retarded (Fig. 3.11A).

Irrespective of the concentration of glycogen present in the gels, the electrophoretic migration of SSIIa was unmodified by glucan (Fig. 3.11B); hence the dissociation constant for rabbit liver glycogen could not be determined (Fig. 3.12B).

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Figure 3.11: Determination of dissociation constant of maize SSIIa for amylopectin and glycogen by affinity gel electrophoresis. After incubation with ATP (1mM) or APase (100U), amyloplast lysates were electrophoresed in non- denaturing polyacrylamide gels containing various concentrations of (A) corn amylopectin and (B) rabbit liver glycogen. SSIIa protein was detected by probing the immunoblots with maize anti-SSIIa specific antibody. The migration of SSIIa protein is indicated by arrows.

143

APase

Figure 3.12: Effect of phosphorylation on the dissociation constant (Kd) of maize SSIIa for (A) amylopectin and (B) glycogen. The relative migration (Rm) of SSIIa was determined from Fig. 3.11. The reciprocal of Rm was plotted against the substrate concentration [S] and the dissociation constant (Kd) was determined from the X-intercept.

144

The dissociation constant was determined for maize stromal SSIIa under conditions of phosphorylation and dephosphorylation are shown in the Table 3.3. ATP or

APase treatments did not significantly affect the Kd of SSIIa for amylopectin (Table 3.3).

Table 3.3 Dissociation constants (Kd) of maize stromal SSIIa for corn amylopectin under phosphorylating and dephosphorylating conditions

Substrate Untreated ATP APase (1mM) (100U)

Amylopectin Kd (mg/ml) 0.06±0.03 0.06±0.03 0.06±0.03

Results are means ± S.E.M. for five independent experiments; *P value (Students’ T test) = 0.130.

3.3.8 Preparation of radio-labelled ADP-[U-14C] glucose Quantitative estimation of the catalytic activity of maize SSIIa by 14C- labelled

ADP-glucose assay required ADP-[U-14C] glucose as the glucan substrate. Due to the unavailability of a cheaper source of this reagent commercially, ADP-[U-14C] glucose was synthesized from [U-14C] glucose-1-phosphate (PerkinElmer).

3.3.8.1 Production and purification of recombinant AGPase in E coli E. coli-derived AGPase cDNA (Glgc) built in the pETM-11vector was transformed into E. coli Arctic Express cells and the expression of the recombinant

AGPase was induced by 1mM IPTG as described in the section 3.2.6.2. Expression of

AGPase was qualitatively assessed by separating the proteins in the E coli cell lysates by

SDS-PAGE and comparing the expression levels of the induced cell cultures with

145 uninduced controls by Coumassie staining (Fig. 3.13). A significantly higher level of

AGPase expression was observed in induced E coli cell lysates when compared to the uninduced controls (Fig. 3.13). Recombinant AGPase from the induced soluble cell lysate was then purified using the Invitrogen Ni-NTA (Ni2+ -nitrilotriacetate) Purification

System following the manufacturer’s protocol. The purity of the eluted protein from the

His-tag purification column was examined by running the induced soluble fraction and eluates on SDS-PAGE followed by Coumassie staining (Fig. 3.13). Coumassie staining clearly showed that the eluted AGPase was pure and devoid of any contaminating E coli proteins (Fig. 3.13). The pyrophosphorolytic activity of AGPase was measured as described in 3.2.6.2 and the specific activity of the protein was determined. The specific activity of the expressed AGPase protein was 800 nmoles/min/mg of protein.

146

Figure 3.13: Expression and purification of recombinant AGPase from E coli. Approximately 5-10µg of the induced protein in the cell lysate was run on 10%

SDS-PAGE gel and the proteins were visualized by Coumassie staining. The induced cell lysate containing the expressed AGPase protein was subjected to by Ni-NTA (Ni2+ -nitrilotriacetate) His-tag purification and the eluted pure protein was electrophoresed on 10% SDS-PAGE gel along with the induced cell lysates and uninduced controls. The induced AGPase protein in the cell lysate and the eluted pure protein from the His-tag purification column are shown by the arrows.

147

3.3.8.2 Synthesis of ADP- [U14C] Glucose from recombinant AGPase ADP-[U-14C] glucose was synthesized from [U-14C] glucose -1-phosphate

(PerkinElmer) using purified E. coli AGPase as described in section 3.2.5. Following the synthesis assay, ADP-[U-14C] glucose present in the supernatant was collected and analyzed by TLC for efficiency of conversion. Autoradiography of the developed TLC was performed by incubating the TLC sheet on an X-ray film in the dark for 10 days at -

80°C. The developed TLC and its autoradiograph are shown in Fig. 3.14. The synthesized

ADP-[U-14C] glucose migrated to the same position as the standard ADP-glucose disodium salt (≥95% pure) obtained from Sigma (Fig. 3.14 A). [U-14C] Glucose -1- phosphate did not migrate from the origin (Fig. 3.14 A and B). The radioactivity of the synthesized ADP-[U-14C] Glucose was measured using the Beckman LS 6500 Liquid

Scintillation counter and estimated as 0.027 µCi/µl and was equivalent to the radioactivity of the commercial ADP-[U-14C] Glucose (0.025 µCi/µl) formerly manufactured by Perkin Elmer.

148

Figure 3.14: Synthesis of ADP-[U-14C] glucose from [U-14C] glucose -1- phosphate. Following the ADP-[U-14C] glucose synthesis reaction, the ADP-[U- 14C] glucose present in the supernatant was collected and TLC was performed to check the efficiency of conversion as described in section 3.2.4.2. (A) Samples visualized as separate spots on developed TLC sheet following spraying with

Naphthoresorcinol (Dihydroxy-naphthalene) –H3PO4 mixture and incubation at 100°C for few minutes until the spots developed. (B) An autoradiograph was obtained by incubating the TLC sheet on an X-ray film in the dark for 10 days at - 80°C.

149

3.3.9 Activity of immunopurified SSIIa from maize amyloplast stroma under conditions of phosphorylation and dephosphorylation

SSIIa was immunopurified from maize amyloplast stroma which had been pretreated with ATP (1mM) or alkaline phosphatase (100U), using peptide specific anti-

SSIIa antibodies. Protein-A-Sepharose bead- protein complex was analyzed by SDS-

PAGE and immuno-detection with antibodies specific to various maize SSs (for all the biological replicates of this experiment) to confirm that only SSIIa was immunopurified

(Fig. 3.15).

Figure 3.15: Immunopurification of maize SSIIa from amyloplast stroma. SSIIa was immunopurified from maize amyloplast stroma pre-treated with ATP (1mM) or alkaline phosphatase (100U), using peptide specific anti-SSIIa antibodies and Protein -A-Sepharose. Immunopurified SSIIa protein was analyzed by SDS-PAGE and immunoblotting with antibodies specific to various maize SSs (viz., SSI, SSIIa,

SSIII and SSIV).

150

The catalytic activity was measured using ADP-(U-14C) glucose and 0.3% maize amylopectin as the glucan primer as described in the section 3.2.6. ATP treatment enhanced the catalytic activity of immunopurified maize stromal SSIIa significantly, whilst APase reduced it (Fig. 3.16). The effects of ATP and APase on the catalytic activity of SSIIa were statistically significant at a confidence level of α ≤ 0.05. In order to determine whether there was any residual APase activity that might interfere with the

ADP-(U-14C) glucose assay, other control experiments were conducted. Equal volumes of

ATP-treated and APase-treated samples were mixed, as well as untreated and APase treated samples and the catalytic activity of SSIIa measured. The ATP- APase mixture exhibited approximately half the activity (4.4 nmoles/µg/h) compared to the ATP-treated sample (8.5 nmoles/µg/h), as would be expected for a 50:50 mix, and was significantly higher than the APase treated sample (0.7 nmoles/µg/h). Similarly the untreated-APase mixture exhibited activity (1.7 nmoles /µg /h) commensurate with a 50:50 mix obtained by combining the samples (Fig. 3.16). The advantage of using Agarose- conjugated

APase was that it could be removed from the reaction mixture after incubation by centrifugation. Therefore, the results showed that apart from its direct effect on dephosphorylating SSIIa, APase didn’t exhibit any residual activity and did not interfere with the SS activity assay (Fig. 3.16). The catalytic activity of immunopurified untreated

SSIIa (2.3 nmoles/µg/h) was of the same order of magnitude to that previously determined for immunopurified wild type stromal maize SSIIa (4.3 nmoles/µg/h) by Liu et al. (2012b).

151

Figure 3.16: Effect of protein phosphorylation on the catalytic activity of immunopurified maize stromal SSIIa. SSIIa was immunopurified from maize amyloplast stroma, pre-treated with ATP (1mM) or alkaline phosphatase (100U). Catalytic activity was measured using 2mM ADP-(U-14C) glucose and 0.3% maize amylopectin as the glucan primer as described in section 3.2.5. In order to determine whether there was any residual APase activity that could interfere with the assay, equal volumes of ATP-treated and APase- treated, as well as equal volumes of untreated and APase- treated samples were mixed and incubated for 20 min at 25°C and the activity of SSIIa determined. Results are the means ± S.E.M. of three to five biological replicates. The catalytic activity values were significantly different at P≤ 0.05.

152

3.3.10 Kinetics of immunopurified SSIIa under conditions of phosphorylation and dephosphorylation

In order to study the kinetics of the immunopurified maize SSIIa for its substrates under phosphorylating (ATP-) and dephosphorylating (APase- treated) conditions, its catalytic activity was determined at various concentrations of corn amylopectin and

ADP-[U-14C] glucose. SSIIa exhibited Michaelis-Menten kinetics under all conditions for both amylopectin and ADP-glucose (Fig. 3.17 A&B). The Km and Vmax values of untreated, ATP- and APase-treated SSIIa were estimated from Eadie-Hofstee plots by plotting reaction velocity (V0) versus V0/[S] (Fig. 3.18 & 3.19). The y-intercept gives the maximal velocity (Vmax) achieved by the protein under each treatment condition while the x-intercept provided Vmax/Km (Fig. 3.18 &3.19). The Vmax and Km values were estimated from Eadie-Hofstee plots for all treatments and for all the replicates and analyzed statistically. The kinetics of immunopurified maize SSIIa under conditions of phosphorylation and dephosphorylation are summarized in Table 3.4.

APase treatment significantly reduced both the Km value (2.5 fold) and the maximal velocity (Vmax) (four fold) of SSIIa for the glucan primer (corn amylopectin) significantly (Table 3.4 & Fig. 3.17A; 3.18) compared to the untreated enzyme.

Nevertheless, ATP treatment did not affect the Km value of SSIIa (Table 3.4 & Fig.

3.17A; 3.18) for amylopectin. However, the Vmax of the ATP- treated SSIIa for amylopectin was significantly higher (3 fold) compared to the untreated control, (Fig.

3.17A; 3.18) and was 12-fold higher than the sample which was dephosphorylated with

APase (Table 3.4).

153

APase treatment also produced a statistically significant reduction in the Km value of SSIIa for ADP-glucose (Table 3.4). However, the Km value of ATP-treated SSIIa for

ADP-glucose remained unchanged compared to the control (Table 3.4). The Vmax of

SSIIa for ADP-glucose was significantly (four fold) enhanced by ATP (Table 3.4; Fig.

3.17B; 3.19) compared to the untreated enzyme. The p values obtained by paired

Students’ T-test analysis indicated that Km values were significantly different (p value <

0.05) between untreated and APase- (p= 0.003) and between ATP- and APase- (p= 0.004) treated SSIIa for amylopectin, and between untreated and APase-treated SSIIa (p= 0.02) for ADP-glucose. Vmax values were significantly different (p value < 0.05) between all the treatments for both substrates. The Km values of immunopurified, untreated, stromal maize SSIIa for amylopectin and ADP-glucose determined here were comparable to those previously determined for recombinant maize SSIIa (Table 3.1) (Imparl-

Radosevich et al.,1999a) (Table 3.4).

154

Figure 3.17: Michaelis-Menten kinetics of immunopurified maize SSIIa for (A) amylopectin and (B) ADP-glucose under conditions of phosphorylation and dephosphorylation. SSIIa from maize amyloplast stroma, pretreated with ATP (1mM) or APase (100U), was immunopurified using anti-SSIIa specific antibodies and the catalytic activity measured using ADP-[U-14C] glucose assay at various concentrations of corn amylopectin and ADP-[U-14C] glucose. 155

Table 3.4 Kinetic constants of immunopurified maize SSIIa under conditions favouring protein phosphorylation or dephosphorylation

Treatments Km Vmax

Amylopectin

Untreated 0.20±0.05 *3.83 ±0.16

(Km recombinant maize SSIIa – 0.16 mg/ml (Imparl-Radosevich et al., 1999a) ATP (1mM) 0.15±0.02 *12.0±0.07 APase (100U) *0.08±0.04 *0.93±0.07 ADP-Glucose

Untreated 0.30±0.05 *2.2±0.07

(Km recombinant maize SSIIa – 0.11 mM (Imparl-Radosevich et al., 1999a) ATP (1mM) 0.30±0.01 *8.2±0.15 APase (100U) 0.20±0.02 *0.7±0.03

Results are means ± S.E.M. for three independent experiments; For ADP-Glucose, Km is expressed in mM ADPGlc and for amylopectin Km is expressed in mg/ml of gelatinized amylopectin. Vmax values are in nmol/h/µg protein. Paired (Students’ T-test) was conducted and p values were determined. * Km and Vmax values that are significantly different (P value < 0.05) are indicated.

156

Figure 3.18: Effect of protein phosphorylation on the Km and Vmax of SSIIa for amylopectin. SSIIa from maize amyloplast stroma pretreated with ATP (1mM) or APase (100U) was immunopurified and the catalytic activity measured using the ADP-[U-14C] glucose assay at various concentrations of corn amylopectin. The rate of reaction (V0) (nmoles/h/µg protein) was plotted against V0/[S] (S- substrate concentration) under (A) untreated, (B) ATP-treated and (C) APase-treated conditions to obtain the Eadie-Hofstee plot for each replicate. The Vmax and Km values obtained from three independent replications were statistically analyzed using Students’ T- test.

157

Figure 3.19: Effect of protein phosphorylation on the Km and Vmax of SSIIa for ADP- glucose. SSIIa from maize amyloplast stroma pretreated with ATP (1mM) or APase (100U) was immunopurified and the catalytic activity measured using the ADP-[U-14C] glucose assay at various concentrations of ADP-glucose. The rate of reaction (V0) (nmoles/h/µg protein) was plotted against V0/[S] (S- substrate concentration) under (A) untreated, (B) ATP-treated and (C) APase-treated conditions to obtain the Eadie- Hofstee plot for each replicate. The Vmax and Km values obtained from three independent replications were statistically analyzed using Students’ T- test.

158

3.4 Discussion

The previous chapter (Chapter 2) provided evidence that SSIIa in maize amyloplast stroma can be post- translationally modified by protein phosphorylation. The results described in this chapter present direct evidence that protein phosphorylation has a significant effect on the regulation of maize SSIIa activity. Application of exogenous

ATP and APase were shown to have direct influence on the catalytic activity of SSIIa in samples of maize amyloplast stroma, strongly supporting the possibility of its post- translational regulation by protein phosphorylation.

Comparative analysis of SS activity and identification of stromal maize SSIIa pretreated with ATP or APase, using non-denaturing zymograms, revealed that exogenous ATP significantly altered the mobility of SSIIa on native gels, with or without glucan substrates (Fig.3.2 (1-3 B) & Fig. 3.6). During non-denaturing gel electrophoresis, separation of a protein depends on several factors including its size, shape, and native charge. Therefore, any processes that alter either the charge or the conformation of SSIIa in this case, including post translational modifications like protein phosphorylation, self- aggregation, and interactions with other proteins could influence its mobility (Hames,

1990; Tetlow et al., 2004a, 2008; Hennen –Biewagen et al., 2008; 2009; Liu et al., 2009).

Importantly, the ATP-induced mobility shift of SSIIa could be restored when a dephosphorylated (APase-treated) sample was rephosphorylated (ATP-treated) (Fig. 3.6

A&B lane 4). This argues strongly that the observed mobility shift of SSIIa was produced by protein phosphorylation-induced modification and that the effect could be reversed.

159

Moreover, ATP could induce the mobility shift of maize stromal SSIIa in non- denaturing gels even at concentrations as low as 2-5µM (Fig. 3.4 and Fig. 3.7), a concentration comparable to the Km of protein kinases for ATP (Traut, 1994; Hardie,

1996; Huber, 2007; Moorhead et al., 2009). The ATP- induced mobility shift was evident even after a very short period of incubation (as low as 1 min) (Fig. 3.5) suggesting an enzyme mediated activity. The failure of other mono, di and triphosphates (AMP, ADP,

GMP, GDP, GTP, UTP and CTP) to produce mobility shift of native SSIIa (Fig. 3.7) at physiological concentrations (µM) reinforced the hypothesis that the mobility shift of maize SSIIa resulted from the post translational modification of the protein by phosphorylation. It is argued that the observed mobility- shift of SSIIa treated with higher concentration of triphosphates (other than ATP) could be accounted for by the contamination with ATP present in these commercial preparations. Endogenous ATP concentration in a cell typically ranges from 1-10mM (Bies and Newsholme, 1975). Blatt

(1987) estimated an ATP concentration of 1.3mM in plant cytoplasm; whereas Song et al. (2006) found an increase in the extracellular ATP levels of up to 40µM in wounded

Arabidopsis roots. Even though techniques like NMR (Nuclear Magnetic Resonance), micro-dissection and micro-assay were developed in the past to measure subcellular concentrations of metabolites such as nucleotides in leaves (Stitt et al., 1989) and potato tubers (Tiessen, 2000; Farre et al., 2001; Tiessen et al., 2002), not many studies have successfully analyzed the exact concentration ranges of ATP and other triphosphates in developing seed endosperms (Tiessen et al., 2012). Analyzing the effect of illumination on the adenine nucleotide levels in the cytosol, chloroplasts and mitochondria of wheat leaf protoplasts, Stitt et al. (1982) reported that ATP to ADP ratio in the cytosol is

160 significantly higher than that in the mitochondrial matrix or the chloroplast stroma, and that these nucleotides are equilibrated by adenylates kinase in the stroma and cytosol, but not in the mitochondria. It was also found that stromal ATP/ADP quotient (3.14) was significantly higher in light, than in the dark (1.12), while the cytosolic ATP/ADP decreased in the light (6.44 in the light and 9.18 in the dark) (Stitt et al., 1982). This observation contradicted the general assumption that export of ATP from chloroplast photosynthesis, increases the ATP/ADP ratio in the cytosol, which then inhibits mitochondrial oxidative phosphorylation (Stitt et al., 1982). In a recent study, using a non-aqueous fractionation (NAF) method, Tiessen et al. (2012) determined the cytosolic and plastidial concentrations of nucleotide triphosphates, and other metabolites in the pathway of sucrose to starch conversion, in a developing barley endosperm. While ADP and AMP were located mainly in the plastid, most of the other nucleotides and metabolites were located in the cytosol (Tiessen et al., 2012). The observed cytosolic and plastidial concentrations of the various nucleotide phosphates are; ATP (456 µM and 66

µM), ADP (93 µM and 185 µM), AMP (98 µM and 109 µM), UTP (229 µM and 14

µM), GTP (171 µM and 13 µM ) , GDP (81 µM and 6 µM ), and GMP (40 µM and 16

µM), respectively (Tiessen et al., 2012). However, phosphorylation of proteins mediated by endogenous protein kinases is a dynamic process and occurs at lower physiological concentrations (µM) of ATP (Hardie, 1996; Huber, 2007; Moorhead et al., 2009).

Interestingly in the current study, protein kinase inhibitors, FSBA and K252a prevented the phosphorylation-dependent mobility shift of SSIIa on non- denaturing amylopectin containing gels at low ATP concentrations (Fig. 3.9). FSBA (5- fluorosulfonylbenzoyl-5΄adenosine) and K252a (staurosporine) are broad spectrum

161 protein kinase inhibitors that can competitively inhibit major serine/threonine protein kinases (Adams and Parker, 1992; Ohto and Nakamura, 1995; Tapley et al., 1992). While

K252a is a reversible and ATP-competitive inhibitor which acts against multiple protein kinases, FSBA covalently modifies a conserved lysine present in the ATP binding site of most protein kinases and inhibits them competitively (Adams and Parker, 1992). This again suggests that the ATP-induced mobility-shift of stromal maize SSIIa could be a result of the action of endogenous protein kinase(s) present in the amyloplasts.

Among the three components of commercial phosphatase inhibitor cocktail,

Phosphatase ArrestTM viz., sodium fluoride, sodium metavanadate and tetra-sodium pyrophosphate, tetra sodium pyrophosphate alone produced a shift in the electrophoretic mobility of SSIIa, (similar to the ATP-induced mobility shift) even in the absence of exogenous ATP (Fig. 3.10). However, a similar shift in SSIIa mobility was also observed in phosphatase cocktail treated samples in the absence of exogenous ATP. Tetra-sodium pyrophosphate was effective in inhibiting endogenous phosphatases in the amyloplast lysate that might otherwise interfere with the phosphorylation of SSIIa, and prevent its

ATP-induced mobility shift on non- denaturing gels. Hence, subsequently, in most of the experiments conducted in this study, the phosphatase inhibitor cocktail was replaced by

25mM tetra-sodium pyrophosphate which produced similar results to the commercial cocktail.

The fact that ATP could cause this mobility shift independent of the presence of glucan substrate indicates that SSIIa could be subject to conformational changes (Fig.

3.6A). Protein phosphorylation can promote conformational changes of the target protein, and these changes can also alter the surface properties of the protein that affect self-

162 association or association with other proteins, thus affecting its mobility in a polyacrylamide gel (Johnson and Barford, 1993). Experimental evidence from previous studies emphasizes the potential importance of phosphorylation-dependent, functional protein-protein interactions between starch biosynthesizing enzymes (SSs and SBEs) during storage starch biosynthesis in cereal endosperms (Tetlow et al., 2004a; 2008;

Hennen –Biewagen et al., 2008; 2009; Liu et al., 2009; 2012a&b). Among the large number of pair-wise protein interactions detected in a developing maize endosperm during starch biosynthesis, SSIIa was found to strongly interact with SSI, SBEIIa and

SBEIIb (Hennen –Biewagen et al., 2008; 2009). In one of the identified high molecular weight protein complexes (670 kDa), SSIIa associated with SBEIIa and SBEIIb and SSIII interacted with this complex in a phosphorylation dependent manner (Hennen –Biewagen et al., 2008; 2009). Therefore a phosphorylation-induced mobility shift of maize SSIIa could be the result of conformational changes thereby affecting its interaction with other starch biosynthetic enzymes- most probably SSI, SSIII and SBEs (Hennen –Biewagen et al., 2008; 2009; Liu et al., 2009; 2012a). However, lack of co-migration of SSI with

SSIIa observed in this study, (Fig. 3.3) does not suggest that they are associated under the conditions employed.

Furthermore, the possibility of homo-oligomerisation cannot be ruled out.

Homooligomerisation (aggregation of monomeric protein units to form oligomers) has been previously reported in glycosyl transferases that might be critical for their function

(Hashimoto et al., 2010). Protein phosphorylation is found to generate conformational changes in glycosyl transferases, such as glycogen phosphorylase, resulting in the association of a pair of functional dimers to form a tetramer and affecting its enzymatic

163 activity (Barford 1991; Barford and Johnson, 1992). However there is no direct evidence available to date as to whether starch synthases like SSIIa undergo homo-oligomerisation.

Irrespective of the presence of glucan substrate, stromal SSIIa in the untreated and

APase treated amyloplast lysates migrated similarly (Fig 3.6) in non- denaturing gels.

This suggests that most, if not all, of the maize stromal SSIIa exists in a dephosphorylated form following amyloplast purification. However this might not be a true indication of the in vivo state of the protein, as the turnover of the phosphate groups attached to SSIIa is likely to change during amyloplast extraction.

Comparison of the activities of phosphorylated and dephosphorylated forms of

SSIIa in SS activity zymograms suggested that ATP- treated (phosphorylated) SSIIa exhibited higher activity compared to the untreated and APase treated forms (Fig. 3.2 (1

& 3 A)). ATP treated amyloplast samples exhibited multiple activity bands corresponding to SSIIa activity, as compared to the untreated and APase- treated samples in non-denaturing gels with amylopectin and starch (Fig.3.2 (1-3) A). Moreover, the major SSIIa activity band (‘b’) observed in the untreated, wild-type, maize amyloplast sample in amylopectin-containing zymogram gels (Fig. 3.3), was absent in the untreated amyloplast sample from the sugary-2 mutant (lacking SSIIa), which further supports the contention that this particular SS activity is due to SSIIa. In the ATP- treated amyloplast lysates (Fig.3.2 (1 A&B)), another band of SS activity (‘d’) was apparent just below the major SSIIa activity band (‘c’) in the zymograms containing amylopectin and starch.

Interestingly, this band of SS activity showed a minor interaction with SSIIa specific antibody in the corresponding western blot of the amylopectin containing gel. This indicated that the observed activity band (‘d’) could either be due to SSIIa alone or

164 possibly due to other SS isoforms (which are not detected here) that co-migrate. In a study in wheat, Tetlow et al. (2004a) observed that the catalytic activities of SBEIIa and

SBEIIb were substantially enhanced by ATP and reduced by APase (Fig 3.1). In the present study, APase didn’t produce any visible increase or decrease in the intensity of activity bands of SSIIa (Fig. 3.2). However, the activities of ATP and APase-treated

SSIIa could not be compared quantitatively by zymogram analysis.

An additional protein band (‘a’) was detected by anti-SSIIa specific antibodies just above the major SSIIa band (‘b’) (Fig. 3.6 A), in both untreated and APase treated lanes in gels lacking the glucan substrate. These bands might represent different forms of

SSIIa with varied mobility and phosphorylation state under conditions when amyloplasts are purified. Two differentially migrating forms of SSIIa have been previously reported by analyzing starch granule proteins in maize (Zhang et al., 2004; Grimaud et al., 2008), and rice (Umemoto and Aoki, 2005), suggesting that SSIIa (granule bound) is differentially, post-translationally modified by phosphorylation. The present study has demonstrated that starch granule bound SSIIa is phosphorylated (Fig. 2.6). Similar to the report by Grimaud et al. (2008), the differential electrophoretic mobility of the forms of stromal SSIIa observed in the present study, could be the consequence of their differential phosphorylation states. It has been previously demonstrated that in cereal endosperm, the differences in phosphorylation- dependent protein complexes containing SSIIa are mirrored in the supplement of these enzymes detected within the granule, strongly supporting the role of phosphorylation driven functional heteromeric complexes containing SSIIa in amylopectin synthesis (Tetlow et al., 2004a; Grimaud et al., 2008;

Liu et al., 2009).

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In the present study, the SS activity bands on the zymogram gel containing rabbit liver glycogen failed to align with the corresponding protein bands detected by anti-SSIIa antibodies in the immunoblots (Fig. 3.2 (2A&B). Likewise, increasing concentrations of rabbit liver glycogen did not affect the electrophoretic migration of

SSIIa in the non- denaturing gels (Fig.3.11B and 3.12B). In a similar study detecting the activity of SSIIa in the wild type and transgenic rice endosperms, Fujita et al. (2012) observed that it was difficult to detect SSIIa activity by native PAGE and staining, when oyster glycogen was incorporated as glucan substrate in the zymogram gels. Being an animal storage carbohydrate, glycogen might not be a suitable glucan primer for SSIIa.

Previous studies have analyzed the differences in the primer affinity, primer preference and maximal velocities of various SSII isoforms (Imparl-Radosevich et al., 1999 a &b). It was observed that recombinant maize SSIIa exhibited higher Vmax for amylopectin than glycogen as the glucan primer (Table 3.1) (Imparl-Radosevich et al., 1999a).

Observations from zymogram analysis in this study suggest that stromal maize SSIIa has no affinity for rabbit liver glycogen. Glycogen from rabbit liver has been routinely used as the glucan primer for SS activity zymograms in many related studies with starch biosynthetic enzymes up to the present time (Buléon et al., 1997; Imparl-Radosevich et al., 1999 a &b; Cao et al., 2000; Commuri and Keeling 2001). The present study has demonstrated that glycogen is not a good substrate for determining SSIIa activity in non- denaturing zymograms.

Even though phosphorylated (ATP-treated) SSIIa migrated further in non- denaturing gels in the absence of any glucan substrate (Fig 3.6 A), its mobility was noticeably retarded with the incorporation of 0.3% corn amylopectin into the gel (Fig. 3.6

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B). Retardation of proteins in non- denaturing gels containing their oligosaccharide substrate is considered an indication of the affinity of the proteins towards their substrate

(Takeo and Nakamura, 1972; Matsumoto et al., 1990; Commuri and Keeling, 2001).

Analysis of the dissociation constants of ATP-treated and APase-treated forms of stromal maize SSIIa, using glucan affinity gel electrophoresis, showed that this was not affected by phosphorylation (Fig. 3.11A and Fig. 3.12A; Table 3.3). The dissociation constant

(Kd) is the concentration of substrate at which half of the substrate binding sites of an enzyme are bound with the substrate and hence Kd values are indirectly indicative of the affinity of an enzyme towards its substrate. Based on the results, it could be hypothesized that ATP might have caused significant conformational changes to SSIIa which perhaps affected its association with other starch biosynthetic enzymes and thereby resulted in its differential mobility, without significantly affecting its affinity towards amylopectin.

Vmax is the maximal rate of reaction catalyzed by an enzyme when all the enzyme active sites are saturated with substrate. Km is the substrate concentration at which the velocity of reaction reaches half of the maximal velocity (Vmax). APase substantially lowered the Km and Vmax of SSIIa for amylopectin and significantly lowered its Vmax for ADP-glucose (Table 3.4). However, ATP did not alter the Km value of SSIIa for amylopectin and ADP- glucose but significantly increased its Vmax (four fold) towards the glucan primer amylopectin and ADP-glucose (Table 3.4). Previous investigations on the kinetic properties of maize SSIIa (Imparl-Radosevich et al., 1999 a and b) have not taken into consideration the effect of protein phosphorylation on the catalytic activity and glucan affinity of native SSIIa in developing maize endosperm.

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The possibility of the presence of other starch biosynthetic enzymes (SSs and

SBEs) that physically interact and accompany SSIIa (upon phosphorylation or dephosphorylation) has to be accounted for, before concluding that the above observed effects of protein phosphorylation are direct effects of ATP and endogenous protein kinases on SSIIa protein itself. Therefore in order to investigate the consequences of exogenous ATP and APase on the functional aspects of stromal maize SSIIa (free from interfering proteins), SSIIa was immuno-purified from maize amyloplast lysates.

Immunopurification of SSIIa from maize amyloplast stroma using peptide-specific anti-

SSIIa antibody provided a convenient and straight forward method to investigate the implications of protein phosphorylation on the functional activity of maize SSIIa from endosperm tissue (Fig. 3.15). The absence of other starch biosynthetic enzymes that could interact with SSIIa protein on the Protein-A-Sepharose beads indicated that SSIIa was substantially immunopurified from those enzymes present in the amyloplast stroma

(Fig. 3.15).

Phosphorylation of stromal maize SSIIa by exogenous ATP caused more than

12-fold enhancement in its catalytic activity as compared to that of dephosphorylated

(APase-treated) SSIIa (Fig. 3.16) strongly suggesting that protein phosphorylation greatly enhances the catalytic activity of maize SSIIa. The stimulatory effect of ATP on the catalytic functions, and protein – protein interactions, of SBE isoforms during starch biosynthesis has been previously demonstrated in wheat (Tetlow et al. 2004a; 2008) and maize endosperms (Liu et al., 2009, 2012a). The ATP- dependent stimulation of the catalytic activity of maize SSIIa likely arises as a result of the effect of endogenous protein kinase(s) and suggests that protein phosphorylation might have significant

168 implications in regulating SSIIa during the process of starch biosynthesis in developing maize endosperm.

The major findings discussed in this chapter strongly indicate that protein phosphorylation regulates the catalytic function of maize SSIIa during starch biosynthesis. Protein phosphorylation essentially enhances the catalytic activity of stromal maize SSIIa. It also affects it electrophoretic mobility in non-denaturing gels.

Experimental evidence from previous investigations in cereal endosperm emphasizes the potential significance of functional protein – protein interactions between starch biosynthetic enzymes during the synthesis of starch (Tetlow et al., 2008, Hennen –

Biewagen et al., 2008; 2009; Liu et al., 2009; 2012 a&b). Many of the major starch biosynthetic enzymes are capable of associating with each other directly or indirectly, and SSIIa has been found to be a major component in several well defined functional starch biosynthesizing protein complexes in cereal endosperms (Tetlow et al., 2008,

Hennen –Biewagen et al., 2008; 2009; Liu et al., 2009; 2012b). The direct effects of protein phosphorylation on the regulation of SSIIa activity might also impact the physical association of this protein with other proteins in the starch biosynthetic pathway, thereby co-ordinating their functions to synthesize highly organized, semi crystalline amylopectin. Such functional protein interactions of SSIIa with other enzymes might explain the altered mobility and kinetic properties of maize SSIIa under conditions of phosphorylation and dephosphorylation. Biochemical investigations on the effect of protein phosphorylation on the interactions of maize SSIIa with other major starch biosynthetic enzymes to form functional, heteromeric protein complexes during starch biosynthesis are discussed in the following chapter.

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CHAPTER 4

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CHAPTER 4: THE EFFECT OF PROTEIN PHOSPHORYLATION ON THE INTERACTIONS OF MAIZE SSIIa WITH OTHER STARCH BIOSYNTHETIC ENZYMES

4.1 Introduction

The physiological significance of protein phosphorylation on the regulation and activity of SSIIa in wild type maize amyloplast stroma has been demonstrated in the previous chapters. Genetic and biochemical evidence indicate that the highly organized, and conserved architecture of amylopectin is probably the result of the actions of several combinations of interacting enzymes. These enzymes form components of functional heterocomplexes that might be active or inactive at different stages of development, depending on the post translational regulatory mechanisms affecting the individual enzymes (Colleoni et al., 2003; Dinges et al., 2003; Morell et al., 2003; Tetlow et al.,

2004b; 2008). Data from previous investigations on the mechanisms involved in starch biosynthesis in cereal endosperms are consistent with the existence of functional protein - protein interactions among the major starch biosynthetic enzymes, particularly between

SBE and SS isoforms (reviewed in Tetlow, 2011; Geigenberger, 2011). Recent evidence indicates that many of the major starch biosynthetic enzymes, including SSIIa, are capable of associating with each other directly or indirectly to form well defined functional starch biosynthesizing protein complexes in cereal endosperms (Tetlow et al.,

2008, Hennen –Biewagen et al., 2008; 2009; Liu et al., 2009; 2012a and b). Whilst the fundamental mechanisms of protein complex formation are largely unsolved, the evidence suggests that these physical protein-protein interactions are dependent on the

172 phosphorylation status of the participating enzymes (Tetlow et al., 2004a; 2008; Liu et al., 2009; Geigenberger, 2011).

Investigating functional protein-protein interactions among enzymes involved in starch biosynthesis in developing wheat endosperm, Tetlow et al. (2008) identified physical interactions between SBEs and SSs in wild type amyloplasts during the phase of active starch accumulation. Using co-immunoprecipitation and cross-linking strategies, these authors reported the existence of at least two distinct complexes containing SSI,

SSIIa and either of SBEIIa or SBEIIb. Gel permeation chromatography separation of amyloplast extracts demonstrated the presence of a 260 kDa protein complex which was found to contain SSI, SSIIa and SBEII forms, identified by mass spectrometry. Further, in vitro phosphorylation experiments with γ- [32-P] ATP showed that SSIIa was phosphorylated along with SBEII forms and importantly, this 260 kDa SS-SBE assembly was dissociated by dephosphorylation by exogenous APase (Tetlow et al., 2008).

Utilizing yeast two-hybrid assay, affinity purification with immobilized recombinant ligands, and co-immunoprecipitation, Hennen-Bierwagen et al. (2008) detected a large number of pair wise interactions between SSs and SBEs in developing maize endosperm- in particular: SSIIa/SSI, SSIIa/SBEIIa and SSIIa/SBEIIb (in addition to SSI/SSIII,

SSI/SBEIIb and SSIII/SBEIIa). Additionally, SSIIa, SBEIIb, and SBEIIa comigrated with SSIII in a high molecular weight complex (670 kDa) and in another high molecular weight (300 kDa) protein complex lacking SSIII, during GPC analysis (Hennen-

Bierwagen et al., 2008& 2009). Further analysis of the trimeric protein complex consisting of SSI, SSIIa and SBEIIb in maize endosperm revealed that SBEIIb in the complex is phosphorylated (Liu et al., 2009). Importantly, the association of SSIII with

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SSI, SSIIa and SBEIIb in the high molecular weight (670 kDa) complex was phosphorylation dependent (Hennen-Bierwagen et al., 2008& 2009). Analysis of many well characterized mutants in the starch biosynthetic pathway in maize with altered starch phenotypes, (Lee, 2004) strongly suggest the co-ordination and interdependence of the activities of these major enzymes during starch biosynthesis (Boyer and Priess, 1981;

Singletary et al., 1997; Gao et al., 1998; Tetlow et al., 2004a &2008). The mutations that eliminated any one of these starch biosynthetic enzymes (SSs or SBEs) or their activities, prevented others from assembling into high molecular weight multi-enzyme complexes and also resulted in distinct patterns of protein-protein interactions (Liu et al., 2009). For example, in maize, SBEIIb is the most abundant protein in the amyloplast stroma and the most predominant SBE isoform expressed in the endosperm (Mu et al., 2001). In the endosperm of a null mutant of maize, lacking SBEIIb protein (known as the amylose extender ( ae 1.1), the wild-type trimeric protein complex (SSI/SSIIa/SBEIIb) appears to be replaced by a different complex containing SSI, SSIIa, SBEI, SP and SBEIIa in which both SBEI and SP are phosphorylated (Liu et al.,2009). Significantly, the diversity in hetero-protein complexes in the amyloplast stroma is reflected in the complement of these enzymes within the starch granule matrix (Tetlow, 2011) e.g. in the ae 1.1 null mutant SBEI, SP and SBEIIa are found in the granules in addition to SSI and SSIIa.

These phosphorylated granule associated proteins are also the components of phosphorylation-dependent stromal hetero-protein complexes (Tetlow et al., 2004a;

Grimaud et al., 2008; Tetlow, 2011). This again strengthens the concept that phosphorylation-dependent, functional protein complexes comprise the functional units involved in amylopectin biosynthesis that become trapped within granules later during

174 the process (Liu et al., 2009). Recently, biochemical analysis of starch from an allelic variant of the ae- mutation, ae1.2, expressing a catalytically inactive form of SBEIIb

(catalytically inactive SBEIIb in ae1.2 lacks a 28 amino acid peptide (Val272–Pro299) and is unable to bind to amylopectin) showed altered starch granule morphology and physicochemical characteristics different from those of the ae1.1 mutant and the wild- type (Liu et al., 2012a). The organization and association of protein complexes of starch biosynthetic enzymes in ae1.2 endosperm were also distinct from that observed in wild type and ae1.1 amyloplasts, and this difference was also reflected in the composition of starch granule bound proteins. Unlike the ae 1.1 mutant, the presence of SP in the enzyme complexes and starch granule was not detected in ae1.2. Importantly, the formation of stromal hetero- protein complexes in the wild-type and ae1.2 endosperms was significantly enhanced by ATP, and reversed by APase, indicating a role for protein phosphorylation in their assembly. Even though the inactive SBEIIb failed to bind starch directly, it was strongly associated with the starch granule, as a result of physical association with other starch biosynthetic enzymes (Liu et al., 2012a). Interestingly, the granule bound SBEIIb was phosphorylated along with SBEI in ae1.2 endosperm (Liu et al., 2012a). These observations strongly support the hypothesis that the specific complement of heteromeric complexes of starch biosynthetic enzymes dictates the fine structure of the starch granule.

Recently in a study investigating the physicochemical properties of starch in maize sugary 2 mutant, (with a catalytically inactive SSIIa) Liu et al. (2012b) observed that the altered granule morphology and crystallinity and other physiochemical properties exhibited by these mutants was due to the loss of glucan binding ability of SSIIa. The

175 mutation in SSIIa also resulted in the inability of SSI and SBEIIb (the major trimeric protein complex in maize endosperm) to become granule bound (Fig. 2.1). In cross- linking experiments, SSIIa was found to be in close physical proximity to SBEIIb and

SSI, possibly being the core of the complex and influencing the ability of SSI and SBEIIb to bind amylopectin. This is strong evidence that these starch biosynthetic enzymes function as protein complexes during amylopectin synthesis, and that SSIIa is critical in guiding the other components of the complex towards the granule matrix (Fig. 2.1) (Liu et al., 2012b).

A diagrammatic representation of the functional protein- protein interactions between major starch biosynthesizing enzymes in cereal endosperm is given in Figure

4.1. It is postulated that the SS/SBE protein complexes function at the periphery of the growing starch granule, producing an assembly of short- to intermediate-sized glucan chain clusters which eventually form the semi-crystalline lamellae of starch granules within which the enzymes get entrapped. However there is currently no evidence as to whether they remain as protein complexes within the granular matrix (Tetlow, 2011).

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Figure 4.1: Protein–protein interactions between the major starch biosynthetic enzymes during starch granule formation in cereal endosperms (derived from Tetlow, 2011). The major amylopectin synthesizing enzymes in the amyloplast stroma are associated to form different functional protein complexes (described in the text). One such important trimeric protein complex (in wheat and maize) consisting SSIIa as the critical component (highlighted in colour) (Liu et al., 2009; 2012b) and SSI and SBEIIb is shown and its assembly is dependent on protein phosphorylation (Hennen-Bierwagen et al., 2008; Tetlow et al., 2004a; 2008). In the maize trimeric complex, SBEIIb is phosphorylated (denoted as ‘P’ symbols) (Liu et al., 2009) and in vitro dephosphorylation with alkaline phosphatase disrupts this protein assembly. The diagram also demonstrates how these proteins, which are present in the amyloplast stroma as heteromeric complexes, are partitioned within the starch granule matrix as ‘granule-associated’ proteins along with granule-associated GBSS.

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While the physiological importance of functional protein complexes and their direct influence on starch biosynthesis in cereal endosperms is not fully understood, it is believed that these heteromeric complex assemblies between the starch biosynthetic enzymes might improve the efficiency of amylopectin polymer synthesis (reviewed in

Tetlow, 2011; Geigenberger, 2011). This is because in a highly concerted process like starch biosynthesis, which involves the action of several enzymes, physical interactions between these enzymes, could facilitate substrate channeling, so that the product of one reaction becomes the substrate for the other within the complex. Liu et al. (2012b) proposed that the reduced SBEIIb activity observed in the maize sugary -2 mutant trimeric complex (containing SSI and inactive SSIIa) could therefore be a result of reduced substrate supply, possibly because the branching enzyme activity is dependent on the availability of elongated glucan chains supplied by SSI and SSIIa (Guan et al., 1997;

Liu et al., 2012b). This highly organized and coordinated action of starch biosynthetic enzymes, via functional protein complexes, might thus promote a favored three dimensional structure within the growing amylopectin polymer maintaining its crystallinity (Tetlow et al., 2008; Tetlow, 2011). These heterocomplexes of starch biosynthetic enzymes along with the amylopectin polymer might represent “carbohydrate chaperonins” during starch biosynthesis (Tetlow et al., 2008; Tetlow, 2011). The protein complexes also might possibly protect the growing glucan polymer from degradative enzymes present within the amyloplast (Tetlow, 2011).

Formation of heterocomplexes between starch biosynthetic enzymes through their interactions might also influence the kinetic properties of the individual participating

178 enzymes through conformational changes (Tetlow, 2011). The activities of SSs and SBEs were found mutually dependent during such physical interactions and the loss of the function of one isoform significantly decreased the activity of the other (Boyer and

Priess, 1981; Gao et al. 1998; Cao et al., 1999, 2000; Nishi et al., 2001). Again, loss of

SSIIa in wheat, barley and rice endosperm abolished the granule association of SSI,

SBEIIa and SBEIIb and caused a significant reduction in the synthesis of amylopectin molecules (Yamamori et al.,2000; Morell et al.,2003; Umemoto and Aoki, 2005) further indicating the functional interdependence of the specific enzymes within these protein complexes (Tetlow et al., 2008). Even though ATP-stimulated association of SSI, SSIIa and SBEIIb was consistent in both the wild type and sugary-2 maize amyloplasts, the presence of a catalytically inactive SSIIa is likely responsible for the reduction in the SS and SBE activities of the sugary-2 trimeric complex (Liu et al., 2012b). SSIIa being the central core of the complex might have had a physical influence on the conformation of both SSI and SBEIIb and perhaps played a key role in the functioning of this protein complex (Liu et al., 2012b). These findings are indicative of the physiological significance of multi-enzyme complexes in starch metabolism.

The major findings of the current study (presented in the previous chapters) clearly reveal the significant role of protein phosphorylation in regulating the catalytic function of maize SSIIa during starch biosynthesis. Phosphorylation by exogenous ATP in the presence of endogenous protein kinases in the amyloplast significantly enhances the catalytic activity of maize SSIIa. It also affects the electrophoretic mobility of SSIIa in non-denaturing gels, suggesting that significant changes in its configuration and/ or its interaction with other starch biosynthetic enzymes might have occurred by this post-

179 translational modification. Therefore, determining the relationship between SSIIa, its regulation by protein phosphorylation and the formation of functional protein complexes between SSIIa and other biosynthetic enzymes will provide a better understanding of how

SSIIa functions during starch biosynthesis in developing maize endosperm. It is hypothesized that SSIIa being the core and critical component of the functional trimeric protein complex (SSI/SSIIa/SBEIIb), its phosphorylation state affects its association with other enzymes of starch biosynthesis that function in the organization of the crystalline amylopectin structure. Chapter 4 therefore examines the effect of protein phosphorylation and dephosphorylation of maize SSIIa on its interactions with other starch biosynthetic enzymes.

4.2 Materials and Methods

4.2.1 Immunoprecipitation of SSIIa from maize amyloplast lysates SSIIa from wild type maize amyloplasts pretreated with ATP (1mM) or APase

(100U) was immunoprecipitated using peptide-specific anti maize SSIIa antibodies following the method described previously by Tetlow et al. (2004a). Purified maize SSIIa antibodies were added (30 µg/mL) to 1mL of maize amyloplast lysates (1-1.5mg/mL) and incubated for an hour on a rotator at room temperature. SSIIa was immunoprecipitated by adding 80-100 μL of 50% (w/v) Protein A-Sepharose slurry. The Protein A-Sepharose slurry was made by adding phosphate buffered saline, PBS (137 mM NaCl, 10 mM

Na2HPO4, 2.7 mM KCl, 1.8 mM KH2PO4, pH7.4), to the Protein A-Sepharose beads and incubating for 1hr at room temperature. The Protein A-Sepharose- antibody / protein complex was then centrifuged at 100 g for 2 min at 4°C in a refrigerated micro centrifuge. The resulting pellet was washed three times (1.3mL each) with PBS followed

180 by three washes with 10 mM HEPES/NaOH, pH 7.5 (at 100 g, 2 min centrifugation). The immunoprecipitated pellet, containing Protein-A-Sepharose- antibody/protein complex, was boiled in 2X SDS loading buffer for 8 min. The protein bound to the sepharose beads was analysed by SDS-PAGE and immuno-blotting. To detect the co-immunoprecipitation of SSIIa with other major starch biosynthetic enzymes including SSI and SBEIIb, the immunoblots were probed with anti- peptide antibodies specific for the major starch biosynthetic enzymes.

4.2.2 Chemical cross-linking to detect protein – protein interactions of SSIIa Chemical cross-linking using wild type maize amyloplast lysates was conducted following the procedure described by Tetlow et al. (2008). For cross-linking experiments amyloplasts were prepared in EDTA- and DTT- free buffers. After incubating with ATP

(1mM) or APase (100U) for 30-40 min at room temperature, amyloplast lysates were immediately incubated with 1 mM of the homo-bifunctional cross-linking reagent Bis-

(sulfosuccinimidyl) suberate (BS3), (Pierce/BioLynx) on a rotator at room temperature for 30 min. The cross-linking reaction was quenched by the addition of 1M Tris, pH 8, to the reaction mix. The cross-linked samples were then mixed with SDS sample buffer, the proteins separated by 10% SDS-PAGE, and immunoblots probed with peptide specific antibodies to SSIIa, SSI, SBEIIb and SBEI.

4.2.3 Gel permeation chromatography (GPC) Wild type maize amyloplast lysates pretreated with ATP (1mM) or APase (100U) were separated through a Superdex 200 10/300GL gel permeation column pre- equilibrated with rupturing buffer containing 5mM DTT (section 2.2.2.1), using an

AKTA-FPLC system (Amersham Pharmacia Biotech) at 4°C. Amyloplast lysates (1.5-2 mg/ml total protein concentration) were loaded onto the GPC column (flow rate of 0.5

181 mL/min) in a final volume of 500 µL and fractions of 500 µL were collected using Frac

950 fraction collector (Amersham Pharmacia Biotech). The column was routinely calibrated with commercially available protein standards of range 13.7 kDa to 440 kDa purchased from GE Healthcare as Gel Filtration calibration kits containing low and high molecular weight standard proteins. Following GPC, the fractions were boiled in 2X SDS loading buffer for 8 min and proteins separated by SDS-PAGE. Western blots were probed with peptide specific antibodies for various starch biosynthetic enzymes including

SSIIa to detect their elution profiles under untreated, ATP- and APase- treated conditions.

4.2.4 Phospho- protein affinity (Phos-tagTM) acrylamide gel electrophoresis

Phosphorylation state of maize SSIIa in the high and low molecular weight fractions of amyloplast lysates separated previously by GPC (section 4.2.3) was detected by using affinity-based Mn2+ Phos-tagTM (Wako chemicals Ltd.) poly-acrylamide electrophoresis according to the method described in section 2.2.5.2. Stromal SSIIa from amyloplast lysates pre-treated with ATP (1mM) or alkaline phosphatase (APase) (100U) were separated by GPC as described in section 4.2.3. The high molecular weight (HMW)

(Fraction Nos: 19, 20, 21, 22) and low molecular weight (LMW) (Fraction Nos: 23, 24,

25, 26) forms of SSIIa (detected from GPC results Fig. 4.5) from the untreated, ATP- and

APase-treated samples were pooled and loaded on an 10% SDS gel containing 50µM

Phos-tag ligand (Mn2+ Phos-tagTM (Wako chemicals Ltd.) and electrophoresed. The phosphorylation-dependent mobility shift of SSIIa was observed by immuno-blot analysis using SSIIa- specific antibodies.

4.2.5 Substrate affinity electrophoresis of GPC fractions

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Following GPC separation, SSIIa in the HMW and LMW fractions (from untreated GPC samples) were separated on non-denaturing native 5% (w/v) polyacrylamide gels (Table 3.2) containing the α-amylase inhibitor Acarbose (Prandase,

Bayer) (80 µL in 10 mL gel solution), and different concentrations of corn amylopectin as the glucan primer. The migration distance of native maize SSIIa was measured after immunoblotting and probing with anti-SSIIa specific antibodies. Dissociation constants of the enzyme-substrate complex (Kd) and affinity (1/Kd) were calculated from the retardation of the electrophoretic mobility of SSIIa in the HMW and LMW forms from the untreated, ATP and APase GPC fractions, following the methods described in section

3.2.3. The experiment was repeated three times with different biological replicates.

Students’ paired-sample t tests were used to compare the Kd and affinity constants.

4.2.6 Co-immunoprecipitation of SSIIa in the HMW and LMW GPC fractions with other major starch biosynthetic enzymes SSIIa was immunoprecipitated from the HMW fractions 20 and 22 and the LMW fractions 25 and 26 using anti- SSIIa specific antibodies as described in section 4.2.1. To detect the co-immunoprecipitation of SSIIa with other major starch biosynthetic enzymes in these GPC fractions from the untreated, APase- and ATP-treated samples, the immunoblots were probed with anti- peptide antibodies specific for SSI and SBEIIb following SDS-PAGE.

4.3 Results

4.3.1 Determining the effects of protein -phosphorylation on protein–protein interactions of SSIIa SSIIa from wild type maize amyloplast stroma pretreated with ATP (1mM) or

APase (100U) was immunoprecipitated using peptide-specific, anti-maize SSIIa

183 antibodies following the method described previously by Tetlow et al. (2004a) (Fig.4.2).

The supernatant was collected and denatured by boiling with the SDS loading buffer to use as an indicator of the immunoprecipitation efficiency. The SSIIa antibodies successfully immunoprecipitated native SSIIa in the maize amyloplast stroma on Protein-

A-Sepharose beads under untreated, ATP and APase conditions. Even though SSIIa antibodies immunoprecipitated a significant proportion of the SSIIa protein from the untreated, ATP and APase treated amyloplast lysates on Sepharose beads, some unbound protein remained in the supernatant following centrifugation (Fig. 4.2). The results indicated that SSIIa antibodies specifically immuno-precipitated SSIIa from the plastid lysates and that pre-incubation with ATP or APase did not affect the ability of the antibodies to bind to the respective protein (SSIIa) (Fig. 4.2). This immunoprecipitation method was then utilized to investigate protein –protein interactions between SSIIa and other starch biosynthetic enzymes.

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Figure 4.2: Immunoprecipitation of stromal SSIIa from wild type maize amyloplasts using peptide specific anti- SSIIa antibodies. Wild type maize amyloplast lysates (1.5 mg/ml) were pretreated with 1 mM ATP or 100 U APase and SSIIa was immunoprecipitated using anti-SSIIa specific antibodies followed by washing the Protein-A-Sepharose beads with 1X PBS and 10 mM HEPES/ NaOH as explained in section 4.2.1. The protein -bead complexes were then separated by SDS-PAGE and SSIIa was identified by immunoblotting with anti-SSIIa specific antibodies.

4.3.2 Phosphorylation dependence of protein-protein interactions between SSIIa and other starch biosynthetic enzymes

To examine the protein- protein interactions of SSIIa with other SSs and SBE enzymes in the amyloplast stroma, and to investigate the possible role of phosphorylation in facilitating such interactions, co-immunoprecipitation was performed using amyloplast lysates under conditions which would enhance phosphorylation and dephosphorylation.

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SSIIa from wild type maize amyloplast stroma pretreated with ATP (1mM) or APase

(100U) was immunoprecipitated using peptide-specific anti maize SSIIa antibodies following the method described in the section 4.2.1. SSIIa antibodies specifically immunoprecipitated SSIIa present in the amyloplast stroma (Fig. 4.3) irrespective of the treatment with ATP and APase. The interaction of SSIIa with SBEIIb was more pronounced when the lysates were pre-incubated with ATP, as compared to the untreated and the APase-treated samples. This was evident from the more intense SBEIIb band in

Fig. 4.3. Again, APase-treated samples exhibited less co-immunoprecipitation of SBEIIb

(Fig. 4.3). Co-immunoprecipitation of SSI was observed in the untreated, ATP- and

APase-treated samples (Fig. 4.3) although was slightly reduced following treatment with

APase. SSIV (MW= 104 kDa) was not detected following immunoprecipitation with

SSIIa under untreated, ATP- or APase- treated conditions (Fig. 4.3).

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50kDa 50 kDa 50 kDa

Figure 4.3: Co-immunoprecipitation of SSIIa with other starch biosynthetic enzymes. Wild type maize amyloplast lysates (1.5 mg/ml) were pretreated with 1mM ATP or 100U APase, and stromal SSIIa, was immunoprecipitated. The protein -bead complexes were separated by SDS-PAGE and immunoblots were probed with peptide-specific antibodies for SSIIa, SSI, SBEIIb and SSIV. The co- immunoprecipitated protein bands are indicated by arrows. Note: SSIV was not detected in any immuno-precipitates. The molecular weight of the enzymes are; SSI at 74 kDa, SSIIa at 86 kDa, SBEIIb at 85 kDa and SSIV at 104 kDa. The IgG is approximately at 50kDa appearing as a thick band.

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4.3.3. Analysis of the phosphorylation -dependent interactions of SSIIa with other starch biosynthetic enzymes by Gel Permeation Chromatography (GPC)

Maize amyloplasts lysates (22 DAA) (1.5-2 mg/ml total protein concentration) pretreated with 1mM ATP or 100U APase (500 μL of each) were eluted through a

Superdex 200 10/300GL gel permeation column to investigate whether ATP or APase treatment influenced the interactions of SSIIa with other starch biosynthetic enzymes in the amyloplast stroma. Fractions collected were separated by SDS-PAGE, and the elution pattern of the major starch biosynthetic enzymes (SSI, SSIIa and SBEIIb) was analyzed by immuno-detection with respective peptide specific antibodies (Fig. 4.5). Prior to GPC, the column was calibrated using commercial protein standards (Fig. 4.4).

Figure 4.4: Calibration of GPC for the determination of molecular weight. Superdex 200 10/300GL gel permeation column was calibrated using commercial protein standards of molecular weight ranging from 13.7kDa to 440kDa. The standard curve shows the relationship between log10 values of the molecular weight of the commercial standard proteins versus the fraction numbers. 188

Under untreated and APase-treated conditions, SSIIa eluted predominantly at high molecular weight fractions (Fraction 19 (740 kDa) to 27 (158 kDa) (Fig. 4.5). Upon treatment with ATP there was a shift in the SSIIa elution profile. SSIIa from ATP-treated samples eluted in lower molecular weight fractions (fraction 23 (260- 300 kDa) to 27

(85kDa)) as compared to the APase-treated and untreated samples (Fig. 4.5). The elution profile of SSI and SBEIIb were identical regardless of ATP or APase treatments. SSI eluted at fractions from 19 – 26 under all treatment conditions (Fig 4.5). SBEIIb mostly eluted from fractions 20 to 28 with the peak elution between 24 and 26 (Fig. 4.5). SSI and

SSIIa co-eluted in fractions 20-24 (670-260 kDa range) under untreated and APase treated conditions. However, SBEIIb mostly co-eluted with SSIIa (under all treatment conditions) in fractions 23 to 24 (300- 260 kDa range) (Fig. 4.5). SSIIa co-eluted with

SSI and SBEIIb at fractions 23 to 25 (300- 230kDa). Following GPC, the fractions designated as “high” molecular weight (HMW) and “low” molecular weight (LMW) were used to evaluate the phosphorylation state (Phos-tag-PAGE) and glucan affinity

(substrate affinity electrophoresis) of SSIIa in these fractions, and to investigate the interactions of SSIIa with other starch biosynthetic enzymes (co-immunoprecipitation) as described in the following sections.

189

Figure 4.5: GPC separation of amyloplast stromal proteins under conditions of phosphorylation and dephosphorylation. Maize amyloplasts lysates (22 DAA) pretreated with 1mM ATP or 100U APase (500 μL of each) were separated through a Superdex 200 10/300GL gel permeation column. The fractions were separated by SDS-PAGE and immunoblots probed with anti- SSIIa (86 kDa), anti-SSI (74 kDa) and anti-SBEIIb (85 kDa) antibodies. The arrows on the left hand side mark the position of 75 kDa protein marker. The molecular weight of the proteins eluting in each fraction was estimated from the standard curve developed by calibrating the Superdex 200 10/300GL gel permeation column using commercial protein standards of molecular weight ranging from 13.7kDa to 440kDa. The estimated molecular weights of the HMW and LMW fractions are indicated by arrows on the top with their corresponding molecular weights. The HMW fraction (670 kDa range) is shown in the red box and the LMW (260 kDa range) is shown in the blue box. 190

4.3.4 Phosphorylation status of SSIIa in the GPC fractions

The phosphorylation status of SSIIa in fractions designated as high molecular weight (HMW) (Fraction Nos: 19, 20, 21, 22 pooled) and low molecular weight (LMW)

(Fraction Nos: 23, 24, 25, 26 pooled (Fig. 4.5) was determined by detecting the differential mobility of SSIIa in SDS-PAGE gels containing Mn2+-Phos-tagTM phospho- protein affinity ligand as described previously (section 2.2.5.2 in Chapter 2) (Fig. 4.6).

Immunodetection of SSIIa indicated that LMW fractions from the untreated and ATP- treated samples were significantly retarded when compared to the LMW forms from

APase-treated samples (note that ATP-treated SSIIa only eluted in the LMW range) (Fig.

4.6). The HMW fractions from both the untreated and the APase samples migrated to similar electrophoretic distances as the LMW fractions from APase-treated samples, and were less retarded compared to the LMW fractions from the untreated and ATP- treated samples (Fig. 4.6). However, the LMW fraction from the APase-treated sample migrated similarly to the HMW fractions from the untreated and APase-treated samples (Fig. 4.6).

These results indicated that SSIIa in the LMW fractions from the untreated and ATP- treated samples could be in a (more) phosphorylated state, when compared to SSIIa in the

HMW fractions and the LMW fractions from the APase samples.

191

Figure 4.6: Phosphorylation state of SSIIa in the HMW and LMW fractions obtained by GPC separation of maize amyloplast lysates pretreated with ATP and APase. Maize amyloplasts lysates (22 DAA) pretreated with 1mM ATP or 100U APase (500 μL of each) were separated through a Superdex 200 10/300GL gel permeation column. The HMW (670 kDa) and LMW (260 kDa) fractions were separated by affinity-based Mn2+– Phos-tagTM SDS-PAGE and the immunoblots were probed with anti- SSIIa antibodies. The differential mobility of the different fractions is indicated by arrows.

4.3.5 Glucan binding properties of HMW and LMW fractions containing SSIIa

The dissociation constant (Kd) of SSIIa for amylopectin in the HMW (670 kDa) and LMW (260 kDa) fractions (HMW and LMW fractions from untreated lysates) were determined by affinity gel electrophoresis as described in section 4.2.6. The migration distance of native maize SSIIa in the HMW and LMW fractions was measured after immunoblotting and probing with anti-SSIIa specific antibodies (Fig. 4.7). Irrespective of the presence of glucan substrate in the gels, the LMW (260 kDa) form of SSIIa migrated further on non-denaturing gels compared to the HMW (670 kDa) form (Fig.

4.7). As the concentration of corn amylopectin was increased in the gels, both HMW and

192

LMW forms of SSIIa were retarded (Fig. 4.7). The relative migration (Rm) of native

SSIIa in non-denaturing gels in the presence of different concentrations of α-(1-4) – linked glucan (amylopectin) was calculated with reference to the mobility of SSIIa in the absence of the substrate for a given form (HMW or LMW) (Fig.4.7 and Fig. 4.8). The 1/

Rm values of the HMW and LMW forms of SSIIa in gelatinized amylopectin containing gels were linearly related to the concentration of the glucan substrate in the gels and the dissociation constants (Kd) were determined and compared (Fig 4.8 and Table 4.1).

It is notable that the relative electrophoretic migration of the HMW and LMW forms of SSIIa appears very similar to that observed for the untreated and ATP-treated forms of SSIIa when studying maize amyloplast lysates, as described in the previous chapter (section 3.3.7). Table 4.1 presents the dissociation constants determined for the

HMW and LMW forms of SSIIa from the GPC samples. There was no significant difference between the affinity constants of HMW (670 kDa) and LMW (260 kDa) for corn amylopectin (Table 4.1). The dissociation constants of the HMW and LMW forms of SSIIa were identical to those calculated for the untreated, ATP and APase-treated

SSIIa from maize amyloplast lysates (Table 3.3).

193

Figure 4.7: Substrate affinity electrophoresis of HMW (670 kDa) and LMW (260 kDa) forms of maize SSIIa. Following GPC separation, SSIIa in the designated HMW and LMW fractions were electrophoresed in non-denaturing polyacrylamide gels containing various concentrations of corn amylopectin. SSIIa was detected by probing the immunoblots with anti-maize SSIIa specific antibody. The migration of SSIIa protein bands is indicated by arrows.

194

Figure 4.8: Dissociation constants of HMW (670 kDa) and LMW (260 kDa) forms of SSIIa. Following GPC separation, SSIIa in the HMW and LMW fractions were electrophoresed in non-denaturing polyacrylamide gels containing various concentrations of corn amylopectin. The mobility of SSIIa protein in the HMW and LMW forms was detected by probing the immunoblots with maize anti-SSIIa specific antibody. The relative migration of SSIIa on the immunoblots at various glucan concentrations was determined by dividing the distance travelled by SSIIa protein band at various amylopectin concentrations by the distance travelled in the absence of the substrate in the HMW and LMW forms separately. The reciprocal of the relative migration (1/Rm) of SSIIa was plotted against the concentration of corn amylopectin in the non- denaturing gels. The dissociation (Kd) constant was determined from the intercept on the X-axis.

195

Table 4.1 The dissociation (Kd) constants of HMW and LMW forms of SSIIa for corn amylopectin following GPC separation of wild type maize amyloplast lysate

Substrate HMW (670 kDa) LMW (260 kDa) form of SSIIa form of SSIIa

Amylopectin Kd 0.06±0.02 0.06±0.02

Results are means ± S.E.M. for three independent experiments ; *P value (T test) = 0.1090.

4.3.6 Relative electrophoretic mobility of SSI, SBEIIb and SSIIa in the HMW and LMW fractions following non-denaturing PAGE

In order to investigate whether or not SSIIa co-migrates electrophoretically with other starch biosynthetic enzymes (Liu et al., 2009), the relative mobility of SSI and

SBEIIb with respect to that of SSIIa was determined on non- denaturing gels. Following

GPC separation, the designated HMW (Fractions: 19-22, pooled) and LMW (Fractions

23- 26, pooled) fractions from the untreated, ATP and APase treated GPC samples were electrophoresed on non-denaturing polyacrylamide gels containing varied concentrations of corn amylopectin. The mobility of maize SSIIa, SBEIIb and SSI was detected by western blotting (Fig. 4.9).

196

Immunoblots probed with anti-SBEIIb antibodies showed the presence of SBEIIb protein bands only in the LMW fractions from the untreated, ATP- and APase-treated samples. Similarly, SSI antibodies detected SSI protein bands mostly in the LMW fractions of the ATP- and APase-treated samples (Fig. 4.9). It was also difficult to detect prominent SSIIa bands in the HMW fractions (lane 1 and 4). The protein bands are labelled alphabetically for convenience (Fig. 4.9). In the absence of amylopectin, SBEIIb

(mostly in LMW fractions lanes 2, 3 and 5) and SSI (in LMW fractions lanes, 3 and 5) migrated significantly further in the gels. SBEIIb migrated approximately 4.5 cm down from the top of the gel and is indicated as protein band “a”, while SSI migrated 3.8 cm down and is denoted as band “b”. Alternatively, this SSIIa protein band in the HMW fractions (lanes 1 and 4) from the untreated and APase-treated samples shifted less far

(“e” 2.2 cm) compared to SSIIa in the LMW fractions from the untreated, ATP and

APase-treated samples (lanes 2,3 and 5) (Fig. 4.9). The immunoblots probed with anti-

SSIIa antibodies exhibited multiple protein bands especially in the LMW fractions (Fig.

4.9). The results indicated that SSIIa in the LMW fractions (untreated, ATP- and APase- treated samples) exhibited a considerable shift in its mobility similar to the ATP-treated forms of SSIIa from maize amyloplast lysates as described in the previous chapter

(section 3.3.7) (Fig. 4.9). In the LMW fractions (lanes 2, 3 and 5) two prominent bands of

SSIIa protein (band “a” (4.5cm) and “b” (3.8cm)) were detected in the immunoblots of gels without the substrate. Interestingly, in the absence of amylopectin, some co- migration of SBEIIb with SSIIa was observed as protein band “a” (4.5cm) and as very faint bands, “b” (3.8 cm) in the LMW fractions (lanes 3 and 5). Similarly, SSI also co-

197 migrated with SSIIa, and (possibly SBEIIb) at 3.8cm (band “b”) in the LMW fractions

(lanes 3 and 5) in the absence of amylopectin (Fig. 4.9).

In the presence of 0.1% or 0.3% amylopectin, all three enzymes, SSI, SSIIa and

SBEIIb were substantially retarded (“c” (0.1cm)) and remained near the top of the gel, with the same very faint bands ( “a” and “b”) observed at the lower portion of the gels without amylopectin (Fig. 4.9). The SSIIa protein band in the HMW fractions was also considerably retarded in its mobility from “e” to “d” (0.2 cm), when the concentration of amylopectin in the gel was increased to 0.3% (Fig. 4.9; lanes 1 and 4; more clearly in lane 4). It is important to note that, in gels with 0.3% amylopectin, SSIIa co-migrated with SBEIIb (in the LMW fractions from all three treatments; lanes 2, 3 and 5) and SSI

(in the LMW fractions from ATP and APase-treated forms; lanes 3 and 5) showing significant retardation in their electrophoretic mobility (the protein barely entering the gels) (Fig. 4.9).

198

Figure 4.9: Co-migration of SSI and SBEIIb relative to SSIIa from HMW (670 kDa) and LMW (260 kDa) GPC fractions on non-denaturing gels. Following GPC separation, the HMW and LMW fractions from the untreated, ATP and APase treated GPC samples were electrophoresed in non-denaturing polyacrylamide gels containing various concentrations of corn amylopectin. Native SSIIa, SBEIIb and SSI present in the HMW and LMW forms were detected by probing the immunoblots with respective maize antibody specific for each protein. The start of each gel is indicated by a black arrow. The protein bands are labelled alphabetically and indicated by red arrows, and their migration distances from the top of the gel given in ‘cm’ in parentheses. 199

4.3.7 Protein –protein interactions of SSIIa following GPC of amyloplast lysates

In order to examine possible protein -protein interactions of SSIIa with SSI and

SBEIIb, SSIIa was immunoprecipitated from the high (Fractions 20 and 22) and low molecular weight (Fractions 25 and 26) GPC fractions using anti- SSIIa specific antibodies. Co-immunoprecipitation of SSIIa with SSI and SBEIIb in these GPC fractions from the untreated, APase and ATP samples was investigated by probing the immunoblots with anti- SSIIa, SSI and SBEIIb antibodies.

The results showed that SSIIa could be immunoprecipitated from both HMW and

LMW fractions in the untreated (Fraction Nos: 20, 22, and 25) and APase-treated

(Fraction Nos: 20, 22, 25 and 26) samples; whereas in the ATP treated samples, SSIIa could be immunoprecipitated only from the LMW fractions (Fraction Nos: 25 and 26), not the HMW fractions (Fig. 4.10). SSI co-immunoprecipitated with SSIIa mostly in

HMW fractions in the untreated (Fraction No: 22) and APase-treated (Fraction Nos: 20 and 22) samples. However, in ATP treated samples, SSI –SSIIa interaction was not observed in the LMW fractions (Fig. 4.10). A weak SSI protein band was found co- immunoprecipitated in HMW fraction No: 22 from ATP-treated GPC sample (Fig. 4.10).

This could more likely be a non-specific interaction of SSI with the sepharose beads as

SSIIa was not found immunoprecipitated on the beads from that fraction (Fig. 4.10). SSI-

SSIIa interaction was detected in the LMW fraction No: 25 in the untreated samples.

SBEIIb co-immunoprecipitated with SSIIa on sepharose beads only in the LMW fractions (Fraction No: 25 and 26) in untreated, ATP and APase treated samples (Fig.

4.10). Interestingly, the interaction between SSIIa and SBEIIb was found to be enhanced under ATP treated conditions (LMW fractions 25 and 26 from ATP sample). This was

200 indicated by comparatively more intense bands of SBEIIb on the beads in the LMW fractions 25 and 26 in the ATP-treated GPC sample as compared to the untreated sample

(Fig. 4.10). Furthermore, the SSIIa-SBEIIb interaction observed in the LMW fraction 26 was significantly reduced under APase-treated conditions as evident from the less intense

SBEIIb protein band which co-immunoprecipitated (Fig. 4.10). Useful “control” experiments would have been to conduct co-immunoprecipitation with fractions where, monomeric forms of each enzyme would be expected, and also with unfractionated amyloplasts. Unfortunately, such controls were not included in this study.

201

Figure 4.10: Protein-protein interactions of SSIIa from the designated HMW (670 kDa) and LMW (260 kDa) GPC fractions with other starch biosynthetic enzymes. Following GPC separation, SSIIa in the HMW (Fraction Nos: 20 & 22), and LMW (Fraction Nos: 25 & 26) fractions from untreated, ATP and APase treated GPC samples was immunoprecipitated using anti-SSIIa specific antibodies followed by washing Protein - A-Sepharose beads with 1X PBS and 10mM HEPES/ NaOH as described in section 4.2.7. The protein -bead complexes were then separated by SDS-PAGE. The immunoblots were probed with peptide specific antibodies for SSIIa, SSI, and SBEIIb. The co-immunoprecipitated protein bands on the sepharose beads are indicated by arrows. The molecular weight of the enzymes are; SSI at 74 kDa, SSIIa at 86 kDa, SBEIIb at 85 kDa. The IgG is approximately at 50kDa appearing as a thick band.

202

To summarize, possible protein interactions of SSIIa with SSI and SBEIIb were investigated. Following treatment with ATP or APase, maize amyloplast lysates were separated by GPC. Following GPC analysis, the relative electrophoretic migration of SSI,

SSIIa and SBEIIb were determined on non-denaturing gels. Likewise, co- immunoprecipitation of these proteins with SSIIa from HMW and LMW fractions was investigated. The salient findings of these experiments are as follows.

 SSIIa in the LMW fractions (260 kDa) from the untreated, ATP- and APase-

treated samples, migrated markedly further than the HMW fractions, in non-

denaturing gels in the absence of glucan substrate.

 SSIIa co-migrated with SBEIIb and separately, with SSI in the LMW fractions

(260 k Da) in non-denaturing gels. This electrophoretic co-migration of SSIIa

with either SSI or SBEIIb was more evident in the ATP-treated samples.

 Electrophoretic migration of SSIIa, SSI and SBEIIb (in LMW fractions) was

significantly retarded in non-denaturing gels (0.3% amylopectin), compared to

those lacking glucan substrate, and appeared co-incident, though the migration

distances were small.

 SBEIIb co-immunoprecipitated with SSIIa in the LMW (260 kDa) fractions

(Fraction Nos: 25 and 26).

 The interaction between SBEIIb and SSIIa was enhanced by ATP and reduced

by APase treatments (Lanes 5 & 6 in Fig. 4.10), when examining the subsequent

LMW fraction post-GPC, indicating that their interaction could be more likely be

dependent on the phosphorylation state of SSIIa.

203

 SSIIa interacted with both SSI and SBEIIb only in the LMW fraction (Fraction

No: 25) from the untreated sample. However, SSI interacted with SSIIa in the

HMW fractions from the untreated and APase-treated samples.

4.3.8 Detection of protein –protein interactions of SSIIa with other major starch biosynthetic enzymes by chemical cross-linking

Stromal proteins from maize amyloplast lysates pretreated with ATP (1mM) or APase (100U) were incubated with 1 mM of the homo-bifunctional cross-linking reagent Bis-(sulfosuccinimidyl) suberate (BS3) (Pierce/BioLynx), and the proteins separated by SDS-PAGE, immunoblotted and probed with various anti-SS (SSI and

SSIIa) and anti- SBE (SBEIIb and SBEI) antisera as described in section 4.2.3. Figure

4.11 shows the immunoblots of the cross linked proteins.

Cross-linking detected a product of approximately 250 kDa recognized by anti-

SSIIa antibodies which disappeared in the ATP treated samples (Fig. 4.11). This cross- linked product displayed a very minor cross reactivity with SBEIIb and SBEI mostly in untreated samples (Fig. 4.11). The monomeric form of each protein was detected by the respective antibodies in the immunoblots (Fig. 4.11).

204

Figure 4.11: Cross-linking of stromal proteins in wild type maize amyloplasts under conditions of phosphorylation and dephosphorylation. Stromal proteins from maize amyloplast lysates pretreated with ATP (1mM) or APase (100U) were incubated with 1 mM of the homo-bifunctional crosslinking reagent Bis- (sulfosuccinimidyl) suberate (BS3) (Pierce/BioLynx) and the proteins separated by SDS-PAGE. Immunoblots were probed with peptide specific antibodies for SSIIa, SSI, SBEI and SBEIIb. The high molecular weight (250 kDa) cross-linked product is shown by black arrows and the monomeric forms of proteins are indicated by red arrows.

205

4.4 Discussion

The experimental results discussed in the previous chapters (Chapter 2 and 3) of this thesis clearly demonstrated that stromal SSIIa in wild type maize amyloplasts could be post -translationally regulated by protein phosphorylation. The present chapter investigated the effect of protein phosphorylation on the ability of SSIIa to interact with other starch biosynthesizing enzymes (mainly SSs and SBEs) to form functional multi- enzyme complexes. Co-immunoprecipitation of other SSs and SBEs with SSIIa, changes in the elution profile of SSIIa pretreated with ATP or APase following GPC, and the differential electrophoretic mobility of the HMW (670 kDa) and LMW (260 kDa) GPC fractions of SSIIa, provide substantial evidence for the existence of functional interactions between SSIIa and other SSs (SSI) and SBEs (SBEIIb) in the amyloplasts of developing maize endosperm.

Pretreatment with ATP or APase did not affect the immunoprecipitation of SSIIa from maize amyloplast stromal lysates by anti-SSIIa specific antibodies (Fig. 4.2).

Notably, co- immunoprecipitation of SBEIIb with SSIIa was significantly enhanced by exogenous ATP (Fig. 4.3). Similarly, the interaction of SSIIa with SBEIIb was substantially reduced when amyloplasts were pretreated with APase (4.3). However,

SSIIa- SSI interaction was seemingly unaffected by prior incubation with ATP or APase

(Fig. 4.3). There is abundant evidence for the operation of functional, physical associations between SS and SBE forms in cereal endosperms, and these interactions are driven by protein phosphorylation (Tetlow et al., 2008, Hennen –Biewagen et al., 2008;

206

2009; Liu et al., 2009; 2012a). Pair-wise interaction of SSIIa with SSI, SBEIIb and

SBEIIa was previously reported in wild type maize amyloplasts (Hennen –Biewagen et al., 2008) and, importantly, the SSIIa – SBEII interaction was also found to be phosphorylation- dependent in wheat (Tetlow et al., 2004a; 2008). Co- immunoprecipitation results indicated that SSIIa formed a protein complex with SSI and

SBEIIb, and that the interaction between SSIIa and SBEIIb in this complex was greatly influenced by the phosphorylation state of SSIIa (Tetlow et al., 2008). In a similar study in wild type maize amyloplasts, Liu et al. (2009) postulated that a trimeric protein complex containing SSI, SSIIa and SBEIIb is involved in the organisation of short and intermediate glucan clusters during amylopectin synthesis, and that the association of this complex was strongly enhanced by the phosphorylation of SBEIIb. The present results demonstrated that the association of SSIIa with SBEIIb to form this functional protein complex is noticeably enhanced by ATP and disrupted by APase again suggesting a likely role of protein phosphorylation in their assembly.

Co- elution of SBEIIb with SSIIa following GPC occurred mostly in the LMW

260 kDa range (Fractions 23 - 26) (Fig. 4.5). The significant reduction in the interaction between SSIIa and SBEIIb by APase treatment of lysates prior to GPC was evident when

SBEIIb was co-immunoprecipitated with SSIIa from these LMW fractions (260 kDa)

(Fig.4.10). A similar 260 kDa protein complex containing SSI, SSIIa and SBEII forms in wheat amyloplasts was found disrupted by dephosphorylation by exogenous APase

(Tetlow et al., 2008). This observation further supported the proposal that their interaction is phosphorylation dependent. Co-elution of SSI and SBEIIb with SSIIa from

GPC in the 260 -300 kDa range (fractions 23 - 26) is consistent with the hypothesis that

207 these three proteins could co- exist as a multi-enzyme complex (Fig. 4.5). It could also be possible that SSIIa perhaps associate with other SBE isoforms (SBEIIa or SBEI) (which is not investigated in this study), or SBE dimers, or SSI dimers, or even other non- identified interacting proteins and associate as multi-enzyme complexes in this LMW range (260 -300 kDa). Furthermore, the electrophoretic co- migration (identical mobility) and retardation of SSI, SSIIa and SBEIIb in the LMW GPC fractions (260 kDa) in amylopectin (0.3%) containing non-denaturing gels (Fig. 4.9) strongly suggested that

SSIIa could form a functional protein complex with SSI and SBEIIb showing considerably greater affinity towards amylopectin. However, an investigation of the electrophoretic mobility of “monomeric” forms of SSIIa (which wasn’t examined in this study) could have further confirmed the existence of such a functional complex between

SSIIa with SSI and SBEIIb. An increased affinity for glucan substrates was exhibited by hetero-trimeric complexes (260 kDa) in wheat consisting of SSIIa, SSI and SBEII forms compared to the monomeric SBEII forms as a result of the functional interaction between these SS and SBE forms (Tetlow et al., 2008).

A significant shift in the elution profile of SSIIa from HMW range (740 kDa -

158 kDa) towards the LMW range (260- 85 kDa) was observed when amyloplast lysates were treated with ATP prior to GPC analysis (Fig. 4.5). This observation indicated that when ATP was present, SSIIa might exist in lower molecular weight protein complexes

(LMW fractions) as compared to the untreated and APase-treated SSIIa that interact with other enzymes to form comparatively larger protein complexes (HMW fractions).

Exogenous ATP could perhaps induce the dissociation of other enzymes which might otherwise have interacted with SSIIa (under untreated and APase treated conditions). As

208 observed previously by Hennen –Biewagen et al. (2008), in the absence of any pretreatment of amyloplast extracts, maize SSIIa predominantly eluted in the high molecular weight fractions while the majority of SBEIIb eluted in the monomeric state. In maize amyloplasts, SSIII was found to interact with SSIIa -SBE complex (670 kDa) in a phosphorylation dependent manner (Hennen –Biewagen et al., 2008; 2009). The phosphorylation or dephosphorylation of one or more of the participating proteins in this

670 kDa complex is therefore essential for its stability. This suggests the possibility that

SSIIa could perhaps be interacting with SSIII, contributing to a high molecular weight protein complex. Therefore the possibility of SSIIa –SSI-SBEII forms interacting with

SSIII in the HMW range (670 kDa) to form larger heterocomplexes could not be ruled out. Due to technical difficulties (lack of specific antibodies, reliably able to detect maize

SSIII), immuno-detection with anti- maize SSIII antibodies was not successful for any of the GPC, native -PAGE and co-immunoprecipitation experiments described.

Another group of highly conserved and physiologically significant proteins, called

14-3-3 proteins has previously been found to be involved in the phosphorylation and consequent regulation of starch biosynthetic proteins (Sehnke et al., 2001). 14-3-3 proteins are involved in the regulation of several enzymes in the eukaryotic system by forming functional protein complexes, with target proteins, following protein phosphorylation (Sehnke and Ferl, 2002; Comparot et al., 2003; Roberts, 2003). The observation by Alexander and Morris (2006) in barley endosperm indicating the association of SSI, SSIIa and SBEIIa with amyloplast 14-3-3 proteins, in a phosphorylation-dependent manner, points to the possibility that 14-3-3 proteins might also be interacting with SSIIa (depending on its phosphorylation state) in the high

209 molecular weight fractions. However, the direct role of 14-3-3 proteins in starch metabolism has not been established. Although there is no evidence available to date for homo-oligomerisation of SS forms, the possibility of existence of protein complexes consisting of homo or hetero dimers of SS forms with SBE isoforms in the high molecular weight GPC fractions could not be ruled out. Exogenous ATP might catalyze the dissociation of high molecular weight complexes (670 kDa range) involving SSIIa though this remains speculative.

Following GPC, SSI co-eluted with SSIIa from 440-260 kDa, with a peak of elution at 440 kDa range (4.5). It could be more likely that SSI formed a complex with

SSIIa and SBEIIb at the 260 kDa range (LMW) since SSI co-existed with SSIIa and

SBEIIb and migrated to the same distance in non-denaturing amylopectin containing gels

(Fig.4.9; lanes 3 and 5). However, co-immunoprecipitation of SSI with SSIIa from the

HMW fractions 20 (APase-treated sample) and 22 (untreated sample) (670 kDa) showed that SSI tended to interact with SSIIa predominantly in the high molecular weight fractions (Fig. 4.10). Given the molecular masses of SSI (74 kDa), SSIIa (86 kDa) and

SBEIIb (85 kDa), the proposed 260 kDa protein aggregation product might possibly contain either the hetero trimeric complex of SSI-SSIIa-SBEIIb or might contain SSIIa-

SBEIIb/SSI homodimers. In the present study, the molecular masses of the proposed protein complexes (shown in Fig. 4.5) were estimated from the GPC standard curve, and the individual components of the suggested protein aggregates (viz., HMW (670 kDa),

LMW (260 kDa) were proposed based on their respective GPC elution profiles, migration on native –PAGE gels, and co-immunoprecipitation from the high and low molecular weight fractions. Analysis of the proposed protein complexes utilizing cross-linking

210 techniques was not informative. In this study, chemical cross-linking of stromal proteins from maize amyloplasts pretreated with ATP or APase did not provide any clear indication about the possible interactions of SSIIa with other starch biosynthetic enzymes

(Fig.4.11).

The co-migration of SSI and SBEIIb with SSIIa in the LMW fractions (260 kDa) on non-denaturing gels containing corn amylopectin (0.3%) resulted in significant retardation of all three near the top of the gel. Given their limited PAGE migration under these conditions, it is not possible to draw any firm conclusions, especially as the electrophoretic mobility of GPC fractions containing monomeric forms of SSIIa was not investigated, and should be in future. In the present study, SSI and SBEIIb protein bands were not detected in the HMW GPC fractions in the immunoblots of non-denaturing amylopectin containing gels (Fig. 4.9) even though they were detectable following SDS-

PAGE (Fig. 4.5). However, where SSIIa could be detected in the HMW fractions following electrophoresis, it was strongly retarded by amylopectin (Fig. 4.9), consistent with the results observed in Figure 4.7.

In Figure 4.9, the anti-SSIIa antibodies detected multiple protein bands in the

LMW fractions (lanes 2, 3 and 5) including a prominent band barely entering the gel

(band “c”). Results from Figure 4.7 indicated that, even though SSIIa in the LMW (260 kDa) fractions (from untreated sample) migrated further in non- denaturing gels with and without corn amylopectin, as compared to those present in the HMW (670 kDa) fractions, the dissociation constants of both forms of SSIIa towards corn amylopectin were similar

(Fig. 4.8 and Table 4.1). However, in these experiments, a prominent protein band detected by anti-SSIIa antibodies was not detected near the top of the gel (Fig. 4.7).

211

Following GPC of amyloplast lysates, the calculated Kd for glucan following substrate affinity electrophoresis (Fig. 4.8) gave similar results to those obtained with unfractionated amyloplast lysates (Fig. 3.10, 3.11 and Table 3.3 in the previous chapter).

The Phos-tag -PAGE results imply that SSIIa is phosphorylated in the LMW fractions (from the untreated and ATP-treated samples) as compared to SSIIa present in the HMW fractions from the untreated and the APase-treated GPC samples (Fig. 4.6).

Following Phos-tag electrophoresis, SSIIa present in the LMW fractions from the APase- treated amyloplast lysates appeared to be in a less phosphorylated or dephosphorylated state as compared to those present in the LMW fractions from the ATP-treated and untreated samples (Fig. 4.6). These results indicated that ATP treatment might have no significant effect on the substrate affinity of SSIIa for amylopectin. This is consistent with the results from Chapter 3 where ATP treatment substantially enhanced the Vmax of stromal maize SSIIa, without significantly affecting its Kd or Km for amylopectin. It might be possible that ATP treatment (phosphorylation) could affect which other proteins that associate with SSIIa to form different multi-enzyme complexes of varying molecular masses (as compared to the untreated and APase treated forms) without affecting its relative affinity for the growing amylopectin cluster. This could explain the presence of multiple bands of SSIIa activity with differential mobility observed in the amyloplast lysates pretreated with ATP on non-denaturing amylopectin containing gels, compared to the untreated and APase-treated forms (Fig. 3.2 in the previous chapter). It could only be speculated that under untreated and APase-treated conditions, SSIIa might be interacting with other proteins from the endosperm (possibly SSIII), possibly recruiting them to the already existing trimeric complex of SSI-SSIIa-SBEIIb (260 kDa-300 kDa) to form

212 higher molecular weight multi-enzyme complexes (670 kDa range) (see the proposed model given in Figure. 4.12). Unfortunately, in this study, during co- immunoprecipitation, interaction between SSIIa and SSI was not detected in the LMW fractions from the ATP-treated samples (Fig. 4.10), even though SSI co-migrated with

SSIIa in non-denaturing amylopectin gels in these LMW fractions (Fig. 4.9; lane 3).

In a recent study, Liu and co-workers (2012b) proposed a model, whereby the granule associated proteins, SSI, SSIIa and SBEIIb, involved in the amylopectin synthesis are partitioned into the developing starch granule, as a result of their association to form functional, phosphorylation-dependent trimeric protein complex. Importantly, it was shown that SSIIa forms the core of this complex with SSI and SBEIIb, fundamentally dictating the glucan binding ability of this complex within the growing clusters of amylopectin (Liu et al., 2012b). The results presented in this chapter confirm that SSIIa could interact and physically associate with other starch biosynthetic enzymes mainly SSI and SBEIIb depending on its phosphorylation state. The GPC separation of stromal proteins from amyloplast lysates pretreated with ATP or APase suggested the existence of SSIIa at least in two distinct forms - HMW (670 kDa) and LMW (260 k Da) protein aggregates. Following ATP treatment, SSIIa eluted in relatively higher proportion in LMW fractions (260 kDa) along with SSI and SBEIIb, as compared to the untreated and APase-treated samples, where the relative proportion of SSIIa was found higher in the HMW fractions (670 kDa) again co-eluting with SSI and SBEIIb (Fig. 4.5).

Pretreatment with ATP could have caused SSIIa to exist in LMW protein complexes with other starch biosynthetic enzymes (possibly, SSI and SBEIIb) as compared to the untreated and APase-treated forms, where SSIIa existed mostly in the HMW forms by

213 recruiting other proteins to these already existing LMW complexes. Phosphorylation might also have caused the dissociation of HMW complexes along with the formation of

LMW complexes. These two processes might be linked or independent. Besides, the possibility of SSIIa forming homo-or hetero oligomers in the absence of ATP, could not be ruled out. Functional hetero-complexes comprising of SSI, SSIIa and SBEIIa or

SBEIIb have been detected in cereal endosperms and the association of these proteins depended on the phosphorylation state of the individual SS and SBE isoforms (Tetlow et al., 2004, 2008; Hennen –Biewagen et al., 2008; Liu et al., 2009). Therefore the ability of

SS and SBE isoforms to associate with each other is more likely to be conserved in endosperms and other starch synthesizing tissues (Hennen –Biewagen et al., 2008;

Tetlow et al., 2008). Under untreated or APase- treated conditions, the 260 kDa complex of SSIIa – SSI-SBEIIb might possibly associate with other starch synthesizing enzymes

(like SSIII) and or itself, to form HMW complexes of 670 kDa or more. The proposed protein – protein interactions of SSIIa with other starch biosynthetic enzymes based on the results obtained from the present study are shown in Figure 4.12.

214

Figure 4.12: Proposed interactions of SSIIa with other starch biosynthetic enzymes. The proposed multi-protein complexes of SSIIa were derived from the GPC analysis of wild type maize amyloplast stroma pretreated with 1mM ATP or 100U APase and also from the NATIVE-PAGE analysis and co-immunoprecipitation experiments with the designated HMW (670 kDa) and LMW (260 kDa) GPC fractions. Under ATP treated conditions, SSIIa co-eluted in LMW fractions with SSI and SBEIIb and therefore might form LMW complexes (260- 300 kDa range) as shown on the right hand side- (c) hetero- trimeric protein complexes with SSI and SBEIIb or (d) complexes with SSIIa/SBE dimers or (e) SSIIa/SSI dimers. Under untreated and APase treated conditions, SSIIa eluted in HMW fractions and might thus recruit other proteins (a) ‘X’or ‘Y’ or (b) both (in addition to the already existing trimeric complex) to form larger protein complexes as shown on the left hand side. The unknown protein components ‘X’ and ‘Y’ could be any other protein that is expressed in the amyloplast during the active starch accumulation period such as SSIII, SBEI, SBEIIa, SP or even the regulatory 14-3-3 proteins.

215

It is therefore hypothesized that SSIIa associates with different starch biosynthetic proteins in a phosphorylation dependent manner resulting in the formation of different heteromeric protein complexes with varying molecular weights. The high (670 kDa) and low molecular weight (260 kDa) forms of SSIIa showed similar affinity towards amylopectin. This suggests that the phosphorylation status of SSIIa might affect interactions with other starch biosynthetic proteins, without affecting the relative affinity of the enzyme for its substrate, although Chapter 3 indicated that its Vmax could be significantly affected. Functional assemblies of different starch biosynthetic enzyme isoforms like those described above would presumably improve the efficiency of starch biosynthesis as it would facilitate substrate channeling and encourage the formation of the highly organized three dimensional crystalline structure of amylopectin polymer

(Tetlow, 2011 and Geigenberger, 2011). The formation of various heteromeric enzyme complexes, including SSIIa, as a function of phosphorylation state might suggest that, the unique three dimensional crystalline structure of amylopectin molecule could probably be the result of the action of multiple, varied, functional protein complexes during starch biosynthesis.

216

CHAPTER 5

217

CHAPTER 5: GENERAL DISCUSSION

Storage starches produced from cereal endosperms have diversified uses and, being a major dietary component, there is increasing recognition that some starches have significant human health benefits (Keenan et al., 2006; Clarke et al., 2008; Asare et al.,

2011). The research presented in this thesis investigated the regulation of starch synthase

IIa (SSIIa) in amyloplasts of developing maize (Zea mays L.) endosperm. A detailed biochemical analysis was conducted to examine whether maize SSIIa is phosphorylated, whether this post-translational modification affects its catalytic activity, and whether its physical association with other starch biosynthetic enzymes is altered.

5.1 Post-translational modification of maize SSIIa by protein phosphorylation

The significance of SSIIa in the biosynthesis of starch, its important role in the synthesis of glucan intermediates, and in maintaining the crystalline structure of amylopectin in cereal endosperms is well established from previous studies (Fontaine et al., 1993; Craig et al., 1998; Morell et al., 2003; Zhang et al., 2004; Shimbata, 2005) as discussed in this thesis (sections 1.5 and 2.1). Current understanding suggests that in developing maize endosperm, SSIIa forms the core of a trimeric, functional enzyme complex with SSI and SBEIIb, the formation of which is phosphorylation-dependent. In doing so, SSIIa facilitates trafficking of SSI and SBEIIb into the starch granule through their physical interaction (Tetlow et al., 2008; Hennen-Bierwagen et al., 2008; Liu et al.,

2009; 2012a & b). The presence of catalytically active SSIIa is critical for the catalytic activity of the complex, and to maintain the structural organization of amylopectin (Liu et

218 al., 2012b). These observations reinforce the physiological significance of functional protein complexes containing SSIIa, the formation of which is highly dependent on post- translational modification by protein phosphorylation.

Protein phosphorylation of maize SSIIa was investigated using various biochemical and proteomic techniques, as described in Chapter 2. Examination of wild- type maize kernels (22 DAA) indicated that SSIIa is partitioned between the amyloplast stroma and the starch granule (Fig. 2.2), as observed previously (Li et al., 1999). Maize amyloplast lysates or immunopurified SSIIa were incubated with ATP or APase (in the presence of protein kinase(s) from amyloplast lysates) in order to phosphorylate, or dephosphorylate the protein respectively. The use of Phos-tag TM phospho affinity acrylamide gel electrophoresis demonstrated that SSIIa in maize amyloplast stroma can be phosphorylated by endogenous protein kinase(s) in the plastids and that the process is reversible (Fig. 2.5A). Interestingly, starch granule bound SSIIa migrated similarly to the phosphorylated (ATP-treated) stromal SSIIa on Phos-tag gels, suggesting that maize

SSIIa bound to the granular matrix exists in the phosphorylated state (Fig. 2.6). However, stromal SSIIa was found in a dephosphorylated state under the conditions used when amyloplasts were purified (Fig. 2.6). Phosphorylation of granule bound form of SSIIa was reported earlier in wheat using other methods (Tetlow et al., 2004a), and has been previously observed in maize endosperm during granule phospho-proteome analysis

(Grimaud et al., 2008). In fact, the presence of phosphorylated forms of proteins (like

SSIIa) in starch granules, which are also components of phosphorylation-dependent, protein complexes (Tetlow et al., 2004a; Grimaud et al., 2008), supports the hypothesis that heteromeric complexes involved in starch biosynthesis form functional units, and get

219 entrapped in the granular matrix (Liu et al., 2009). The patterns of electrophoretic mobility of SSIIa observed in the Phos-tag gels indicated that SSIIa might be subject to phosphorylation only on one site, in contrast to maize SBEIIb (three sites) in an earlier study (Liu et al., 2012a; Makhmoudova et al., 2014).

In the current study, immunopurification using peptide specific, anti-SSIIa antibodies, (Liu et al., 2012b) provided highly purified active SSIIa from maize amyloplast stroma, and served as a useful technique to investigate the consequences of protein phosphorylation, without the interference of other starch biosynthetic enzymes.

The increased staining intensity of immunopurified SSIIa following Pro-Q diamond phospho-protein gel staining (Fig. 2.7A), and the radiolabelling of immunopurified SSIIa, following incubation with γ- [32-P] ATP and maize amyloplast lysates, provided clear evidence for direct phosphorylation of SSIIa by endogenous protein kinase(s) present in the amyloplasts (Fig. 2.8). The dependence of phosphorylation of recombinant SSIIa on the presence of amyloplast lysates (Fig. 2.11) further suggested a role for one or more amyloplast protein kinase(s) in this post-translational modification. The effect of protein kinase inhibitors, FSBA and K252a in preventing the phosphorylation-dependent, mobility shift of ATP-treated SSIIa on Phos-tag gels reinforced the above argument.

Together, the results presented in Chapter 2 strongly suggest that maize SSIIa can be post-translationally regulated by protein phosphorylation by one or more protein kinase(s) present in the amyloplasts. Based on the patterns of electrophoretic migration observed (in Phos-tag gels), it is also suggested that SSIIa might be subject to phosphorylation only on one site. However, this could only be confirmed by a further detailed investigation on the putative phosphorylation sites of this protein, which was not

220 conducted in this study. The significant role of protein phosphorylation in regulating physical associations between SSs and SBEs to form functional multi-enzyme-complexes during starch biosynthesis in cereal endosperms has been well documented (Tetlow et al.,

2004a; Tetlow et al., 2008; Hennen-Bierwagen et al., 2008 & 2009; Liu et al., 2009).

Based on the results presented in Chapter 2 it was proposed that protein phosphorylation could regulate enzyme conformation affecting the catalytic activity, and enhancing physical associations of starch biosynthetic enzymes, increasing the efficiency of starch biosynthetic machinery.

5.2 Regulation of the catalytic functions of maize SSIIa by protein phosphorylation

Protein phosphorylation has been previously shown to regulate the catalytic activity of enzymes in the starch biosynthetic pathway (e.g. starch branching enzyme activity) (Tetlow et al., 2004a) and the formation of a trimeric protein complex with SSI and SBEIIb (Tetlow et al., 2008; Hennen –Biewagen et al., 2008; 2009; Liu et al., 2009).

Importantly, the total SS activity associated with the complex was enhanced by exogenous ATP (Liu et al., 2012b). Chapter 3 presents direct evidence that protein phosphorylation has a significant effect on the activity and conformation of maize SSIIa.

Independent of the presence of glucan substrates, exogenous ATP significantly altered the electrophoretic mobility of SSIIa in non-denaturing gels, at concentrations comparative to the physiological concentrations of ATP used by protein kinases (Chapter

3). Interestingly, the reversibility of the ATP-induced mobility shift of SSIIa strongly supports the view that the observed effect was a consequence of protein phosphorylation

(Fig. 3.6). Further, this ATP-induced shift in the electrophoretic mobility of SSIIa, was inhibited by protein kinase inhibitors FSBA and K252a (Fig. 3.9), further suggesting that

221 this phenomenon could be a result of the action of endogenous protein kinase(s) present in the amyloplasts.

Post translational modifications like protein phosphorylation have been shown to cause changes in the conformation of the target protein, and its surface properties, that affect self-association or association with other proteins, thus influencing their electrophoretic mobility (Hames, 1990; Johnson and Barford, 1993; Tetlow et al., 2004a,

2008; Hennen –Biewagen et al., 2008; 2009; Liu et al., 2009). Following non-denaturing

PAGE in the absence of glucan substrate, SSIIa was detected as two forms with different mobility in the untreated and dephosphorylated (APase-treated) amyloplast samples, suggesting that these forms could represent SSIIa with varied phosphorylation or in a different oligomeric state. Differentially migrating forms of granule bound SSIIa, have been previously reported in maize (Zhang et al., 2004; Grimaud et al., 2008), wheat

(starch granule proteins (SGP) (Rahman et al., 1995) and rice (Umemoto and Aoki, 2005) using denaturing SDS-PAGE, that were proposed to be differentially, post-translationally modified by phosphorylation. In the current study, phosphorylated (ATP-treated) SSIIa exhibited multiple activity bands on SS activity zymograms containing amylopectin and starch. All these observations imply that differential electrophoretic migration of SSIIa protein could be the result of conformational changes affecting its association with itself

(homo-oligomers), and /or other starch biosynthetic enzymes, consequent to its post translational modification by protein phosphorylation.

Observations from the SS zymogram analysis, and substrate affinity electrophoresis (Chapter 3), clearly demonstrated that SSIIa had no affinity for rabbit liver glycogen (a routinely used glucan primer in SS zymograms, in many starch

222 biosynthesis related studies), and therefore it is not a suitable primer for determining

SSIIa activity in non-denaturing zymograms. Analysis of the glucan affinities of phosphorylated (ATP-treated), and dephosphorylated (APase-treated) stromal maize

SSIIa showed that neither state had a significant effect on the Kd of the enzyme for amylopectin (Chapter 3). Similarly, ATP did not affect the Km value of SSIIa for amylopectin and ADP-glucose significantly, but substantially enhanced the Vmax of the enzyme towards the two substrates (Chapter 3, Table 3.4). The results presented in

Chapter 3 clearly demonstrated that the catalytic activity of stromal maize SSIIa is substantially enhanced (12-fold) by phosphorylation (ATP), and reduced by dephosphorylation (APase) (Fig. 3.16). Previous investigations on starch biosynthesis in cereal endosperms emphasize the significant role of protein phosphorylation on the catalytic functions of starch biosynthetic enzymes, and their interactions to form functional protein complexes facilitating biosynthesis of starch. For example, a stimulatory effect of protein phosphorylation was inferred by the ATP dependent stimulation of total SS and SBE activities of the well-defined trimeric complex (SSI-

SSIIa-SBEIIb) in maize (Tetlow et al. 2004a; 2008; Liu et al., 2009, 2012a &b). Protein phosphorylation is also been found to significantly affect the catalytic functions of glycosyl transferases like glycogen synthase (GS) and glycogen phosphorylase (GP), through conformational changes, enzyme associations, and allosteric activation (Barford and Jonson, 1992; Lawrence et al., 1997; Cohen, 1999; Jope and Johnson, 2004;

Hashimoto et al., 2010). Protein phosphorylation by glycogen synthase kinase (GSK3) has been found to decrease the sensitivity of glycogen synthase to allosteric activators, and inactivate the enzyme (Lawrence et al., 1997; Jope and Johnson, 2004). Likewise,

223 conformational changes following protein phosphorylation resulted in the association of a pair of functional dimers of glycogen phosphorylase to form a catalytically inactive tetramer (Barford, 1991; Barford and Jonson, 1992). During glycogen synthesis, both these enzymes (GS and GP) are enzymatically interconverted between active and inactive forms, with distinct kinetic and allosteric properties, via covalent modifications like protein phosphorylation (Voet and Voet, 2004).

The results shown in Chapter 3 clearly demonstrated that protein phosphorylation stimulates the catalytic activity (Vmax) of stromal maize SSIIa substantially, and affects its electrophoretic mobility. The enhancement of the activity of immunopurified SSIIa by protein phosphorylation suggests that SSIIa doesn’t have to be associated with other enzymes in a protein complex to be stimulated. The differential electrophoretic mobility of SSIIa following protein phosphorylation or dephosphorylation also indicates that major changes in protein conformation, and/ or its association with itself or other enzymes has occurred, possibly resulting in the formation of homo- or hetero-oligomers.

It is yet to be investigated as to whether the activity of maize SSIIa could be stimulated when it exists as a monomer or as a homo-oligomer. Chemical-crosslinking studies have shown that in wheat endosperm, in addition to forming hetero-protein complexes with SS isoforms, SBEII isoforms also formed homo-dimers in HMW forms resulting in their higher affinity for glucan substrates compared to the monomeric SBEII forms (Tetlow et al., 2008). Even though there is no evidence to date for homo-oligomerisation of SS isoforms, the possibility of homo-oligomerisation of SSIIa cannot be ruled out. The change in the elution profile of SSIIa following GPC analysis (Fig. 4.5) (discussed in the following section), upon pre-treatment of amyloplast lysates with ATP, also hints to the

224 possibility that SSIIa might exist as a homo- or hetero-oligomer in the absence of ATP. A detailed investigation of the monomeric forms of SSIIa, and the HMW and LMW protein aggregates containing SSIIa, following GPC analysis, utilizing chemical cross-linking techniques and mass spectrometry, could provide more direct evidence for the homo or hetero-oligomerisation of maize SSIIa. Further detailed analysis of the kinetics of these forms (monomers, homo or heteromers) of SSIIa could reveal whether a homo-oligomer might exhibit subunit co-operativity that perhaps resulted in an allosteric kinetics for

SSIIa in the untreated amyloplast sample as indicated by Figure 3.17. The data presented in Chapter 3 therefore signify major implications of protein phosphorylation on the regulation of SSIIa, during starch biosynthesis in developing maize endosperm.

5.3 Interactions of SSIIa with other starch biosynthetic enzymes is regulated by protein phosphorylation A great deal of experimental evidence indicate the operation of functional, physical interactions between SS and SBE isoforms in cereal endosperms which are regulated by protein phosphorylation of the participating enzymes (Tetlow et al., 2008,

Hennen –Biewagen et al., 2008; 2009; Liu et al., 2009; 2012a). Such phosphorylation- dependent, functional, protein-protein interactions of SSIIa with other starch biosynthesizing enzymes were therefore examined in Chapter 4. The co- immunoprecipitation of SBEs and other SSs with SSIIa, altered GPC elution profile of

SSIIa under conditions favouring phosphorylation or dephosphorylation, and the differential electrophoretic mobility of the designated HMW (670 kDa), and LMW (260 kDa) GPC fractions of SSIIa, provide substantial proof for the existence of phosphorylation-dependent, functional, protein-protein interactions of SSIIa with other

SS, and SBE isoforms in amyloplasts of developing maize endosperm (Chapter 4). Co-

225 immunoprecipitation of SBEIIb with SSIIa was significantly enhanced by protein phosphorylation (ATP) and reduced by dephosphorylation (APase), while, SSIIa-SSI interaction was apparently unaffected by exogenous ATP or APase (Fig. 4.3). In maize endosperm, pair-wise interaction of SSIIa with SSI, SBEIIb and SBEIIa have been previously reported (Hennen –Biewagen et al., 2008) and, the SSIIa – SBEII interaction was found to be phosphorylation dependent in wheat (Tetlow et al., 2004a; 2008).

The differential elution profile of SSIIa following GPC separation of stromal proteins from amyloplast lysates, pretreated with ATP or APase, suggested the existence of SSIIa at least in two distinct - HMW (670 kDa) and LMW (260 kDa) - protein complexes. This was reinforced by their differential mobility in non-denaturing PAGE

(Fig. 4.7). It could also be possible that SSIIa perhaps exists as a homo or hetero oligomer in the HMW (670 kDa) fractions in the absence of ATP. Unfortunately, the homo- or hetero-oligomerisation of SSIIa following phosphorylation or dephosphorylation of amyloplast lysates was not investigated in detail in this study.

Phosphorylated stromal SSIIa eluted in relatively higher proportion in LMW fractions

(260 kDa), along with SSI and SBEIIb, whereas stromal SSIIa from the untreated, and dephosphorylated lysates, eluted mostly in the HMW fractions (670 kDa) with SSI and

SBEIIb (Fig. 4.5). Furthermore, results from Phos-tag SDS-PAGE indicated that SSIIa is phosphorylated in the LMW fractions (in untreated and ATP-treated amyloplast samples). Co-elution of SBEIIb with SSIIa, following GPC, occurred mostly in the LMW

(260 kDa) fractions (Fig. 4.5), and co-immunoprecipitation of SBEIIb with SSIIa, from these LMW fractions was significantly reduced by dephosphorylation by APase

(Fig.4.10). These observations are consistent with the hypothesis that these three proteins

226

(SSIIa-SSI-SBEIIb) co-exist as a multi-enzyme complex, and that their interaction is phosphorylation dependent. However, co-immunoprecipitation results showed that SSI tended to interact with SSIIa predominantly in the HMW fractions (670 kDa) (Chapter 4).

Moreover, the co- migration (identical mobility) of SSI, SSIIa and SBEIIb in the LMW fractions (260 kDa) in non-denaturing amylopectin -containing gels (Fig. 4.9) further suggested that SSIIa could more likely form functional protein complexes with SSI and/or SBEIIb. Consistent with the glucan affinity data observed for SSIIa in the phosphorylated (ATP-treated) and dephosphorylated (APase-treated) amyloplast lysates

(Chapter 3), the dissociation/affinity constants of SSIIa in HMW (670 kDa) and LMW

GPC fractions (untreated) remained similar (Table 4.1). This indicated that protein phosphorylation might have influenced the association of SSIIa with other enzymes to form different multi-enzyme complexes (260-300 kDa) of varying molecular masses, without significantly affecting its affinity for the growing amylopectin cluster. An assessment of the electrophoretic mobility of “monomeric” SSIIa following GPC, (not examined in this study), could be performed to investigate whether SSIIa affinity towards amylopectin is different between the monomeric and heteromeric forms as observed by

Tetlow et al. (2008) for SBEII isoforms in wheat.

Together, the data from Chapter 4 indicate that SSIIa interacts and physically associates with other starch biosynthetic enzymes mainly SSI and SBEIIb, depending on its phosphorylation state. The differential elution of stromal maize SSIIa from phosphorylated and dephosphorylated amyloplast lysates following GPC, indicated the existence of SSIIa in two distinct protein aggregates - HMW (670 kDa) forms under conditions favouring dephosphorylation, and LMW (260 kDa) forms under conditions

227 favouring phosphorylation. It is proposed that, pretreatment with ATP might catalyze the formation of LMW protein complexes with other starch biosynthetic enzymes (possibly,

SSI and SBEIIb). This possibility was reinforced by co-immunoprecipitation studies, although these were not effective in detecting other proteins in the HMW fraction, the reasons for which are not clear, but could arise from SSIIa interacting with other proteins such as SSIII in the HMW fraction. It is inferred that phosphorylated, stromal maize

SSIIa, associates with SSI and SBEIIb in LMW (260 kDa) protein complexes, whereas under conditions favouring dephosphorylation, this 260 kDa complex of SSIIa-SSI-

SBEIIb possibly associates with other starch synthesizing enzymes and/or itself to form

HMW complexes of 670 kDa or more. The proposed protein-protein interactions of SSIIa with other starch biosynthetic enzymes, under conditions that favour protein phosphorylation and dephosphorylation, are shown in Figure 4.12. A more specific, and detailed analysis of the proposed protein complexes, utilizing chemical cross-linking techniques and mass spectrometry, could expose the interactions of SSIIa with other component proteins in these hetero-protein complexes. Such functional assemblies of different starch biosynthetic enzyme isoforms are postulated to improve the efficiency of the starch biosynthesis machinery resulting in the unique, highly organized, three dimensional crystalline structure of starch (Tetlow, 2011 and Geigenberger, 2011).

In summary, the results presented in this thesis demonstrate that stromal maize

SSIIa can be phosphorylated by one or more protein kinase(s) present in the amyloplasts of developing maize endosperm. The results also suggest that SSIIa bound to the starch granular matrix, exists in the phosphorylated state. The data generated suggest that protein phosphorylation regulates the catalytic function of maize SSIIa during starch

228 biosynthesis, and significantly affects the conformation of the enzyme, and its electrophoretic mobility. Most importantly, protein phosphorylation causes a significant enhancement in (12-fold) the catalytic activity (Vmax) of stromal maize SSIIa. Moreover, the direct effects of protein phosphorylation on the regulation of SSIIa activity were also found to have an impact on the functional protein –protein interactions of SSIIa with other enzymes (SSI and SBEIIb) in the pathway. The present study generates further insight into our growing knowledge of how the activities of different enzymes associated in starch synthesis, are coordinated through phosphorylation-dependent functional protein-protein interactions in developing maize endosperm. Further, this research could highlight novel targets for genetic manipulation, which is an essential prerequisite for rational manipulation of the quality, and quantity of starch produced by crop plants. Our understanding of the roles of protein phosphorylation in plant systems lags appreciably behind the significant progress made with animal systems. Hence this investigation will also generate new insights into a universal, regulatory mechanism controlling diverse biological functions in organisms, and is of potential biotechnological significance.

5.4 Future directions of research

In this thesis, the post translational regulation of maize SSIIa by protein phosphorylation and its physiological significance in the regulation of starch biosynthesis in developing maize endosperm has been investigated. Future investigations should be focussed on understanding the various processes involved in this post-translational modification, e.g. identification of the specific protein kinase(s) and phosphatase(s) involved, and their direct and indirect effects on the physical and functional properties of the enzyme. Prediction of putative phosphorylation sites on maize SSIIa using Net-Phos

229

2.0 Server indicated that SSIIa can be phosphorylated on 8 serine (Ser), 4 threonine (Thr) and 7 tyrosine (Tyr) residues (Fig. 2.15). Further research exploring the possible phosphorylation site(s) on maize SSIIa protein, based on the available bioinformatics database, will throw more light on how this post translational modification influences the function of SSIIa in starch biosynthesis. Similarly, further research using mutants and genotypes with modifications in the specific protein –protein interactions, will help unveil the physiological significance of these functional protein complexes in starch biosynthesis.

As a long term perspective, the results from the investigations conducted in vitro, could be correlated to explore the physiological significance of this regulatory process in controlling and coordinating the activities of starch biosynthetic enzymes including

SSIIa, under various physiological conditions of plant growth in vivo. Some aspects that can be investigated include the effect of protein phosphorylation on the catalytic function of maize SSIIa in relation to the different stages of endosperm development, changes in the concentration of metabolites like sugars (sucrose) and in relation to diurnal cycles.

Time-based detection of protein phosphorylation of starch biosynthetic enzymes including SSIIa, utilizing phospho-peptide specific antibodies that specifically detect phosphorylated proteins will throw more light on how these enzymes are post- translationally regulated during different stages and conditions of plant growth described above.

230

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