Luận án Tiến sĩ: Vai trò của Chu trình Protein trong Tích lũy Protein Hạt Lúa mì

The Contribution of Protein Turnover to the Accumulation of Wheat Grain Proteins Hui Cao Master of Science This thesis is presented for the degree of Doctor of Philosophy (Biochemistry) of The University of Western Australia ARC Centre of Excellence in Plant Energy Biology School of Molecular Sciences April 2021 Thesis Declaration I, Hui Cao, certify that: This thesis has been substantially accomplished during enrolment in this degree. This thesis does not contain material which has been submitted for the award of any other degree or diploma in my name, in any university or other tertiary institution. In the future, no part of this thesis will be used in a submission in my name, for any other degree or diploma in any university or other tertiary institution without the prior approval of The University of Western Australia and where applicable, any partner institution responsible for the joint-award of this degree. This thesis does not contain any material previously published or written by another person, except where due reference has been made in the text and, where relevant, in the Authorship Declaration that follows.

This thesis does not violate or infringe any copyright, trademark, patent, or other rights whatsoever of any person. Third party editorial assistance was provided in preparation of this thesis by Professor A.Harvey Millar, Dr. Owen Duncan and Dr Nicolas Taylor. Technical assistance was kindly provided by Owen Duncan for LC-MS/MS that is described in Chapter 2 and 3.

Shahidul Islam for grain protein content measurement by Near Infrared Transmission Spectrophotometry that is described in Chapter 2. Ricarda Fenske and Glenda Oh for metabolomics analysis by GC-MS that is described in Chapter 3. This thesis contains published work and/or work prepared for publication, some of which has been co-authored. Signature: Date: 15/10/2020 I Publications Manuscript prepared for publication Chapter 2 Increased wheat protein content via introgression of a HMW glutenin selectively reshapes the grain proteome Hui Cao, Owen Duncan, Shahidul Islam, Jingjuan Zhang, Wujun Ma, A.Harvey Millar Preliminary acceptance in revision for Molecular & Cellular Proteomics; Available on bioRxiv - doi: 10.

Chapter 3 The grain protein turnover atlas of hexaploid wheat Triticum aestivum Hui Cao, Owen Duncan, A.Harvey Millar In preparation II Authorship Declaration: Co-authored Publications This thesis contains work that has been prepared for publication. Details of the work: Increased wheat protein content via introgression of a HMW glutenin selectively reshapes the grain proteome Hui Cao, Owen Duncan, Shahidul Islam, Jingjuan Zhang, Wujun Ma, A. Harvey Millar Location in thesis: Chapter 2 Student contribution to work: Together with A. Harvey Millar and Wujun Ma, we conceived and planned the project.

I performed the experiment and data analysis, and wrote the manuscript. Co-author signatures and dates: 13/10/2020 14/10/2020 III Details of the work: The grain protein turnover atlas of hexaploid wheat Triticum aestivum Hui Cao, Owen Duncan, A. Harvey Millar Location in thesis: Chapter 3 Student contribution to work: Together with A. Harvey Millar and Owen Duncan, we conceived and planned the project.

I performed the experiment and data analysis, and wrote the manuscript. Co-author signatures and dates: 13/10/2020 14/10/2020 Student signature: Date: 15/10/2020 I, Harvey Millar works listed above are correct. Coordinating supervisor signature: Date: 13/10/2020 IV Acknowledgements As I am heading towards the end of the journey of my PhD, I would like to express my sincere gratitude to everyone who has helped me. I could not have reached this far without their supports.

First of all, I would like to thank my supervisor Professor A. Harvey Millar for opening me the door towards the world of PhD right before my IELTS test expired, and for all the valuable guidance, advice and discussion throughout my entire PhD. I would also like to thank my co-supervisors Dr. Owen Duncan and Dr.

Nicoles Taylor for their excellent technical support and encouragement that helped me find my confidence and complete the PhD. A special thank you must go to Owen for all the help with the QTOF-6550A. Many thanks also go to past and present members of the Millar lab for being so nice to me all the time, and for the wonderful help both in and out the lab. For which, I would like to mention Lei, Ricarda, Ting, Adriana, Xiao, Jakob, Martyna, Bhagya, Shaobai, Elke, Brendan, Santi, Nathan, and all other PEB members.

I would like to give a special thank you to Alex for his help in finding chemicals and tools in the lab whenever it was needed, and for the great conversations we had throughout the years. A special thank you should also go to Xuyen, Glenda, Alika and Lilian, for creating such a friendly and amazing office space, an I pleasure to work with you all in such a wonderful lab and centre. Thank you to everyone in the Admin team, including Geetha, Deborah, Jennifer, Olga, Karina and Rosie, for their efforts in keeping our academic life easy by creating an amazing workplace and helping us through tonnes of paperwork and orderings. This research was supported by an Australian Government Research Training Program Fee Offset (RTP) and an International Student and UWA Safety-Net Top-Up Scholarship, both of which were gratefully appreciated.

My greatest thanks must go to Doris, my wife, who has a great contribution to my PhD. Her help was always there whenever the help was needed, as well as her love that always encouraged me and helped me for overcoming all the challenges that I have faced. I also want to thank you for bring our little boy into the family who has brought so many happiness and wonderful memories to me. Many thanks must go to my parents and parents-in-law for their supports in so many ways.

I am so grateful to have you all in my life. V Abbreviations RP 19S regulatory particle CP 20S core protease CME Caveolin-mediated endocytosis CMA Chaperone-mediated autophagy DPA Days post anthesis DUB Deubiquitylating enzyme DEP Differentially expressed protein EF Elongation factor ER Endoplasmic reticulum EUE Energy use efficiency FDR False discovery rate FCP Fold change in protein abundance FW Fresh weight FHB Fusarium head blight GC-MS Gas chromatography mass spectrometry GS Glutamine synthetase GPC Grain protein content HSP Heat shock proteins HMW-GS High molecular weight glutenin subunits HPLC High-performance liquid chromatography iBAQ Intensity based absolute quantification iTRAQ Isobaric tags for relative and absolute quantitation ICAT Isotope-coded affinity tag LFQ Label free quantification LPF Labelled protein fraction LEA Late embryogenesis abundant proteins LC-MS Liquid chromatography mass spectrometry LMW-GS Low molecular weight glutenin subunits MS Mass spectrometry MALDI-TOF MS Matrix-assisted laser desorption/ionisation-time of flight mass spectrometry NIL Near-isogenic line VI NR Nitrate reductase NiR Nitrite reductase PTM Post-translational modification PCA Principal component analysis KD Protein degradation rate KS/A Protein synthesis rate RSD Relative standard deviation by mean SDS Sodium dodecyl sulfate SILAC Stable isotope labelling with amino acid in cell culture PSV Storage protein vacuole TMT Tandem mass tags TFBSs TF binding sites TPP Trans Proteomic Pipeline TF Transcription factors TGN Trans-Golgi network TCA cycle Tricarboxylic acid cycle UPS Ubiquitin-proteasome system VII Abstract Wheat (Triticum aestivum L.) is one of the three most important grain crops and provides approximately 20% of the calories consumed by humans. Due to the negative correlation between wheat grain yield and grain protein content, low grain protein content has become a significant issue in some countries due to the breeding focus on the improvement of grain yield. It has been shown that the introgression of a high molecular weight glutenin subunit (HMW-GS) gene, Glu-1Ay, into commercial wheat cultivars can add 1% more protein to total grain content on a grain weight basis and improve bread-making quality without lowering grain yield.

Results within this thesis revealed the quantitative proteomic changes present in introgressed Glu-1Ay gene lines that underpin bread wheat protein content without changing other agronomic traits. It showed that the abundance of the 1Ay21* HMW-GS protein contributed less than 20% of the total protein increase observed. Of the 115 differentially expressed proteins (DEPs) found in Glu-1Ay lines, the majority are other storage proteins, and proteins involved in protein synthesis, protein folding, protein degradation, stress response and grain development. Gene co-expression analysis demonstrated that nearly half the DEP encoding genes strongly co-expressed during grain development.

Promoters of these genes are enriched in elements associated with transcription initiation and light response, such as CAAT-box, G- box, GATT-motif and the Gt1-motif. The final protein abundance in wheat grain comes from the relative rates of protein synthesis and protein degradation during grain development. To gain a more comprehensive and quantitative understanding on how different sets of proteins accumulate, a protein turnover atlas of wheat grain proteins during grain development was generated in this thesis by combining in vivo stable isotope labelling (15N) and in-depth quantitative proteomics. This atlas illustrated that although large variations of protein turnover rates were observed among individual proteins, they are tightly associated with protein abundance accumulation over time in different functional protein categories and different wheat grain tissues.

Functional category analysis showed that storage proteins are rapidly cycled, having both higher synthesis and degradation rates than other functional categories. By contrast, those proteins that participate in photosynthesis, DNA synthesis and glycolysis showed low synthesis and degradation rates at all times. Calculations from these rates show that 18% of total grain ATP production is used for grain proteome biogenesis and 2% for maintenance, and storage proteins alone consumed nearly half of these budgets. Due to the relatively high protein degradation rate of storage proteins as a class of grain proteins, it was calculated that 25% of newly synthesized storage proteins were degraded during grain development and thus were absent at harvest.

These VIII data show that stabilization of the wheat grain proteome could improve grain protein content without introducing extra protein synthesis costs. Overall, the findings obtained from this thesis provide a quantitative insight into which sets of wheat proteins accumulate in high protein genotypes and a new perspective on the energy use efficiency of protein synthesis and maintenance (PEUE). The data helps fill the knowledge gap of the spatiotemporal variation of wheat grain protein abundance that cannot be directly explained by transcript abundance and can also direct selection of storage protein candidates with higher PEUE for future wheat quality breeding programmes. IX Contents Thesis Declaration.

II Authorship Declaration: Co-authored Publications. VIII List of Figures. XII List of Tables. XIV Chapter One:.

1 Wheat: a critical economic crop. 2 Wheat grain development and storage protein biosynthesis. 2 Mature wheat grain and storage protein content. 7 Proteomics in plants.

11 Protein related challenges in modern wheat agriculture. 17 Aims of this project. 30 Increased wheat protein content via introgression of a HMW glutenin selectively reshapes the grain proteome. 32 Materials and Methods.

62 The grain protein turnover atlas of hexaploid wheat Triticum aestivum. 64 Materials and Methods. 102 Futile grain protein turnover causes a remarkable loss in wheat grain protein content. 104 Ay HMW-GS introgression improved energy use efficiency in grains.

105 Protein turnover rates regulated by multiple factors. 106 Conclusions and future directions. 111 XI List of Figures Chapter 1 Figure 1 Model of the phylogenetic history of bread wheat (Triticum aestivum L; AABBDD). Figure 2 The structure of wheat grain.

Figure 3 Main components of wheat grain and main storage protein fractions. Figure 4 The key components of a mass spectrometer Figure 5 Protein turnover contributors in protein abundance changes. Chapter 2 Figure 1 HMW-GSs abundance in parental and Ay integrated lines. Figure 2 Principal component analysis of protein abundances from parental and Ay integrated lines.

Figure 3 Differentially expressed proteins following integration of Ay HMW-GS across three wheat cultivars. Figure 4 Correlation coefficients of gene expression profiles of the 114 common differentially expressed proteins. Figure 5 Model of a putative pathway for grain protein accumulation triggered by the introgression of 1Ay21* HMW-GS. Supplemental Figure S1 Peptide mass spectral data quality controls.

Supplemental Figure S2 Volcano plot for commonly detected proteins.