Proteomic study on the starch synthesis and regulation in developing hybrid rice seeds.

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