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

The peculiarities of gravisensing of hypocotyls and roots of the same seedling for the first time were compared under different gravistimulation conditions. Indirect experimental method, i.e. quantitative analysis of the dependence of amyloplast statics and kinetics on the direction and magnitude of gravitational force, was applied to assess the cytoskeleton role in the maintenance of polarized root and hypocotyl statocyte structure and amyloplast movements during seedling gravistimulation. The experiments were performed with garden cress (Lepidium sativum L.) seedlings in the dark by using original devices ‒ in-flight centrifuge ‘Neris-5’ (biosatellite ‘Bion-10’) in space and centrifuge-clinostat (a device with two orthogonal axes) in the Earth for modelling of altered gravity and gravitropic stimulation conditions. The magnitude of the gravitational force was changed from microgravity (real in space or simulated by horizontal clinostat) to 1-g (simulated by centrifugation in space or natural Earth's gravity) and its action direction was changed at 90° or 180° inversion with respect to the longitudinal axis of the seedlings. It was determined that the action of gravitational force is not an essential factor for the formation of gravisensing tissues in hypocotyl and root of garden cress seedlings; however, under real and simulated microgravity the growth of endodermal cells in hypocotyls is slower, the location of amyloplasts changes significantly with respect to the... [to full text] / Pirmą kartą buvo palyginti to paties daigo šaknies ir hipokotilio gravitacijos jutimo savitumai skirtingomis gravitacinio dirginimo sąlygomis. Netiesioginiu eksperimentiniu metodu, t.y. kiekybiškai analizuojant amiloplastų statikos ir kinetikos priklausomybę nuo gravitacinės jėgos veikimo krypties ir dydžio, buvo įvertinta citoskeleto reikšmė poliarizuotos šaknų ir hipokotilių statocitų struktūros palaikymui ir viduląsteliniams amiloplastų judesiams daigų gravitacinio dirginimo metu. Eksperimentai atlikti su sėjamosios pipirnės (Lepidium sativum L.) daigais tamsoje, gravitacijos pokyčių generavimui naudojant originalius prietaisus – borto centrifugą „Neris-5“ kosminio skrydžio sąlygomis (biopalydovas „Bion-10“) ir dviejų ortogonalių ašių centrifugą-klinostatą. Gravitacinės jėgos dydis buvo keičiamas nuo mikrogravitacijos lygmens (reali kosmose arba imituota horizontaliu klinostatavimu) iki 1 g (imituota centrifugavimu arba natūrali Žemės gravitacija), o jos veikimo kryptis buvo keičiama 90° arba 180° kampu daigo išilginės ašies atžvilgiu. Nustatyta, kad gravitacinės jėgos buvimas nėra būtina sąlyga sėjamosios pipirnės daigų šaknų ir hipokotilių gravisensorinio audinio formavimuisi, bet jai neveikiant (reali ir imituota mikrogravitacija) sulėtėja hipokotilių endodermio ląstelių augimas, pakinta gravisensorinių ląstelių poliškumas dėl esminio amiloplastų pakilimo ląstelių centro kryptimi. Nuolatinio gravitropinio dirginimo metu, kuomet sėjamosios pipirnės daigus šaknies... [toliau žr. visą tekstą]

Pirmą kartą buvo palyginti to paties daigo šaknies ir hipokotilio gravitacijos jutimo savitumai skirtingomis gravitacinio dirginimo sąlygomis. Netiesioginiu eksperimentiniu metodu, t.y. kiekybiškai analizuojant amiloplastų statikos ir kinetikos priklausomybę nuo gravitacinės jėgos veikimo krypties ir dydžio, buvo įvertinta citoskeleto reikšmė poliarizuotos šaknų ir hipokotilių statocitų struktūros palaikymui ir viduląsteliniams amiloplastų judesiams daigų gravitacinio dirginimo metu. Eksperimentai atlikti su sėjamosios pipirnės (Lepidium sativum L.) daigais tamsoje, gravitacijos pokyčių generavimui naudojant originalius prietaisus – borto centrifugą „Neris-5“ kosminio skrydžio sąlygomis (biopalydovas „Bion-10“) ir dviejų ortogonalių ašių centrifugą-klinostatą. Gravitacinės jėgos dydis buvo keičiamas nuo mikrogravitacijos lygmens (reali kosmose arba imituota horizontaliu klinostatavimu) iki 1 g (imituota centrifugavimu arba natūrali Žemės gravitacija), o jos veikimo kryptis buvo keičiama 90° arba 180° kampu daigo išilginės ašies atžvilgiu. Nustatyta, kad gravitacinės jėgos buvimas nėra būtina sąlyga sėjamosios pipirnės daigų šaknų ir hipokotilių gravisensorinio audinio formavimuisi, bet jai neveikiant (reali ir imituota mikrogravitacija) sulėtėja hipokotilių endodermio ląstelių augimas, pakinta gravisensorinių ląstelių poliškumas dėl esminio amiloplastų pakilimo ląstelių centro kryptimi. Nuolatinio gravitropinio dirginimo metu, kuomet sėjamosios pipirnės daigus šaknies... [toliau žr. visą tekstą] / The peculiarities of gravisensing of hypocotyls and roots of the same seedling for the first time were compared under different gravistimulation conditions. Indirect experimental method, i.e. quantitative analysis of the dependence of amyloplast statics and kinetics on the direction and magnitude of gravitational force, was applied to assess the cytoskeleton role in the maintenance of polarized root and hypocotyl statocyte structure and amyloplast movements during seedling gravistimulation. The experiments were performed with garden cress (Lepidium sativum L.) seedlings in the dark by using original devices ‒ in-flight centrifuge ‘Neris-5’ (biosatellite ‘Bion-10’) in space and centrifuge-clinostat (a device with two orthogonal axes) in the Earth for modelling of altered gravity and gravitropic stimulation conditions. The magnitude of the gravitational force was changed from microgravity (real in space or simulated by horizontal clinostat) to 1-g (simulated by centrifugation in space or natural Earth's gravity) and its action direction was changed at 90° or 180° inversion with respect to the longitudinal axis of the seedlings. It was determined that the action of gravitational force is not an essential factor for the formation of gravisensing tissues in hypocotyl and root of garden cress seedlings; however, under real and simulated microgravity the growth of endodermal cells in hypocotyls is slower, the location of amyloplasts changes significantly with respect to the... [to full text]