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Long Xiaohang. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 132-155). / Abstracts in English and Chinese. / Thesis/Assessment Committee --- p.I / Statement from Author --- p.II / Acknowledgements --- p.III / Abstract --- p.V / 摘要 --- p.VII / Table of Contents --- p.IX / List of Tables --- p.XV / List of Figures --- p.XVI / List of Abbreviations --- p.XVIII / Chapter Chapter 1 --- General Introduction and Literature Review --- p.1 / Chapter 1.1 --- General introduction --- p.1 / Chapter 1.2 --- Literature review --- p.5 / Chapter 1.2.1 --- Rice --- p.5 / Chapter 1.2.1.1 --- Classification of rice --- p.5 / Chapter 1.2.1.2 --- Rice grain quality --- p.5 / Chapter 1.2.2 --- Overview of current information on the starch biosynthesis and regulation during seed development --- p.7 / Chapter 1.2.2.1 --- Starch property --- p.7 / Chapter 1.2.2.1.1 --- Structure of rice starch granules --- p.7 / Chapter 1.2.2.1.2 --- Properties of rice starch --- p.7 / Chapter 1.2.2.2 --- Starch synthesis related proteins --- p.8 / Chapter 1.2.2.2.1 --- The formation of ADP-glucose through AGPase --- p.10 / Chapter 1.2.2.2.2 --- The synthesis of starch by starch synthases --- p.10 / Chapter 1.2.2.2.2.1 --- Amylose biosynthesis --- p.10 / Chapter 1.2.2.2.2.2 --- Amylopectin biosynthesis --- p.11 / Chapter 1.2.2.2.3 --- Branching of the glucan chain by starch branching enzymes --- p.12 / Chapter 1.2.2.2.4 --- The role of debranching enzymes in polymer synthesis --- p.13 / Chapter 1.2.2.2.5 --- Starch degradation in plastids --- p.13 / Chapter 1.2.2.2.6 --- Other enzymes involved in starch synthesis pathway --- p.13 / Chapter 1.2.2.3 --- Starch biosynthesis regulation --- p.14 / Chapter 1.2.2.3.1 --- Developmental regulation --- p.14 / Chapter 1.2.2.3.2. --- Diurnal regulation --- p.15 / Chapter 1.2.2.3.3 --- 3-PGA/Pi regulation --- p.16 / Chapter 1.2.2.3.4. --- Sugar signaling --- p.17 / Chapter 1.2.2.3.5. --- Hormonal signaling --- p.18 / Chapter 1.2.2.3.6 --- Post translational modification regulation --- p.18 / Chapter 1.2.2.3.6.1 --- Post translational redox modulation --- p.18 / Chapter 1.2.2.3.6.2 --- Protein phosphorylation --- p.19 / Chapter 1.2.2.4 --- Rice grain quality improvement by genetic engineering --- p.20 / Chapter 1.2.2.4.1 --- Cooking and eating quality improvement --- p.20 / Chapter 1.2.2.4.1.1 --- Manipulation of starch content --- p.20 / Chapter 1.2.2.4.1.2 --- Manipulation of amylose/ amylopectin ratio --- p.20 / Chapter 1.2.2.4.2 --- Other targets for manipulating starch quality and quantity --- p.21 / Chapter 1.2.3 --- Proteomics --- p.23 / Chapter 1.2.3.1 --- General introduction --- p.23 / Chapter 1.2.3.2 --- Current technologies of proteomics --- p.25 / Chapter 1.2.3.2.1 --- Protein separation by 2D or non-2D method --- p.25 / Chapter 1.2.3.2.2 --- Protein visualization --- p.26 / Chapter 1.2.3.2.3 --- Computer-assisted image analysis --- p.27 / Chapter 1.2.3.2.4 --- Protein identification by mass spectrometry --- p.28 / Chapter 1.2.3.2.5 --- Database search --- p.28 / Chapter 1.2.3.2.5.1 --- Database searching software --- p.29 / Chapter 1.2.3.2.5.2 --- Protein sequence database --- p.29 / Chapter 1.2.3.2.5.3 --- Evaluating database hits --- p.30 / Chapter 1.2.3.2.6 --- Bioinformatics involved in proteomics --- p.31 / Chapter 1.2.3.2.7 --- Post translational modification --- p.32 / Chapter 1.2.3.2.7.1 --- Glycosylation --- p.32 / Chapter 1.2.3.2.7.1.1 --- N-linked glycosylation --- p.33 / Chapter 1.2.3.2.7.1.2 --- O-linked glycosylation --- p.33 / Chapter 1.2.3.2.7.2 --- Phosphorylation --- p.33 / Chapter 1.2.3.2.7.3 --- Strategies for studying PTMs --- p.34 / Chapter 1.2.3.2.8 --- Other aspects of proteomics --- p.36 / Chapter 1.2.3.2.8.1 --- 2D DIGE --- p.36 / Chapter 1.2.3.2.8.2 --- LC/LC-MS/MS --- p.36 / Chapter 1.2.3.2.8.2.1 --- MudPIT --- p.36 / Chapter 1.2.3.2.8.2.2 --- ICAT --- p.37 / Chapter 1.2.3.3 --- Plant proteomics --- p.37 / Chapter 1.2.3.3.1 --- Proteome analysis of plant tissues and organs --- p.38 / Chapter 1.2.3.3.2 --- Plant organelle proteomics --- p.39 / Chapter 1.2.3.3.3 --- Post translational modifications in plant --- p.41 / Chapter 1.2.3.4 --- Recent progress in rice proteomics --- p.42 / Chapter 1.2.3.4.1 --- General introduction of rice proteomics --- p.42 / Chapter 1.2.3.4.2 --- Rice proteome database construction --- p.43 / Chapter 1.2.3.4.3 --- Comparative proteomics --- p.43 / Chapter 1.2.3.4.4 --- Post translational modification study of rice proteome --- p.44 / Chapter Chapter 2 --- Materials and methods --- p.45 / Chapter 2.1 --- Materials --- p.45 / Chapter 2.1.1 --- Plant materials --- p.45 / Chapter 2.1.2 --- Chemical reagents and commercial kits --- p.46 / Chapter 2.1.3 --- Instruments --- p.46 / Chapter 2.1.4 --- Software --- p.46 / Chapter 2.2 --- Methods --- p.47 / Chapter 2.2.1 --- Fractionation of amyloplast and amyloplast membrane proteins --- p.47 / Chapter 2.2.2 --- Marker enzyme assays --- p.47 / Chapter 2.2.3 --- 2D gel electrophoresis --- p.48 / Chapter 2.2.4 --- Silver staining of 2D gel --- p.49 / Chapter 2.2.5 --- In-gel digestion of protein spots --- p.49 / Chapter 2.2.6 --- Desalination of the digested sample with ZipTip --- p.49 / Chapter 2.2.7 --- Protein identification by mass spectrometry and database searching --- p.50 / Chapter 2.2.8 --- Image and data analysis --- p.50 / Chapter 2.2.9 --- Extraction of starch granule associated proteins --- p.51 / Chapter 2.2.10 --- Western blot analysis --- p.51 / Chapter 2.2.11 --- Sample preparation for N terminal sequencing --- p.51 / Chapter 2.2.12 --- Phosphorylation and glycosylation assays --- p.52 / Chapter Chapter 3 --- Results --- p.53 / Chapter 3.1 --- Protein identification by ID and 2D PAGE --- p.53 / Chapter 3.1.1 --- Isolation and purification of amyloplasts from rice seeds --- p.53 / Chapter 3.1.2 --- Identification of amyloplast and amyloplast membrane proteins by MS/MS --- p.54 / Chapter 3.1.2.1 --- Sample preparation --- p.54 / Chapter 3.1.2.2 --- 2D and ID gel electrophoresis --- p.55 / Chapter 3.1.2.3 --- Protein identification by MS and MS/MS --- p.56 / Chapter 3.1.3 --- Functional classification of identified proteins --- p.69 / Chapter 3.1.3.1 --- Metabolism proteins --- p.71 / Chapter 3.1.3.2 --- Non starch synthesis metabolism proteins --- p.73 / Chapter 3.1.3.3 --- Protein destination --- p.73 / Chapter 3.1.3.4 --- Proteins with other functions --- p.74 / Chapter 3.1.4 --- Cross-correlation of experimental and calculated Mw of proteins --- p.74 / Chapter 3.1.5 --- Granule bound starch synthase (GBSS) --- p.75 / Chapter 3.1.5 --- N-terminal sequencing --- p.77 / Chapter 3.2 --- Protein profiling --- p.80 / Chapter 3.2.1 --- Seed collection and stages chosen --- p.80 / Chapter 3.2.2 --- The proteomic profiles of rice amyloplasts at different developing stages --- p.81 / Chapter 3.2.4 --- Comparing the proteome of three rice lines --- p.85 / Chapter 3.2.4.1 --- Spot number analysis --- p.85 / Chapter 3.2.4.2 --- Functional distribution analysis --- p.86 / Chapter 3.2.4.3 --- Protein amount analysis --- p.87 / Chapter 3.2.5 --- Comparison of expression pattern: cluster analysis (SOM) --- p.88 / Chapter 3.2.5.1 --- Cluster analysis of rice amyloplast proteome --- p.88 / Chapter 3.2.5.2 --- Three major categories of rice amyloplast proteome expression patterns --- p.91 / Chapter 3.2.6 --- Scatter plots analysis --- p.94 / Chapter 3.2.7 --- Comparison of changes in proteins related to starch synthesis --- p.96 / Chapter 3.2.7.1 --- GBSS --- p.96 / Chapter 3.2.7.2 --- AGPase --- p.98 / Chapter 3.2.7.3 --- SSS --- p.98 / Chapter 3.2.7.4 --- SBE --- p.98 / Chapter 3.2.7.5 --- SP --- p.98 / Chapter 3.3 --- Study on protein post translational modifications --- p.102 / Chapter 3.3.1 --- Post translational modifications that potentially regulate starch synthesis --- p.102 / Chapter 3.3.2 --- Post translational modifications at different developing stages --- p.104 / Chapter 3.3.2.1 --- Profiling of post translational modifications of rice amyloplast proteome --- p.104 / Chapter 3.3.2.2 --- Starch synthesis related proteins --- p.106 / Chapter 3.3.2.2.1 --- GBSS --- p.106 / Chapter 3.3.2.2.2 --- SSS --- p.108 / Chapter Chapter 4 --- Discussion --- p.111 / Chapter 4.1 --- Methodology --- p.111 / Chapter 4.1.1 --- Amyloplast isolation --- p.111 / Chapter 4.1.2 --- Protein extraction from amyloplasts --- p.111 / Chapter 4.1.3 --- Protein identification by PMF and MS/MS --- p.112 / Chapter 4.1.4 --- Methods used to study protein expression patterns --- p.113 / Chapter 4.1.5 --- New methods introduced to study post translational modifications --- p.114 / Chapter 4.2 --- Characteristics of rice amyloplast proteins --- p.115 / Chapter 4.2.1 --- Amyloplast proteins associated with starch granules --- p.116 / Chapter 4.2.2 --- Most proteins in amyloplast proteome contain the transit peptide --- p.116 / Chapter 4.2.3 --- Multiple isoforms of starch synthesis related proteins --- p.117 / Chapter 4.2.3.1 --- Multiple spots of GBSS --- p.118 / Chapter 4.2.4 --- Expression patterns of amyloplast proteome --- p.120 / Chapter 4.2.5 --- Post translational modifications potentially regulate starch synthesis --- p.122 / Chapter 4.3 --- Other characteristic aspects of amyloplast proteome --- p.123 / Chapter 4.3.1 --- Comparison between the rice and wheat amyloplast proteomes --- p.123 / Chapter 4.3.2 --- Proteomic comparisons among the three rice lines --- p.124 / Chapter 4.3.3 --- Comparison of starch synthesis enzymes at protein and transcript levels --- p.124 / Chapter 4.3.4 --- Comparison of the starch synthesis related proteins among the three rice lines --- p.126 / Chapter 4.4 --- Limitations of proteomic approach in directly answering the question on how to improve eating and cooling quality --- p.126 / Chapter Chapter 5 --- Conclusion --- p.128 / Chapter Chapter 6 --- Future perspectives --- p.130 / References --- p.132

Collection of the author's previous publications. Includes bibliographies.

Bibliography: leaves 174-192. This research aims to obtain detailed knowledge on the effects of a period of high temperature on the accumulation of grain dry matter and endosperm starch, protein and B-glucan in the developing grain of the malting barley variety Schooner. Bbarley plants are exposed to high temperatures during mid grain filling for 5 days. Grain growth characteristics are measured prior to, during and following the high temperature period, with the aim of characterising the high temperature response in developing grain. The activities of several enzymes and metabolities of the pathway of starch synthesis are monitored and compared to those in grains maintained at a lower temperature. In addition, grain structure is also compared between control and heat treated grain during development, at maturity and following malting.

Four studies were conducted to determine the effect of synchronization of protein and starch degradation on nutrient utilization, microbial protein synthesis and milk production in dairy cows. In Experiment 1, five cereal grains and five protein supplements were compared for extent of solubility and degradability of their starch and nitrogen fractions. Results indicated large differences which permitted their ranking from high to low degradability as follows: grains, oats > wheat > barley > corn > milo protein supplements, soybean meal > cottonseed meal, (CSM) > corn gluten meal > brewers dried grains, (BDG) > blood meal. In Experiment 2, the five grains were incubated for varying times in vitro (with added amylase) or in situ to determine rate and extent of degradation of dry matter, crude protein and starch. Results showed that rate of starch degradation followed a similar, but slightly different trend than in trial 1 (wheat > barley > oats > corn > milo). Rates for DM and CP degradation were similar than those for starch. In Experiment 3, high (barley, HS) and low (milo, LS) degradable starch sources were combined with a high (CSM, HP) and a low (BDG, LP) degradable protein sources to formulate four diets; HSHP, HSLP, LSHP and LSLP. Diets were fed to 32 cows, starting two to four weeks postpartum, for a 60-d milk production and digestibility study. Apparent digestibility was calculated using chromium oxide. Organic matter digestibility was higher (P < .05) was found in nutrient output to the small intestine among diets and microbial CP synthesis was higher (P > .05) for barley diets.

Collection of the author's previous publications / Includes bibliographies / 1 v. (various pagings) : / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (D. Sc.)--University of Adelaide, 1989

Includes bibliographical references (leaves 175-196). / xii, 196 leaves : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / The influence of starch in grain-based diets on fat development in cattle and sheep is investigated. / Thesis (Ph.D.)--University of Adelaide, Dept. of Animal Sciences, 1995

Post-translational redox-regulation is a well-known mechanism to regulate enzymes of the Calvin cycle, oxidative pentose phosphate cycle, NADPH export and ATP synthesis in response to light. The aim of the present thesis was to investigate whether a similar mechanism is also regulating carbon storage in leaves. <br><br> Previous studies have shown that the key-regulatory enzyme of starch synthesis, ADPglucose pyrophosphorylase (AGPase) is inactivated by formation of an intermolecular disulfide bridge between the two catalytic subunits (AGPB) of the heterotetrameric holoenzyme in potato tubers, but the relevance of this mechanism to regulate starch synthesis in leaves was not investigated. The work presented in this thesis shows that AGPase is subject to post-translational redox-regulation in leaves of pea, potato and Arabidopsis in response to day night changes. Light was shown to trigger posttranslational redox-regulation of AGPase. AGPB was rapidly converted from a dimer to a monomer when isolated pea chloroplasts were illuminated and from a monomer to a dimer when preilluminated leaves were darkened. Conversion of AGPB from dimer to monomer was accompanied by an increase in activity due to changes in the kinetik properties of the enzyme. Studies with pea chloroplast extracts showed that AGPase redox-activation is mediated by thioredoxins f and m from spinach in-vitro. In a further set of experiments it was shown that sugars provide a second input leading to AGPase redox activation and increased starch synthesis and that they can act as a signal which is independent from light. External feeding of sugars such as sucrose or trehalose to Arabidopsis leaves in the dark led to conversion of AGPB from dimer to monomer and to an increase in the rate of starch synthesis, while there were no significant changes in the level of 3PGA, an allosteric activator of the enyzme, and in the NADPH/NADP+ ratio. Experiments with transgenic Arabidopsis plants with altered levels of trehalose 6-phosphate (T6P), the precursor of trehalose synthesis, provided genetic evidence that T6P rather than trehalose is leading to AGPase redox-activation. Compared to Wt, leaves expressing E.coli trehalose-phosphate synthase (TPS) in the cytosol showed increased activation of AGPase and higher starch level during the day, while trehalose-phosphate phosphatase (TPP) overexpressing leaves showed the opposite. These changes occurred independently of changes in sugar and sugar-phosphate levels and NADPH/NADP+ ratio. External supply of sucrose to Wt and TPS-overexpressing leaves led to monomerisation of AGPB, while this response was attenuated in TPP expressing leaves, indicating that T6P is involved in the sucrose-dependent redox-activation of AGPase. To provide biochemical evidence that T6P promotes redox-activation of AGPase independently of cytosolic elements, T6P was fed to intact isolated chloroplasts for 15 min. incubation with concentrations down to 100 µM of T6P, but not with sucrose 6-phosphate, sucrose, trehalose or Pi as controls, significantly and specifically increased AGPB monomerisation and AGPase activity within 15 minutes, implying T6P as a signal reporting the cytosolic sugar status to the chloroplast. The response to T6P did not involve changes in the NADPH/NADP+ ratio consistent with T6P modulating redox-transfer to AGPase independently of changes in plastidial redox-state. <br><br> Acetyl-CoA carboxylase (ACCase) is known as key-regulatory enzyme of fatty acid and lipid synthesis in plants. At the start of the present thesis there was mainly in vitro evidence in the literature showing redox-regulation of ACCase by DTT, and thioredoxins f and m. In the present thesis the in-vivo relevance of this mechanism to regulate lipid synthesis in leaves was investigated. ACCase activity measurement in leaf tissue collected at the end of the day and night in Arabidopsis leaves revealed a 3-fold higher activation state of the enzyme in the light than in the dark. Redox-activation was accompanied by change in kinetic properties of ACCase, leading to an increase affinity to its substrate acetyl-CoA . In further experiments, DTT as well as sucrose were fed to leaves, and both treatments led to a stimulation in the rate of lipid synthesis accompanied by redox-activation of ACCase and decrease in acetyl-CoA content. <br><br> In a final approach, comparison of metabolic and transcript profiling after DTT feeding and after sucrose feeding to leaves provided evidence that redox-modification is an important regulatory mechanism in central metabolic pathways such as TCA cycle and amino acid synthesis, which acts independently of transcript levels. / Es ist bereits seit längerem bekannt, dass viele Enzyme des Calvinzyklus, des oxidativen Pentosephosphatwegs, des NAD(P)H-Exports und der ATP-Synthese durch post-translationale Redox-Modifikation in Antwort auf Licht reguliert werden. In der vorliegenden Arbeit sollte untersucht werden, ob ein ähnlicher Mechanismus auch die Kohlenstoffspeicherung in Blättern reguliert. <br><br> Vorangegangene Studien mit Kartoffelknollen zeigten, dass das Schlüsselenzym der Stärkesynthese ADP-Glukose-Pyrophosphorylase (AGPase) durch die Bildung einer Disulfidbrücke zwischen den zwei kleinen Untereinheiten (AGPB) des tetrameren Proteins inaktiviert wird, die Bedeutung dieses Mechanismus für die Stärkesynthese in Blättern blieb jedoch bislang ungeklärt. Die vorliegenden Arbeiten zeigen, das AGPase in Erbsen-, Kartoffel- und Arabidopsis-Blättern über post-translationale Redox-Modifikation in Antwort auf Tag-Nacht Änderungen reguliert wird. Dies erfolgt über ein Licht-abhängiges Signal, da, erstens, AGPB in isolierten Chloroplasten durch Belichtung sehr schnell von Dimer zu Monomer umgewandelt wird und, zweitens, ein Abdunkeln der Blätter zu einer schnellen Umwandlung von AGPB von Monomer zu Dimer führt. Die Monomerisierung von AGPB ging mit Änderungen in den kinetischen Eigenschaften des Enzyms einher, die zu einer Aktivierung führten. Studien mit Extrakten aus Erbsenchloroplasten zeigten, dass die AGPase-Redoxaktivierung in-vitro durch die Thioredoxine f und m aus Spinat vermittelt wird. In einem weiteren experimentellen Ansatz konnte gezeigt werden, dass auch Zucker zu Redox-Aktivierung der AGPase und erhöhter Stärkesynthese in Blättern führen, und dass diese unabhängig von Licht wirken. Externe Zugabe von Zuckern wie Saccharose oder Trehalose an Arabidopsis-Blätter im Dunkeln führten zu Monomerisierung von AGPB und einer Erhöhung der Stärkesyntheserate / während die Spiegel des allosterischen Aktivators 3PGA unverändert blieben und keine Änderungen im NADPH/NADP+-Verhältnis auftraten. Experimente mit transgenen Arabidopsis-Pflanzen mit veränderten Spiegeln des Vorläufers der Trehalosesynthese, Trehalose-6-phosphat (T6P), zeigten, dass T6P und nicht Trehalose zu Redox-Aktivierung von AGPase führt. Expression einer E. coli T6P synthase (TPS) im Zytosol führte zu erhöhter Redox-Aktivierung von AGPase und erhöhter Stäreksynthese in Blättern, während die Expression einer T6P-Phosphatase (TPP) gegenteilige Änderungen bewirkte. Diese Auswirkungen erfolgten unabhängig von Änderungen in den Spiegeln von Zuckern und Zuckerphosphaten oder im NADPH/NADP+-Verhältnis. Externe Zugabe von Saccharose führte zu Monomerisierung von AGPB in Wildtyp und TPS exprimierenden Blättern, während diese Antwort in TPP exprimierenden Blättern stark abgeschwächt war. Dies zeigt, dass T6P eine wesentliche Komponente darstellt, die die Redox-Aktivierung der AGPase in Antwort auf Saccharose vermittelt. T6P wurde auch für 15 min direkt an intakte, isolierte Erbsenchloroplasten gefüttert. T6P Konzentrationen im Bereich von 100 µM bis 10 mM führten zu einem signifikanten und spezifischen Anstieg der AGPB-Monomersierung und der AGPase Aktivität. Dies zeigt, dass T6P auch ohne zytosolische Elemente die Redox-Aktivierung der AGPase stimuliert und somit ein Signal zwischen Zytosol und Plastid darstellt. Diese Antwort erfolgte ohne Änderungen im NADPH/NADP+-Verhältnis, was zeigt, dass T6P eher den Redox-Transfer zu AGPase als den Redoxzustand des Chloroplasten moduliert. <br><br> Acetyl-CoA-Carboxylase (ACCase) ist als Schlüsselenzym der Fettsäure- und Lipidsynthese in Pflanzen bekannt. Zu Beginn der vorliegenden Arbeit lagen hauptsächlich in-vitro Befunde vor, die zeigten, dass ACCase durch DTT und thioredoxine f und m über Redox-Modulation reguliert wird. In der Arbeit sollte daher die in-vivo Relevanz dieses Mechanismus für die Regulation der Lipidsynthese in Blättern untersucht werden. ACCase zeigte einen höheren Redox-Aktivierungszustand in Arabidopsis-Blätter, die während des Tages im Vergleich zur Nacht geerntet wurden. Die Redox-Aktivierung der ACCase wurde von Änderungen in den kinetischen Eigenschaften begleitet und führte zu einer erhöhten Affinität des Enzymes gegenüber Acetyl-CoA als Substrat. <br><br> In weiteren Versuchen wurde sowohl DTT als auch Saccharose an Blätter gefüttert, und beide Behandlungen führten zu Redox-Aktivierung von ACCase, was mit erhöhten Lipidsynthesraten und einem Rückgang des Acetyl-CoA-Spiegels einherging.