Seed Dormancy in Malting Barley Sarah Karen Osama Msc. Plant Breeding and Genetics Bsc Hons Agriculture, Plant Science Major

Seed dormancy in malting barley Sarah Karen Osama MSc. Plant Breeding and Genetics BSc Hons Agriculture, Plant Science Major

A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in (2020) Centre for Nutrition and Food Sciences Queensland Alliance for Agriculture and Food Innovation The University of Queensland Brisbane, QLD 4072, Australia

Abstract Barley is an important crop ranking fourth among cereal crops produced worldwide. Two thirds of barley produced is used as either food or feed, while a third goes into the malting and brewing industry. Importantly for the latter, barley is stored after harvest, prior to being used to make malt which is the primary raw material in producing beer. The barley storage conditions may negatively affect the quality of grain for malting. Dormancy is critical to prevent loss of germination prior to malting. However, when the grain is delivered to the maltsters it must be at maximum germination. Industrial malting relies heavily on rapid and uniform germination to yield maximum quantities of malt. Low germination rates result in financial constraints to maltsters in terms of losses associated with the quality of malt and increased storage costs. Malting plants must optimise protocols for different barley cultivars, to attain the desired germination rate of 98% or higher, and so maximise yield. Therefore the long term storage and dormancy implies differences in malt quality which can affect aspects of the end product, such as flavour. Hence emphasis on improving the quality of malt, is just as desirable as malt yield.

Protein composition in malting barley is important. It varies between grain types and stages of growth. Therefore to study protein composition of seeds at different stages of growth, five replicates of seeds were used before germination, 24 hours after the onset of germination and 72 hours after the onset of germination. Late germinating and dormant seeds were also sampled. These were subjects to HPLC MS/MS and MSstats. From the study 128 proteins were identified and of these, 15 proteins showed significant differences at different times across the course of germination when analysed using ANOVA. Proteins clustered into three groups, (i) proteins important during the seed resting period including but not limited to late embryogenesis (LE19A), defensin and dehydrin, (ii) proteins important in early germination including beta amylase, gamma hordein 3, C hordein fragment and (iii) proteins important for embryo growth and development including but not limited to actin, protein H2A, and malate dehydrogenase.

The second goal of the study was planned around monitoring dormancy of barley varieties for a period of 12 months with proteome changes between dormant and germinating seeds. The storage temperature was at 22 oC. Protein and RNA was extracted from single seeds using modified TRIzol extraction protocol. Using mass spectrometry, 278 proteins were identified and their relative abundances measured. From the experiment we discovered i) the germination

I percentage (GP) declined with storage where the three varieties showed low GP after harvest, it increased quickly in four months to 100% in both Stirling and Gairdner and 97% in Schooner. But there was slow decline after that with Schooner showing the lowest GP of 84% ii) at harvest, proteins with relative abundance which were significantly higher at <0.05 in dormant samples included desiccation tolerance proteins such as the LEA proteins and dehydrin among others, iii) after 10 months storage, proteins such as serpins, aspartic proteinase and serine carboxypeptidase showed significantly higher relative abundance in dormant samples than in germinating samples, iv) comparing protein abundances from dormant samples at 10 months storage vs at harvest showed desiccation tolerance proteins and, protein disulphide isomerase as well as other were significantly higher at harvest. Conversely proteins such as serine carboxypeptidase, aspartic proteinase, and heat shock proteins were significantly higher after 10 months storage.

The next stage was to assess changes during commercial malting. A single malt variety was sampled from two commercial malt plants in Australia to explore the protein profile of germinating barley over time during the malting process, including comparisons between the malt plants. From this experiment, 618 proteins were identified. Analysis of relative abundances of the proteins identified 277 significant changes over the stages of malting. Gene ontology (GO) analysis showed proteins overexpressed in functions related to redox state, dormancy, jasmonic acid metabolism, and amylopectin metabolism, among others. An assessment of the gene functions identified 38 proteins that were directly linked to dormancy maintenance. Proteins such as 1-cysperodoxin (PER 1) identified has been associated with dormancy maintenance in previous studies. Further analysis showed that protein profiles differed during the malting process. There was evidence to show that variation in malt quality and hence beer, may be due to the proteins that are expressed during various stages of the malting process, hence the use of proteomics in investigating the protein profiles of malting grains, this included proteins such as lipid transfer protein, proteinases and lipoxygenase (LOX), among others. Heat map clustering showed protein increases in abundance from the seed through the malting stages to reach the peak at day four germination then lowered significantly by kilning. A critical aspect of the research was experimental design during various stages of the experiments to ensure more robust research outcomes, all the chapters had a given design to ensure precise measurements and reduced variability.

Important proteins to the study which have been identified in all three chapters include LEA proteins which was identified to be involved in both desiccation tolerance and dormancy regulation. LEA are a group of proteins classified under desiccation tolerance which protect the embryo during the dry seed phase. Another important group to dormancy, germination and malting quality is the serpin proteins. They are germination inhibitors through their serine-type endopeptidase inhibitor activity. These were found to increase with germination and were detected even after kilning hence end up in beer. The overall results will assist barley breeders and those in the malting and brewing industry to understand the proteome changes in barley grains in a dormant stage, all the way through to beer production.

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Declaration by author

This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis.

I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, financial support and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my higher degree by research candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award.

I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School.

I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis and have sought permission from co- authors for any jointly authored works included in the thesis.

Publications included in this thesis “No publications included”.

Submitted manuscripts included in this thesis “No manuscripts submitted for publication”.

Other publications during candidature

Anoumaa, M., Yao, N.K., Kouam, E.B., Kanmegne, G., Machuka, E., Osama, S.K., Nzuki, I., Kamga, Y.B., Fonkou, T. and Omokolo, D.N., 2017. Genetic diversity and core collection for potato (Solanum tuberosum L.) cultivars from Cameroon as Revealed by SSR markers. American Journal of Potato Research, 94(4), pp.449-463.

Ayagirwe, B., Meutchieye, F., Djikeng, A., Skilton, R., Osama, S. and Manjeli, Y., 2017. Genetic diversity and structure of domestic cavy (Cavia porcellus) populations from smallholder farms in southern Cameroon. Animal production, 19(1), pp.1-12.

Mekuriaw, G., Mwacharo, J.M., Dessie, T., Mwai, O., Djikeng, A., Osama, S., Gebreyesus, G., Kidane, A., Abegaz, S. and Tesfaye, K., 2017. Polymorphism analysis of kisspeptin (KISS1) gene and its association with litter size in Ethiopian indigenous goat populations.

Tarekegn, G.M., Tesfaye, K., Mwai, O.A., Djikeng, A., Dessie, T., Birungi, J., Osama, S., Zergaw, N., Alemu, A., Achieng, G. and Tutah, J., 2018. Mitochondrial DNA variation reveals maternal origins and demographic dynamics of Ethiopian indigenous goats. Ecology and evolution, 8(3), pp.1543-1553.

Contributions by others to the thesis 1 Prof Glen Fox my principal advisor who helped with the design of the projects, thesis writing guidance and thesis and manuscript modification process 2 Dr. Ben Schulz (co-supervisor) who helped with projects redesigning, running samples through the HPLC, methods generation and thesis and manuscript modification 3 Dr. Alison Kelly (co-supervisor) who helped with statistical designs of the projects, data manipulation, thesis and manuscript modification 4 Dr. Shirley Jones who helped run the experiments, co-ordinated all lab requirements, offered training on equipment use and co-ordinated my field work and brainstormed all research problems and helped come up with solutions 5 Ed Kerr who offered training on MSstats 6 Katelynn Hadzi who offered training on equipment use at the USQ lab

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Statement of parts of the thesis submitted to qualify for the award of another degree No works submitted towards another degree have been included in this thesis.

Research Involving Human or Animal Subjects No animal or human subjects were involved in this research.

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Acknowledgements I would like to thank my principal supervisor Prof. Glen Fox for giving me a chance to do my PhD research under his supervision at the University of Queensland. I thank him for facilitating my candidature and scholarship. From my PhD journey, I have learned to be an independent thinker and logical, attributes which are valuable in research and will give me a great boost in as I move on to the employment world in future. His input in my PhD is immense and I cannot thank him enough. I am extremely grateful to all my supervision team (Prof Glen, Dr Alison Kelly and Dr Benjamin Schulz) for guidance during my PhD journey. I thank them for helping me to put the research in shape with all the time that went into project planning to ensure that we came up with statistically sound research. My great appreciation to Dr Shirley Jones. She not only helped with my studies but in settling into the new way of life in Toowoomba, far from the comfort of the school environment. She facilitated all aspects of my research including setting up the lab at the Leslie Research Centre (LRC, DAF), inductions, training on lab equipment use, planting seeds at Gatton and Warwick, transport to and from Brisbane when needed, conference attendance and also setting up at USQ laboratories for additional work. She also took time to read through all drafted materials and sat through mock presentations to prepare me beforehand for any presentations which I had to make. I am grateful to the LRC team including Dr Kim McIntyre, Drs Mandy and Jack Christopher and the entire ALG team. Thanks for their friendliness and encouragement. Thanks for those who even opened up their home for my family and I. I would also like to acknowledge the USQ team for offering me space in their laboratory to do part of my work. I am particularly grateful to Katelynn Hadzi for her help during this period. I would also like to thank Edward Kerr, a fellow PhD student who trained me on MSstats, his input is highly appreciated. Lastly I would like to appreciate my family. My mum, Florence Osama for having so much faith in me. My husband, James Maina for being my number one supporter. Thanks for agreeing to pack up our lives and come start over in a new country and for working so hard to sustain us during the entire process. My siblings Vera, Fiona and Parton Osama for cheering me on and lastly my children Killian and Avery Maina for making every situation lighter and filling our home with joy.

Financial support I would like to thank The University of Queensland International scholarship for living stipend and tuition fee provision I am also very grateful to the Grains Research and Development Corporation project UMU00047 for project funds provision

Keywords Barley (Hordeum vulgare), Dormancy, Germination, Proteins, malting, Mass Spectrometry, brewing, varieties, pre-harvest sprouting, Desiccation tolerance

Australian and New Zealand Standard Research Classifications (ANZSRC)

ANZSRC code: 100199, Agricultural Biotechnology not elsewhere classified ANZSRC code: 070307, Crop and Pasture Post Harvest Technologies (incl. Transportation and Storage) ANZSRC code: 090899, Food Sciences not elsewhere classified

Fields of Research (FoR) Classification FoR code: 0703 Crop and Pasture Production 50% FoR code: 1001 Agricultural Biotechnology 40% FoR code: 0908 Food Sciences 10%

Dedication

To my children Killian and Avery Maina,

They taught me what strength is.

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Table of Contents Abstract ...... I Declaration by author ...... IV Acknowledgements ...... IX List of Figures & Tables ...... XV List of Abbreviations used in the thesis ...... XIX

CHAPTER 1: Introduction ...... 1 CHAPTER 2: Literature review...... 7

2.1 Barley end uses ...... 8

2.2.1 Malting and brewing process ...... 8

2.2 Barley seed proteins ...... 11

2.2.1 Storage proteins ...... 11 2.2.2 Structural and metabolic proteins ...... 13

2.3 Barley dormancy ...... 14 2.3.1 Classification...... 16

2.3.2 Hormonal control of dormancy ...... 17

2.3.2.1 DELLA proteins inhibition of GA signalling ...... 18

2.3.3 RNA dormancy-germination control ...... 19

2.4 Barley germination...... 22

2.4.1 Starch degradation ...... 23

2.4.1.4 Alpha glucosidase ...... 26

2.4.2 Problem statement and justification ...... 27

CHAPTER 3: Differences in protein expression in dormant and non-dormant barley using SWATH-MS ...... 28

3.1 Introduction ...... 29 3.2 Materials and methods ...... 31

3.2.1 Germination test ...... 31 3.2.2 X-Ray visualisation ...... 31 3.2.3 Protein extraction ...... 32

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3.2.3.1 Mass spectrometry ...... 32

3.4 Results and discussion ...... 34

3.4.1 Changes in the barley seed proteome over the course of germination ...... 34 3.4.2 Proteins abundant before imbibition ...... 38 3.4.3 Proteins that showed initial increase at 24 hrs germination ...... 40 3.4.4 Proteins abundant in the course of germination and during early radicle elongation ...... 42 3.4.5 Proteins present in barley with little change during germination ...... 45

3.5 Conclusion ...... 47

4.0 Genomic and proteomic analysis of barley seeds to better understand dormancy ... 49

4.1 Introduction ...... 49 4.4 Conclusion ...... 73

CHAPTER 5. Protein expression comparison of malt from different malting processes ...... 75

5.1 Introduction ...... 76 5.2 Materials and methods ...... 79

5.2.1 Plant material sampling and sample collection ...... 79 5.2.2 Protein isolation, LC-MS/MS and MS data analysis ...... 79 5.2.3 Data processing ...... 81

5.2.3.1 Hierarchical clustering ...... 81

5.2.3.2 Select proteins of importance to malting and brewing industry ...... 82

5.3 Results and discussion ...... 83

5.3.1 Selected proteins of importance to malting and brewing...... 84 5.3.2. Employing different databases to better understand malting data ...... 99

5.4 Conclusion ...... 107

6. Summary of the thesis ...... 108

6.1 General discussion ...... 108 6.2 Summary of conclusions ...... 108 6.3 Future research plans ...... 110

7. References ...... 111

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List of Figures & Tables

Figure 1.1. Worlds’ top barley producers, Russian Federation was the worlds’ top barley producer followed by Germany derived from FAOSTAT 2015. Figure 1.2. Thesis flow chart indicating how the chapters flow and how they inform each other Figure 2.1. Beer production process in a largescale production facility, showing all the production steps from malting to brewing, until the beer is canned (http://foodtechinfo.com/foodpro/facility_types/311213_malt_beer/). Figure 2.2. Factors affecting seed germinability, including environment, genotypes and their interaction. A seed can overcome primary dormancy in favourable environments and proceed to germinate. If conditions change, however, the seed enters secondary dormancy and will only germinate when the environmental conditions are favourable (Li and Foley 1997). Figure 2.3. The plant germination phases, Phases I and II shows resumption of plant activities such as protein synthesis, translational activities and cell repair functions whereas phase III shows the normal growth and development stages necessary for seedling growth, (Bewley, 1997). Figure 2.4: α amylase structure. Source: http://www.scielo.br/pdf/bjm/v41n4/04.pdf Figure 3.1. Protein abundance patterns from a PCA of 15 proteins measured on 25 seeds. The proteins clustered into three groups, which have been assigned as before germination abundant proteins, initial germination abundant proteins and proteins which increase in abundance over time. The 25 seeds can be grouped into 5 sets;C=before imbibition, N=seeds that germinated after 24 hours imbibition, S=seeds that germinated after 72 hours imbibition, L=seeds that germinated after 24 hours imbibition but sampled after 72 hours and F= failed to germinate after 72 hours imbibition. The protein list is found in Table 2. Figure 3.2. X-ray image of seeds (A) before germination; (B) germinating after 24 hours imbibition. (C) Germinating from 24 hours imbibition after 72 hours imbibition (D) failing to germinate after 72 hours imbibition Figure 3.3. Protein abundance changes over 72 hrs for late embryogenesis abundant protein, defensin-like protein, EM protein and protein dehydrin. Figure 3.4. Protein abundance changes over 72 hrs for Protein disulphide isomerase (PDI), Gamma hordein 3 (HOG3), Beta amylase (AMYB), C hordein fragment (HOR7) and Phytepsin precursor (ASPR)

Figure 3.5. Protein abundance change for proteins Malate dehydrogenase, cytoplasmic (MDHC), Malate dehydrogenase (MDH), homocysteine methyltranferase 1 (METE1), Actin 1 (ACT1), protein H2A.6 (H2A6), 26 kDa endochitinase 2 (CH12) and Alpha amylase (AMY6) Figure 3.6. Protein abundance change for proteins Serpin-Z4, serpin-Z7, Non-specific lipid- transfer protein 1 (NLTP1) and Probable non-specific lipid-transfer protein (LTP2) Figure 4.1 Classification of gene ontologies (GOs) Figure 4.1. Germination percentage of barely varieties Gairdner (Gr), schooner (Sc) and Stirling (St) sampled over twelve months. R1, R2 and R3 representing replicate 1 to 3 for each variety.

Figure 4.1. Germination percentage of barely varieties Gairdner (Gr), schooner (Sc) and Stirling (St) sampled over twelve months. R1, R2 and R3 representing replicate 1 to 3 for each variety.

Figure 4.3. Protein abundance change over 10 months storage for LEA proteins; LEA protein D-34, ABA-inducible protein PHV A1, and LEA protein 1 sampled from Schooner.

Figure 4.4. Protein abundance change over 10 months storage for the protein dehydrin DHN3 sampled from Schooner.

Figure 4.5. Change in protein abundance over 10 months storage period in dormant and germinated grain for proteins serpin Z2A and Z7 sampled from barley variety Schooner.

Figure 4.6 Change in protein abundance over 10 months storage period in dormant and germinated grain for the proteinases aspartic and serine carboxypeptidase 2 sampled from Schooner.

Figure 4.7. Relative protein abundance change over 10 months storage for the protein Late embryogenesis abundant protein D-34 in dormant and germinated samples which were sampled from Schooner.

Figure 4.8. Relative protein abundance change over 10 months storage for the protein Avenin-like a2 and Glutenin, high molecular weight subunit DY10, in dormant and germinated samples which were sampled from Schooner.

Figure 4.9. Relative protein abundance change over 10 months storage for the protein disulphide isomerase, in dormant and germinated samples which were sampled from Schooner.

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Figure 5.1. Schematic presentation of the malting process showing the basic malting steps which include steeping, germination and kilning. The kilned malt is moved to the next brewing process which begins with mashing.

Figure 5.2. The relative abundance of the chloroform methanol (CM a-e) proteins. Data in continuous lines denoting samples from the first plant JP and the dotted lines from the second malting plant BP. The sampling time points correspond to the table 1 above.

Figure 5.3. The effect of malting and kilning on protein Alpha-amylase/trypsin inhibitors CMa, CMb, CMc, CMd and CMe. The first three bars from malting plant JP and the last three from plant BP. Figure 5.4. The effect of malting and kilning on protein Alpha-amylase type A and B isozymes and Beta-amylase. The first three bars from malting plant JP and the last three from plant BP.

Figure 5.5. The relative abundance of the serpin proteins. Data in continuous lines denoting samples from the first plant JP and the dotted lines from the second malting plant BP. The sampling time points correspond to the table 1 above. Figure 5.6. The effect of malting and kilning on Serpin-Z7, the first three bars from malting plant JP and the last three from plant BP. Figure 5.7. The effect of malting and kilning on Non-specific lipid-transfer protein 1, the first three bars from malting plant JP and the last three from plant BP. Figure 5.8. The effect of malting and kilning on putative linoleate 9s-lipoxygenase protein (LOX), the first three bars from malting plant JP and the last three from plant BP

Figure 5.9. The relative abundance of non-specific lipid-transfer proteins 1, 4 and 2 and Putative linoleate 9S-lipoxygenase 3. Data in continuous lines denoting samples from the first plant JP and the dotted lines from the second malting plant BP. The sampling time points correspond to the table 5.1 above.

Figure 5.10. The effect of malting and kilning on proteinases cysteine proteinase EP-B 1 and aspartic proteinase and on proteinase inhibitor cysteine proteinase inhibitor 12, the first three bars from malting plant JP and the last three from plant BP. Figure 5.11. The relative abundance of the proteinases cysteine and aspartic proteinase and cysteine proteinase inhibitor. Data in continuous lines denoting samples from the first plant JP and the dotted lines from the second malting plant BP. The sampling time points correspond to the table 5.1 above.

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Figure 5.12. The relative abundance of the serpin proteins. Data in continuous lines denoting samples from the first plant JP and the dotted lines from the second malting plant BP. The sampling time points correspond to the table 5.1 above. Figure 5.13. Classification of proteins according to their gene ontology terms. The biological process had the number of gene ontologies allocated to it. Figure 5.14. Gene ontology term biological process partitioning. Highest number of GO’s proteins were classified under response to redox state, followed by dormancy process with 85 and 39 respectively.

Figure 5.15. Partitioning of biological process gene ontology showing the different function groups that the proteins are part of dormancy Figure 5.16. Heat map cluster analysis of proteins showing statistical significant change in abundance over the 10 stages of mating. Samples were taken every 12 hrs. G1-G6 indicating the sampling periods during germination process. Figure 5.17. Dendrogram cluster analysis of proteins showing statistically significant change in abundance during malting. The groups indicate the different patterns of change in abundance that the clusters fall into Figure 5.18. The four clusters from the analysis of proteins which showed statistical significant difference in abundance within the different malting steps. Cluster 1, 2, 3 and 4 each have 78, 83, 60 and 56 proteins respectively. Normalised predictions average data was used.

Table 3.1. Seed sampling schedule, five replicate seeds were sampled at each sampling period Table 3.2. List of selected proteins which showed significant variation (p=0.05) from 0-72 hours, their assigned group and their function/role. The accession number and role is based on their identification in the UniProt database Table 3.3: Protein categorised as to have significant high abundance before germination, a, b and c means significantly different at P (0.05). Table 3.4. Protein categorised to have an initial increase in abundance at 24 hours after germination. Superscripts of a, b and c indicate significantly different means at (p=0.05). Table 3.5. Proteins categorised to have significant abundance in the course of germination with maximum level of abundance observed at 72 hours after imbibition. Superscripts of a, b and c indicate significantly different means at (p=0.05) Table 4.1. Grain sampling schedule over a period of 12 months and the germination percentages of barley varieties Schooner, Gairdner and Stirling

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Table 4.2. Proteins with significantly different abundance and large fold change (log2FC) in germinated versus dormant samples at harvest (G0vD0) P≤0.05

Table 4.3. Proteins with significantly different abundance and large fold change (log2FC) in germinated and dormant grain after 10 months storage (G5vD5) P≤0.05.

Table 4.4. Proteins with significantly different abundance and large fold change (log2FC) in dormant grains at harvest versus dormant grain after 10 months storage (G5vD5) P≤0.05

List of Abbreviations used in the thesis ABA - Abscisic acid AMYB- beta amylase ANOVA- Analysis of variance BAC- Bacterial artificial chromosome CV- Cultivar cDNA- complementary DNA DNA- Deoxyribonucleic acid DP - degree polymerisation/maltotetraose ESI MS- Electrospray ionization mass spectrometer FAOSTAT- Food and Agriculture Organization Corporate Statistical Database GA - gibberellic acid GB- Gigabyte GO- Gene ontology HPLC MS- High performance Liquid chromatography mass spectrometry HSP- Heat shock protein LEA - late embryogenesis abundant protein LSD- least significant difference LTP- Lipid transferase protein mRNA- messenger RNA

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MSstats- Mass spectrometry statistics MS/MS- tandem mass spectrometry MTP- Minimum tilling path NaOCl- Sodium hypochlorite NGS - Next Generation Sequencing PCA- Principal Component analysis PDI - protein disulphide isomerase PHS - Pre-harvest sprouting QTLs - Quantitative Trait Loci Qld- Queensland RNA- Ribonucleic acid SAGE- Serial analysis gene expression SD - seed dormancy SNP- Single nucleotide polymorphism SWATH-MS - sequential window acquisition of all theoretical mass spectrometry

CHAPTER 1: Introduction

Barley (Hordeum vulgare subsp. vulgare L.) is the fourth most important cultivated cereal crop in the world after maize, wheat and rice respectively (Schulte et al., 2009). Barley is produced in many regions around the world with the highest producers being the Russian Federation region as indicated in Figure 1, the next large producers of barley are Germany, France and Canada respectively, with Australia coming in at number eight amongst the top ten producers (FAOSTAT 2014).

Figure 1.1. Worlds’ top barley producers, Russian Federation was the worlds’ top barley producer followed by Germany derived from FAOSTAT 2015.

Barley has been cultivated since 8000BC and is therefore, amongst the oldest cultivated food crops in the world (Zohary D, 1993). Archaeological evidence suggests that barley was first cultivated around the “fertile crescent” region where its wild relatives thrive (Zohary D, 1993). Barley was first utilized for beer production by the Chinese. The earliest beer recipes discovered were inscriptions on oracle bones recording the use of both barley, wheat and millet as the main ingredients for beer. This was during the Neolithic Yangshao period, between 5000-2900 BC (Wang et al, 2016).

Cultivated barley is a diploid plant with 2n=14 chromosomes and has a genome size of 5.1 Gb. It is an annual self-pollinated crop grown mainly in the temperate regions. Barley has been used as a model crop for plant genetic research for a long time not only because of its inbred

1 nature (Schulte et al., 2011), but also for its fast growing properties in the temperate regions and its diploidy state, as opposed to polyploids which are complex in genetic research.

The uses of barley are diverse; two-thirds of barley produced is used both as food and feed, while a third goes into the malting and brewing industry (Baik and Ulrich, 2008). Asia and North Africa are the regions where barley is mostly consumed as the main source of food. It is used as a food source especially because of its high dietary fibre which is highly soluble, hence making it suitable for consumption by individuals suffering from type II diabetes, colonic cancer and cardiovascular diseases (Schulte et al., 2011).

The biotic factors affecting barley include pathogens causing disease. Barley production is constrained by over forty pathogens. These bring about an estimated loss of about 19.6% in Australia (Murray and Brennan, 2010) with the highest losses being brought about by net blotch of barley caused by Pytrnophora tore f. maculate, powdery mildew caused by Blumeria graminis f. sp. Hordei and cereal cyst nematodes caused by Heterodera avenae.

Other factors constraining the barley industry include abiotic factors such as, pre-harvest sprouting, a condition whereby the seeds begin to germinate while still in the field as a result of light showers or high humidity around the time of physical maturity (Ullrich et al., 2008; Scursoni and Arnold, 2002; Bonnardeaux et al., 2008) and dormancy (Ullrich et al., 1992).

Seed dormancy can be defined as the inability of a mature viable seed to germinate when presented with favourable environmental factors which would otherwise favour germination (Gubler et al., 2005; Hori et al., 2007; Romagosa et al., 1999). It is an adaptive trait which allows the seed to only germinate when the environment is favourable. Dormancy therefore ensures survival in wild species by ensuring decreased competition amongst the germinated seedlings, hence preventing damage to the seedling from out of season germination (Holdsworth et al., 2008). Dormancy is initiated during seed maturation and maintained in most plants until the seed is completely mature. This type of dormancy is known as primary dormancy and is regulated highly by the plants genetics, the environment and the interactions between the two. Secondary dormancy however, is initiated by environmental factors which do not favour the seed germination requirements, hence seed becomes quiescent (Gubler et al., 2005).

Dormancy is an important trait at harvest in cereals, because it prevents losses associated with pre-harvest sprouting (PHS), a plant response where the seeds will immediately germinate when exposed to some amount of moisture around maturation time when they are still in the field. This is a problem mainly in cereals because it presents quality issues especially with malting barley. PHS is a major problem in wheat and in some species of barley. Some barley species will however, show high levels of dormancy during and after harvest, some of which when used in malting will present losses to the malting companies associated with long storage periods required to overcome the dormancy and have uniform germination (Gubler et al., 2005; Hori et al., 2007).

Barley is stored after harvesting prior to being used in malt production. The storage conditions may affect the quality of barley either positively or negatively with regards to maltability (Reuss et al., 2003). Both PHS and dormancy are presented as problems because they both mean heavy financial constraints to both maltsters and growers (Reuss et al., 2003; Briggs et al., 1994), in terms of losses associated with the quality of malt and increased storage costs. Storage conditions such as temperature, time and seed conditions related to moisture content, eventually determine the quality of malt. Germination is observed to be lost at warmer temperatures and at higher moisture content (Briggs et al., 1994).

Quantitative Trait Loci (QTLs) controlling dormancy have been identified. There are four QTLs which have been widely studied and mapped. These have been labelled as seed dormancy (SD) 1 to 4 (Hans et al., 1996; Romagosa et al., 1999). The study was undertaken using doubled haploid lines of Morex and Steptoe barley varieties and their crosses. In this study, two QTLs were discovered on chromosome 5, one major QTL near the centromere region, and one minor one near the telomere of the long arm. Others were located on chromosomes 4H and 7H. The amount of variation controlled by these four QTLs are 50%, 15%, 5% and 5% for the SD1, SD2, SD3 and SD4 respectively (Hans et al., 1996). SD1 was discovered to be the main QTL which promotes dormancy in order to combat PHS while SD2 mainly regulates when to break dormancy and allow for the plant to germinate.

Seed dormancy in malting barley

1. Introduction 2. Literature review

3. Change of in proteome in the course of

germination in barley and perform a

comparison of the proteome change in

dormant barley using SWATH-MS

4. Gene and protein expression changes as 5. Environmental/site effect to malt proteins. shown by mRNA and protein in quiescent, To determine the protein expression of malt, germinated and non-germinated seed from different malting companies, at the samples over a period of time different stages of malting

6. Summary of thesis

7. References

Figure 1.2. Thesis flow chart indicating how the chapters flow and how they inform each other

This thesis research was conducted with the aim of addressing the following three general objectives:

1. To study the change of in proteome in the course of germination in barley and perform a comparison of the proteome change in dormant barley using SWATH-MS. 2. To assess the gene and protein expression changes as shown by mRNA and protein in quiescent, germinated and non-germinated seed samples over a period of time (9-12 months). This will include the correlation of genes of interest to their translated functional proteins. 3. To determine the environmental/site effect to malt proteins. Furthermore, to determine the protein expression of malt, from different malting companies, at the different stages of malting.

In this study, the first research goal aimed to explore protein expression in germinating and non-germinating barley. To achieve this, a comparison of the proteins expressed during the process of germination was undertaken using the sequential window acquisition of all theoretical mass spectrometry (SWATH-MS). Grains samples exposed to optimal germination conditions were sampled every twenty four hours over a period of four days. Non-germinated samples after seventy two hours imbibition were used to identify protein during the resting/quiescent phase. This provided insight on the different proteins expressed at different time point during germination. An x-ray imager was also used to capture images of germinating barley seeds at the different time points.

As a follow up, the genes and proteins expressed during dormancy and subsequent germination in barley were studied. The varieties proposed for the study were CV Schooner which is susceptible to PHS, CV Gairdner which is intermediate for PHS and CV Stirling which has high levels of dormancy. Barley seeds tend to lose their germinability with prolonged storage. This chapter was therefore aimed at studying the time taken for the seeds to revert to their quiescent state and identifying the genes and proteins expressed to signal this change. Germination tests were conducted on these varieties and samples collected over three days. SWATH-MS analysis was be conducted on the samples to identify the proteins expressed and the differences within the varieties. Next generation sequencing was done using Miseq illumina techniques in order to identify the genes which are up and down regulated at the different time points and within the different time points.

Then gene expression between dormant and non-dormant barley types were to be studied. This was to be accomplished using the sequencing data from the Next Generation Sequencing (NGS), however, due to unforeseeable difficulties this goal was not achieved. Lastly the implications of this entire research was studied. This was done by employing the techniques learnt to samples collected from two different malting industries. The effect of environment on barley germination and the differences in proteins expressed in the samples were explored. The proteins identified were compared for the site differences and temporal differences.

In summary, dormancy in barley was studied by looking at the proteins expressed during germination, and the differences in the proteins expressed in dormant and no-dormant barley types and the effect of the environment on germinating barley.

CHAPTER 2: Literature review

Barley (Hordeum vulgare subsp. vulgare L) belongs to the Poaceae grass family, Triticeae tribe and the genus Hordeum. The genus Hordeum has thirty one different species, most of these species are diploid (2n=14) while others include tetraploids 2n=28 and hexaploids, 2n=28. Barley has two subspecies; cultivated barley, H. vulgare, and the wild form, H. spotanenum. These two species are closely related with respect to biological factors, they however do not inter cross (Nilan and Ullrich, 1993).

Barley is the fourth most produced cereal worldwide with 75% of its production going into fodder and feed, 20% being used in malting industries to make both alcoholic and non-alcoholic beverages and 5% going into a wide array of food production as an ingredient. It is a highly adaptable crop and hence grows in a wide range of environments and tolerates stressful growth conditions better than wheat (Schulte et al., 2011). Cultivated barley types vary greatly and hence have been classified into different groups. The classification includes; hulled versus unhulled, two row versus six row, and spring versus winter and by the end use which is malting versus feed (Baik and Ullrich., 2008).

Over the past five years, barley has been ranked as the fourth highest produced crop in Australia coming in after mixed grasses and legumes, sugarcane and wheat with an average production of 9.14 M tonnes being recorded in 2014 ( FAOSTAT DATA 2014). This rise in production comes after a decline from 10.4 M tonnes produced as recorded in 2003-2004 to 8m tonnes produced in 2010-2011. This was due to the decreased area under production from 5 M hectares to 3.5 M hectares coupled with dry weather conditions faced in the areas where barley is widely produced within Australia (http://www.abs.gov.au/ausstats/[email protected]/Lookup/7124.0Chapter32010-11). The ranking within Australian regions record Western Australia as the highest producer with a recorded high of 2.6 M tonnes followed by South Australia and Victoria with 2.1 M and 1.9 M tonnes respectively.

The types of varieties mainly grown include the two rowed and the six rowed barley. The two rowed barley is used mainly for malting in Australia. Other barley uses include fodder crop where animals are allowed to graze the crop. Rotation cropping is mainly practised in Australia where wheats, oats and pasture are used as the alternative crops

(http://www.abs.gov.au/ausstats/[email protected]/Lookup/by%20Subject/1301.0~2012~Main%20Fe atures~Agricultural%20production~260).

2.1 Barley end uses Beer is one of the oldest beverages produced by humans, as recorded in the ancient Egypt and Mesopotamia history. Beer was regarded as a gift from the Gods and hence used as the most valued gift for the Pharaohs. It was also used for nutrition because, not only did it have a high protein content, but was also sweet, thick and had low alcohol content. Lastly beer was used for medicinal purposes including as a mouthwash (antibacterial properties).

The act of beer drinking was done from a large communal bowl using reeds. Hops, flowers from Humulus lupulus would be added to beer to curb spoilage on unhoped beer and as flavouring. Hops have antibacterial properties which would inhibit the activity of gram positive bacteria hence curbing spoilage. The earliest hops cultivation was documented around 736 in an area known as Hallertsu, Germany whereas the earliest documented use of hops was around the year 822 in a French monastery (Townsend and Shellhammer, 2012; Colgrave et al., 2012).

Currently beer is one of the most popular drinks with the global market revenue of around 500B dollars per annum (Colgrave et al., 2012; Kirin, 2011) with the highest consumers being from the European countries with Germany as the highest consumer. China and USA however showed the largest beer consumption in 2010 per capita with values of 28,640 ML and 23,974 ML respectively (Colgrave et al., 2012; Kirin, 2011).

2.2.1 Malting and brewing process Beer is comprised of mainly cereals, with barley and wheat being the most common grains used in malting. Hops, yeast and water are also added. The malting process begins with the germination of the cereal in use, for example, barley. Germination is under controlled lower temperatures in order to lower the rate of growth of rootlets and sugar reduction through respiratory losses. During germination, starch reduction enzymes known as amylases are activated. These enzymes work to reduce the starch contained in the grains to simple sugars which are necessary for the process of fermentation. The product of this initial process is referred to as malt (Colgrave et al., 2012).

Firstly, the malt is roasted thus stopping the germination process, although amylase activity will be retained. The malt is then milled to form grist, and hot water (approx. 650C) is added to mash the grist. This stops the amylase activity and allows the starch granules to swell. Starch is then converted to oligosaccharides DP4 (four degree polymerisation/maltotetraose) or less as below two DP is the most ideal fermentable sugars through the action of α amylase, β (beta) amylase, α glucosidase and starch debranching enzyme. The resultant product is known as wort and contains the simple sugars. The wort formation process can be prolonged by adding more temperature steps hence allowing continued protease activity (Colgrave et al., 2012).

Wort is then brought to boiling temperature and hops added in order to give beer flavour and aroma. Most of the proteins will precipitate out during this boiling part. This is known as the hot break. It however is also an enrichment step whereby most of the water soluble proteins will go into the wort sugar solution. These water soluble and heat stable proteins are such as serpin Z4 and lipid transfer proteins. The wort will then be filtered through the used up grain husks in order to get rid of the particles, precipitated proteins and debris. The wort is then oxygenated and temperatures brought down rapidly in order to cool it off (Colgrave et al., 2012). The last step is fermentation where yeast is added. The sugars present will be converted to carbon dioxide and alcohol. There are two types of resultant beer, ale or lager. Ale is prepared in warmer temperatures while the lager in cooler temperatures. The process is as outlined in Figure 5.

The malting process is one of the oldest recorded biotechnological processes (Scursoni and Arnold, 2005: Colgrave et al., 2012). Because barley is used widely in the production of alcoholic drinks, it has attracted a lot of interest in its research especially in the biochemistry and physiology of its germination (Nilan and Ullrich, 1993).

Figure 2.1. Beer production process in a largescale production facility, showing all the production steps from malting to brewing, until the beer is canned (http://foodtechinfo.com/foodpro/facility_types/311213_malt_beer/ ).

2.2 Barley seed proteins A dried barley seed contains 8 to 15% protein and, together with starch, this forms about 70% of the dry seed composition. Protein and starch have been studied widely because they directly influence the quality and suitability of the seeds’ end use (Shewry, 1999). The malting and brewing companies require the protein content of barley grain going into beverage production to be between 9-12% while barley grains high in digestible lysine are preferred for food and feed components (Osman, 2002). Seed proteins contained in barley are grouped into four main classes, based on their solubility in different solutions. This classification method is widely known as the ‘Osborne fractions’. The classes include; albumins which are soluble in water and coalesce when they are heated, globulins which are soluble in saline solutions but not in water and could be easily obtained in crystalline form, prolamins which are soluble in alcohols of high percentage e.g. 70 to 80% (They are however insoluble in both water and salt solutions and contain a lot of amides and proline nitrogen), and lastly glutelins which can be extracted using basic solutions but are insoluble in alcoholic, salt and neutral Ph solutions (Shewry and Casey, 1999).

2.2.1 Storage proteins The main storage proteins in most cereal seeds (except rice, oats and their related wild grasses) are prolamins. Hordein is the main barley storage protein. Hordeins in barley are divided into five categories, based on their electrophoretic mobility and their amino acid composition. These categories include; A hordeins which are the smallest polypeptides with an average molecular weight of 15k, are soluble in alcohol. Soluble albumins/globulins and are commonly referred to as a breakdown product of larger hordeins rather than to a true hordein. The B hordeins constitute the bulk of the hordeins, are 70-80% of the total hordein fraction, are sulphur rich proteins, with some appearing as monomers although they are mainly polymers linked with disulphide bonds and are subdivided into B1, B2 and B3 subtypes. The C hordeins, which form the second hordein bulk constituting 10-12% of the total hordein fraction, are sulphur poor and appear as monomers. Lastly the D and γ hordeins form less than 5% of the total hordein fraction. The D hordein is a high molecular weight hordein and is easily degraded during the malting process followed by the B and lastly the C hordeins. The γ hordein is however a sulphur rich hordein (Shewry and Casey, 1999).

In barley, all the hordeins are encoded by structural genes located on chromosome 5 (1H). Genes Hor1 and Hor2 are linked and encode for the C and B hordeins respectively, and are located on the short arm of the chromosome, whereas Hor 3 encodes for the D hordein, and is a loosely linked locus located closest to the long arm. The genes encoding for the γ hordeins haven’t been conclusively decided, although it has been suggested to be HrdF (Hor5) locus located distally to Hor2. The storage proteins in these plant seeds appear as minor component glutelins and have been reported to also be present in wheat, maize and other cereals and are structurally similar to prolamins but differs in their solubility properties. This is due to the existing inter-chain disulphide bonds. In dicotyledonous plants, oats and rice, globulin is the main storage protein. This family has however been classified within glutelins due to its insolubility properties in salt solution. Globulins can be found in small amounts in wheat and are grouped into two categories that is, based on its sedimentation coefficient (S20.w); 7-8s vicilin type and 11-12s legumin type (Shewry and Casey, 1999).

In some species, such as sorghum, storage proteins are clustered within cells as thick deposits know as protein bodies hence ensuring a separation from the metabolic compartment of the cells. These protein bodies seem to be as a result of secretory pathways, as their compartments. It is however suggested that their origin is varied between species at varying stages of development.

Gene expression of seed storage proteins has been found to be tightly regulated hence confined to a specific tissue in the seed. The stage of development is also highly regulated too, therefore, due to these factors, there is no evidence of expression of genes regulating seed storage in any other tissue of the plant. Gene expression is also controlled by the plants nutrition, the storage proteins therefore act as a sink for excess nitrogen and sulphur which is store in cysteine and methionine form. The sulphur is used in protein synthesis regulation hence sulphur rich and sulphur poor storage proteins (Shewry and Casey, 1999).

2.2.2 Structural and metabolic proteins The structural and metabolic proteins are large in number and vary greatly with respect to their properties such as solubility. They are in all the Osborne partitions except the prolamins in the seed. These proteins are found in small proportions and have little individual effect, but when combined, their impact is huge. For example, the albumin and globulin seed fractions in cereals contribute greatly towards the lysine proportions used as nutrition source for monogastric livestock. In barley, beta amylase is one of the main lysine-rich proteins. This protein is synthesised during grain filling stages and is stored in the grain, only to be activated during germination (Shewry and Casey, 1999).

Other proteins include protective proteins whose main function is to confer tolerance/resistance to pests and pathogens, (Shewry and Casey, 1999). Seed material is commonly attacked by pests and pathogens because of their composition which is rich in nutrients including oil, proteins and carbohydrates (Shewry and Casey, 1999). Desiccation tolerance proteins fall into this category and function to protect the seed against the harsh water lacking conditions. As the seed develops and matures, it starts losing its moisture content thereby entering a dry quiescent state. During low water conditions, a number of proteins are activated by response to ABA, such as Aldose reductase, a key enzyme in the polyol pathway whereby glucose is converted to sorbitol, an osmolyte that balances the cell osmotic strength with the environment (Ostergaard et al., 2004). Another group of proteins with a similar desiccation tolerance role is the late embryogenesis abundant protein (LEA). They have been linked to desiccation tolerance because of their amino acid composition which is rich in alanine and glycine and lacking in tryptophan and cysteine. This results in a very highly hydrophilic random coil structure which co-join with hydrophilic side chains of other proteins, thereby compensating for the low water potential. Another group of proteins contributing to low water tolerance is protein sHSP. They do so by binding to other proteins hence preventing the formation of protein aggregates (Ostergaard et al., 2004). Some groups of proteins have been labelled chaperones because of the roles they play, for example, heat shock proteins (HSP) and protein disulphide isomerase (PDI). PDI catalyses proper protein disulphide bond pairing (Ostergaard et al., 2004).

2.3 Barley dormancy Seed dormancy is a desirable trait in cereals because it prevents pre harvest sprouting (PHS). However, dormancy becomes a problem when it is too strong, this is so especially in the malting process, where rapid and uniform germination is desirable (Bonnardeux et al., 2008). It is therefore prerequisite to develop seeds with moderate dormancy levels which enable them to not only withstand PHS, but also germinate rapidly and uniformly when the environmental conditions are favourable.

Dormancy is regulated by three factors. These are; plant genetics, environmental factors or growing conditions, and the interaction between the environment and the genetics (G*E) (Romagosa et al., 1999). It is however suggested in some literature that the embryos’ surroundings initiate and maintain dormancy in most cereals, rather than the embryo itself (Black and Bewley, 1994; Romagosa et al., 1999). This was evident in previous studies carried out whereby excised immature embryos had the capability to germinate in culture. The experiments also confirmed the embryo environment is responsible for the inception and maintenance of dormancy. This inception happens in early stages of seed development, the excised embryos were capable of germinating in artificial media two weeks post anthesis, as the third week approached however, dormancy set in. Many plant species would therefore require a desiccation period to overcome the dormancy (Romagosa et al., 1999).

Seed desiccation is an important step in overcoming dormancy as it allows seed germination and hence growth and development. The desiccation occurring in seeds is suggested to play a very important role in changing the developmental stages in plants (Kermode, 1995). In barley, immature dried seeds will germinate while non dried ones at a similar stage of development will not.

In many plant species, seed dormancy is a quantitatively inherited trait (Foley, 2001). In barley, dormancy QTLs have been identified and extensively researched (Hori et al., 2007; Bonnardeaux et al., 2008: Romagosa et al., 2008: Han et al., 1999: Ullrich et al., 2008) identifying four QTL regions as mention above. These regions include; SD1 which is located on the centromeric region of chromosome 7(5H) in between markers Ale and ABC302 and has a distance of 8.7cM. This QTL displays the largest phenotypic variation of 55% and is most stable over different environments. It however has epistatic effects to the other SD QTLs. The other QTLs include SD2, SD3 and SD4 located in the telomeric region of 5H and controlling

15% phenotypic variation, 4H and 7H controlling 5% phenotypic variation each respectively. The QTLs SD3 and SD4 are minor QTLs, Hori et al., 2007. These QTLs alignment was based on the alignment of linkage maps. Some of the studies on these QTLs include work done by (Oberthur et al., 1995; Hans et al., 1996; takeda, 1996; Romagosa et al., 1999; Zhng et al., 2005). A study conducted by (Romagosa et al.,1999) using excised embryos and artificially dried seeds from doubled haploid lines through the process of seed development, maturation to after- ripening showed that the SD loci introduced dormancy only twenty five days post anthesis and maintained it, only releasing it at the end of the seed development process. They also established that SD1 allele presence inhibited germination to a low extent from twenty five days post pollen shading until seed development is complete. Additionally, they showed that the allelic interactions between the SD alleles determined the classification of genotypes as either early, intermediate or late dormancy release types (Han et al., 1999). Most plant species need an after-ripening conditioning to be able to overcome dormancy and move to a germinable state. The conditions required by different species vary. Some, like rice and wild oats require warm dry conditions while others such as Arabidopsis need cool moist substrate conditions known as stratification. The after-ripening treatment would normally increase the seeds responsiveness to environmental conditions favouring germination. A non-invasive direct method to measure either dormancy or after-ripening state of the seed is currently lacking. While there is some research to address this issue (Hilhorst and Bino, 1999), the methods used involve comparisons between dormant/partially after-ripened seeds and non- dormant/fully after-ripened seeds from the same population to determine the onset and rate of germination. Using this method however has short comings and doesn’t allow exact determination of the dormancy release period. Dormancy release period therefore still remains a mystery. This is because the dormant and partially after-ripened seeds do take some time and eventually germinate. The separation of germination and dormancy break are not easy to differentiate.

Figure 2.2. Factors affecting seed germinability, including environment, genotypes and their interaction. A seed can overcome primary dormancy in favourable environments and proceed to germinate. If conditions change, however, the seed enters secondary dormancy and will only germinate when the environmental conditions are favourable (Li and Foley, 1997).

Evidence exists that there might be aspects lacking in the dormant seeds hence preventing the normal flow of germination events (Cranston et al., 1999). Dormancy studies would be best done by exploring the biochemical, physiological and molecular factors regulating germination using a single seed. This is because of the inherent variation existing within seeds which must be accounted for when estimating dormancy parameters.

2.3.1 Classification Over the years, different methods have been used by researchers to classify dormancy. Some of the systems appear complex but thorough, such as the classification method proposed by (Nikolaeva, 1969) while others are simple. The most common classification method includes classifying dormancy as either primary or secondary. Primary dormancy refers to the occasion where a mature and fully imbibed seed fails to germinate, whereas secondary dormancy refers to when a mature dispersed seed fails to germinate as a result of being exposed to conditions not favouring germination (Bewly and Black, 1994: Foley, 2001). Secondary dormancy occurs in non-dormant seeds and not necessarily all dormant or in completely after-ripened seeds. It

16 can also be induced by application of some enzyme inhibitors, such as, inhibit gibberellins synthesis. After-ripening however breaks both dormancies.

2.3.2 Hormonal control of dormancy Several hormones are known to play a part in dormancy and germination in seeds. During seed maturation, as the embryo matures, dormancy sets in. Abscisic acid, abbreviated ABA has been shown to induce this dormancy (Kermode, 2005). A study undertaken on Arabidopsis seeds derived from a reciprocal cross between wild and ABA deficient mutants showed that ABA rises during seed development (Karssen et al., 1983). This rise was found to be both maternally derived, as determined by the maternal genotype, and embryo regulated, as determined by the embryo genotype. From the study, it was also established that dormancy onset is co-ordinated with the embryo ABA rise. This was mainly due to the reduced ABA level during the late seed development stages. These low levels cannot maintain dormancy in imbibed seed, therefore the deduction of embryo ABA dormancy regulation. ABA synthesis and accumulation begins during the early stages of seed development. The ABA amount in the seed at that stage is low and would increase, reaching its peak during seed reserve synthesis, then, later decline as the seed matures and loses its moisture content. The ABA present may be the main cause of primary dormancy during seed development, however, the seed osmotic surrounding has been shown to be an important factor in maintaining dormancy too and would work together with ABA to initiate and maintain primary dormancy (Bewley, 1997).

One of the effects observed after the application of ABA is the suppression of the expression of enzymes induced by Gibberellic Acid (GA). GA is another well researched plant hormone and seems to work in reverse to ABA in overcoming dormancy and hence promoting germination. It activates enzymes responsible for food reserve mobilization, breaking of the seeds’ cell wall and promoting the multiplication of the embryo cells (Bewley, 1997). These enzymes include (1→3), (1→4) –β-glucanases, α amylases and endopeptidases (Fincher and Stone, 1993). This is thought to occur by ABA boosting the expression of ‘ABA specific’ genes, which includes genes for inhibition of denovo α amylase synthesis, hence lowering its activity, (Foley, 2001). It not only inhibits α amylase synthesis by blocking its gene transcription but also acts by prompting α amylases exact inhibitors within the cells. These ABA inducible genes could also be encoding for other major hydrolytic enzymes, hence lowering the endosperm mobilization in response to environmental stresses. In the advent of

17 unfavourable environment therefore, only minimal endosperm mobilization is observed, as a result of ABA sensitivity in the aleurone cells of mature and germinable seeds (Foley, 2001).

Abscisic acid and gibberellic acid are the two main hormones regulating dormancy and germination (Holdsworth et al., 2008). Other hormones which also play a part in these two processes include ethylene, brassinostreroids and auxins. Ethylene promotes germination by working in an antagonistic manner towards ABA (Beaudoin et al., 2000; Chiwocha et al., 2005). The plant steroidal hormones, known as brassinostreroids, also promote germination by working in a way similar to gibberellic acid. Auxins ensure germination accomplishment and hence seed establishment by building up at the radicle tip and hence growth promotion.

Gibberellins play a major role in production of hydrolytic enzymes from the aleurone cells. This is thought to be so because exogenous application of gibberellic acid (GA) greatly enhance the production of α amylases (Fincher and Stone, 1993). GA is also thought to induce germination and has a role to play in dormancy break because of the high levels of GAs in germinating seeds. The level of GA in dormant seeds has however been discovered to be low. From previous studies, GA deficient mutants remain dormant and would require exogenous application of GA to induce germination. In wild type Arabidopsis, germination is induced by either exogenous GA application or after-ripening. It is therefore thought that GA plays a role in germination (Foley, 2001). This role is however highly debated. This is due to the fact sensitivity to GA and light is only evident, after the after-ripening process is done to lower the levels of ABA in a plant (Kumar et al., 2013). The ratio of ABA to GA present in the seed is important because if ABA is present in high levels, the GA promoting activities in growth are overridden. Although the signalling systems for GA in cereals and dicots have been shown to be similar, some differences are evident. This has been observed in rice where GA mutants exist, denoted ‘gibberellin-insensitive Dwarf 1 (GID1)’. G1D1 leads to the reduction in α amylase production. In Arabidopsis however, mutating the 3 homologue of GID1 will hinder germination (Foley 2001).

2.3.2.1 DELLA proteins inhibition of GA signalling The DELLA proteins are ‘nuclear transcriptional regulators’ which control GA signalling. These regulators are negative regulators. Previous work on Arabidopsis showed that it has five DELLA proteins which include (GA – INSENSITIVE [GA], REPRESOR OF GA1 [RGA], RGA-LIKE1 [RGL1], RGL2, AND RGL3) while rice has SLENDER1 (SLR1) and in barley

SLENDER1 (SLN1). The interaction between the DELLA proteins is brought about from the initial interaction between bioactive GA and GID1. The GID1 interacts with the DELLA domain to form DELLA-GID1-GA complex. In rice the complex promotes affinity of a ‘specific ubiquitin ligase complex SCF E3; SCFSLY1/GID2’ (F box proteins A+SLY1 and OSGID2) also involved in this complex. Following this interaction, the DELLA proteins are destroyed through the 26s proteasome. Transcriptional control of the DELLA genes is done by PIL5, a light sensitive transcriptional factor. PIL5 enhances the transcription of GAI and RGA genes being bound to its ‘promoter site on the G-box’. In barley, DELLA protein is GA dependent and is hindered by ABA. The role that DELLA proteins take in regulation of dormancy is however not known (Kumar et al. 2013).

2.3.3 RNA dormancy-germination control Barley has a large genome size of 5.1GB. However most of this genome is composed of highly repetitive DNA regions. This therefore causes problems when performing sequencing. A full barley sequence is available from bacterial artificial chromosome (BACs) developed from Morex variety (Schulte et al., 2011). This and enrichment by 2M SNPs provide greater detail for 96% of the length of the physical genome which is about 4.96GB.

The Minimum Tilling Path (MTP) for barley is defined and consists of 66,722 clones which overlap only negligibly from roughly 600,000 BACs. The production of a reference barley genome took a leap forward following the MTP 15,622 BACs sequenced representing 72,052 physical map gene bearing BACs from the library developed from Morex hence the generation of approximately 1.7Gb genomic sequence which represents about two thirds of Morex genes, (Schulte et al., 2011).

Gene expression studies in barley were made possible with the development of a chip which is known as Barley1 Gene Chip and was release in 2004 from studies by (Zhang et al., 2004). This chip contains 21,439 genes and has been used in studies of germination and seedling elongation. Recently, full length cDNA libraries have been developed from malting barley varieties known as Huruna Nijo acting as a confirmation model for gene sequences identified and annotated by the barley genome sequence. Of the 26,159 gene sequences identified, 93% were linked to the acquired physical chromosome map assembly. This physical map

19 availability allows the use of NGS to explore gene expression because of high confidence prediction used in the map generation (Schulte et al., 20011).

RNA sequencing as an NGS tool offers greater coverage on the transcripts than the use of microarrays and serial analysis gene expression (SAGE) e.g. the study conducted by (Duan et al., 2015) where cDNA libraries were constructed from caryopsis of two varieties of barley at three weeks after anthesis (Daneri-Castro et al., 2016).

The physiological changes being observed during dormancy and on to germination are subject to both transcription and post transcriptional control. The control level is temporarily co- ordinated and distributed from maturation then to dormancy and lastly to germination. This control is essential for environmental adaptation of the seed and germination timing in order to yield a vigorous seedling (Holdsworth et al., 2008). Mature dry seeds contain large amounts of mRNA which serve as the embryogenesis reservoirs. The genes found in dry mature seeds code mainly for protein regulating protein synthesis and degradation, seed reserve LE19A proteins and proteins regulating metabolism. Cotton studies indicate long lived RNA in the seed contributing to protein synthesis during early germination stages (Dure and Waters, 1965). These genes are also responsible in sending the seed to its transient state (Nakabayashi et al., 2005).

There has been shown to be large increases in RNAs encoding proteins associated with RNA translation, protein degradation and cell wall modification in imbibed and after-ripened seeds. These gene groups have been shown to increase rapidly between 1-3hrs after imbibition. However, in dormant seeds, the gene groups showing increased expression encode for roles which relate to late seed maturation. It has been reported that transcription is not mandatory for the completion of germination. This led to the suggestion that the transcriptome change might be what is needed for a rapid and uniform germination, this is such as the change from the embryonic phase to the plant vegetative state. This has been confirmed by the observed high upregulation of genes such as those needed for auxin transport (AUX1, PIN7, PIN1) which might be in tandem with root growth at phase III onset. Germination is inhibited completely following the application of cyclohexamide which is a translational inhibitor. From this study, it was suggested that protein synthesis is needed in germinating embryos which has been assumed to utilize stored mRNAs as templates (Holdsworth et al., 2008).

Results from another study varying the light requirement of the plant (dark and light) during imbibition using after-ripened Arabidopsis seeds showed that gene expression held up at translation due to varying light treatment. It also showed that the seed expression of non- germinating mutant seeds of COMATOSE (cts-1), a perixomal ABC transporter, was similar to that of the germinating wild type seeds, however, the cts-1 at the proteome level was similar to that of dormant seed. This demonstrated the translational inability of accumulated germination related RNAs (Holdsworth et al., 2008).

Previous studies demonstrated varied gene expression in dormant and non-dormant seeds (Li and Foley, 1997). For example, protein complexes in wheat seeds show an extended synthesis of a variety of heat stable proteins such as dehydrins. In non-dormant seeds however, the synthesis of these heat stable proteins declines after a few hours. ‘cDNA cloning and differential screening techniques’ have enabled the discovery of genes expressed, especially in dormant seeds. These genes were found to be responsive to ABA. The sequence homology indicated most clones correspond to LEA, which has a desiccation protective role of the embryo in periods of repeated desiccation in dormant seeds. This protein however may not be involved in dormancy control and was not uniformly expressed in all the clones.

Another cDNA clone known as pKABA1 whose sequence homology corresponds to serine/threonine protein kinase originated from a library generated from ABA treated wheat embryos. The clones’ dormancy regulation role is not clear since its transcripts decline sharply following imbibition contrasting to its previous high levels before imbibition in dormant seeds. As the non-dormant seed germinates, this transcript increases in quantities leading to the assumption of its role in ABA mediated growth processes in the seed but not in dormancy. Another cDNA clone pBS128 whose sequence homology analysis showed encoded for peroxiredoxins, antioxidants responsible for the protection against reactive oxygen species, was identified in cheat grass. Clone pBS128’s transcript levels were found to increase in dormant seeds during imbibition over four fold, but found to rapidly decline and disappear in non-dormant seeds. Even though many genes have been identified to be differentially expressed in hydrated dormant and non-dormant seeds, there is still need to identify the main ones involved in the maintenance and ending of dormancy. This is because none of the identified genes has a direct role associated with dormancy (Li and Foley, 1997).

2.4 Barley germination During seed development, as the seed matures, it undergoes dehydration, the endosperm cells die. The seed will hence contain starch, proteins and nucleic acids which are important during germination. The aleurone layer however contains living cells which upon activation, their contents undergo a chain of biochemical and morphological changes hence changing it from a dormant to a metabolic active tissue which goes into the synthesis of a varying range of enzymes that will be needed in the process of breaking down of the stored starch in the endosperm.

Scutellum epithelium does the same role as the aleurone layer. The aleurone layer is made up of cells forming a cuboidal layer 3-4 cells thick overlying the starchy endosperm surface. These cells are formed as a result of triploid fusion nucleus similar to the starchy endosperm cells. These cells each have a thick two layered cell wall with a large nucleus and a compactly packed mitochondrion which make their identification at maturity quite clear. The scuttelum however, is a diploid tissue formed from a mass of parenchymatous cells which are arranged as a single layer of elongated cells. Its cells have highly specialized functions in endosperm digestion (Nilan and Ullrich, 1993).

Germination is a process in plant seeds whereby, the quiescent seed takes up water (imbibition) and mobilises the food reserves which eventually leads to the emergence of a radicle (Bewley, 1997). Imbibition occurs in three phases as indicated in figure 3 below, in phase one, water is taken up rapidly by the seed, the seed will then enter phase two which is essentially a plateau phase and lastly, phase three whereby food reserve mobilisation and elongation of the embryonic cells occurs. Water uptake then continues, however, dormant seeds do not enter into phase three.

The water uptake in phase one causes temporary structural disturbances mainly on the membranes which results in solutes and low molecular weight compounds being leaked into the imbibed solution. The seed membranes will therefore return to their normal hydrated liquid-crystalline state (Crowe and Crowe, 1992; Bewley, 1997) from the dry gel like phase that had been maintained during seed maturation and quiescent phases (Bewley, 1997).

Figure 2.3. The plant germination phases, Phases I and II shows resumption of plant activities such as protein synthesis, translational activities and cell repair functions whereas phase III shows the normal growth and development stages necessary for seedling growth, (Bewley, 1997).

After the process of germination starts, the hydrolytic enzymes are synthesized in the aleurone layer and the scutellum, and are transferred into the starchy endosperm where they will lead to the degradation of storage polymers. The degraded food reserves will then move following the diffusion gradient which has been created following their uptake to the epithelial layer of the scutellum and will hence be taken up through the evolving vascular system to the seedling. Here the material will provide energy and nutrients for the developing embryo for the period before the leaves and root system are fully developed to start the process of photosynthesis and active uptake of water.

2.4.1 Starch degradation Starch composition in the starchy endosperm includes 75% amylopectin and 25% amylose. During germination, these are broken down to glucose and simple oligosaccharides by combined action of four enzymes which are; α amylases, β amylases, limit debranching enzymes and α glucosidase. Studies have shown that initial degradation in large starch granules begin from the surface at discreet points on the equatorial axis headed inwards forming large degradation holes. The inner region is hence solubilized therefore prompting the outer shell to collapse. It then gradually disappears. In smaller starch granules however, degradation is by surface erosion (Fincher and Stone, 1993).

2.4.1.1 Alpha amylases Alpha amylases are endohydrolases cleaving internal (1→4)-α- glucosyl linkages of amylose and amylopectin in a random way. They are incapable of hydrolysing (1→6)-α- linkages in amylopectin hence their ability to hydrolyse (1→4)-α- linkages close to (1→6) -α- may vary. Their structure is as shown in figure 4 below. The general structure of alpha amylase is shown in figure 2.4 below.

Figure 2.4. α amylase structure. Source: http://www.scielo.br/pdf/bjm/v41n4/04.pdf

In barley, alpha amylases synthesized are Ca++ metalloproteins with a single polypeptide chain Mr~45,000. They are classified as Amy1 and Amy2 based on their iso-electric points where Amy1 which has a low iso-electric point of pI 4.6 and Amy2 a high pI of 5.9 and is approximately eleven amino acids longer than the low iso-electric amylases. They degrade starch faster, are known for substrate specificity and action patterns. The identity between the two is approximately 80%, however within groups, the amino acid sequence identity of 90- 95% is seen. Principally, α amylases are synthesized in the scuttelum and mainly in the aleurone layer.

2.4.1.2 Beta-amylases Beta-amylases are exohydrolases cleaving (1→4)-α-linkages from the non-reducing end of the (1→4)-α-glucans ending up with maltose, a disaccharide sugar. Principally, they should completely degrade unbranched or unsubstituted amylose molecules, however they also degrade the (1→6)-α-linkages in amylopectin.

Beta-amylases are present in non-germinated barley with concentrations of 1-2% of the total grain protein content. It is located in the starchy endosperm on the surface of starch molecules. They are normally synthesised during seed maturation which contrasts the synthesis of the other enzymes which takes place in either the aleurone of scuttelum during germination. This enzyme is salt extractable, however, some percentage is linked to other protein by disulphide bonds remains in the grain material.

The enzyme is a single chain of Mr 54,000-60,000 with an iso-electric point range of between 5.2-5.7. Their heterogeneity can be generated by limited proteolysis of the COOH- end. Their mature form are normally lacking in NH2-end which is reduced from a member of the family which was generated. Beta-amylase has four glycine-rich repeats which are the main characterisation factor. They are released during germination by proteolic enzymes. The enzyme is however not able to degrade whole starch granules and will only act in synergy with α amylases hence preventing premature starch degradation before germination.

2.4.1.3 Limit dextrinase Limit dextrinase hydrolyses the (1→6)-α-linkages in amylopectin or in subsequent limit dextrins therefore growing the amount of linear (1→4)-α-glucan chains which will in turn be depolymerised by alpha and beta amylases. The action of limit dextrinase is faster in limit dextrins than in amylopectins.

The enzyme is principally synthesised in the aleurone, however, ungerminated grain would contain small amounts of this enzyme. Its concentration increases over time in the course of germination. Results from gel electrophoresis and isoelectric focussing indicate that the limit dextrinases isoforms have a pI value of between 4.2-5.0 and Mr of 80,000-104,000. If mixed with starch, the purified enzyme has no effect, however, when alpha and beta amylases are added to the mixture, it increases the starch degradation rate.

2.4.1.4 Alpha glucosidase This enzyme degrades α-glucosides, maltose and small dextrins to release glucose. It originates from the pericarp, aleurone and the embryo of non-germinated grain. Its synthesis in the embryo and the aleurone layer is induced by GA during germination hence increasing the amount in the grain.

2.4.2 Problem statement and justification The malting and brewing industry prefer barley varieties which maintain a high germination percentage of close to 97%. The varieties should also display rapid but uniform germination. However, most of the seeds will induce dormancy as the storage period increases. This would therefore mean increased loses with increased storage period in order to overcome dormancy.

Dormancy can be defined as failure of a viable mature seed to germinate when provided with all conditions favouring germination (Romagosa et al., 1999). Dormancy is a natural process that the seed undergoes to ensure maturity is attained and regulate germination in order to ensure survival. In barley, seeds which lack dormancy are susceptible to pre-harvest sprouting which is also a big problem in barley production. Striking a balance between the two therefore is most desirable.

Dormancy is a trait which is highly influenced by the environmental factors during seed storage including water content and storage period. Early studies also show that it is regulated by several QTLs which have been studied extensively.

These losses faced by the brewing and malting industry can be evaded by developing mechanisms to monitor the germinability of the seeds during storage. In this project, proposed techniques explore to develop these mechanisms and test their efficiency.

CHAPTER 3: Differences in protein expression in dormant and non- dormant barley using SWATH-MS

Abstract

Barley germination is required for producing malt for brewing. Maltsters require a minimum of 97% germination in each malting batch as well as over the whole processing season which can be more than one year. Seeds in storage over a period of time start losing the ability to germinate. When germination efficiency drops off unexpectedly, there is a major cost to the malting industry and potential for subsequent problems in brewing. To measure this loss of germination viability, we studied the barley seed proteome over the course of germination and in quiescent seed. One barley variety, Stirling, was germinated over a period of three days. Five treatment groups of seeds were tested: before germination, seeds germinated at 24 hours, seeds germinated at 72 hours, seeds germinated at 24 hours but measured at 72 hours, and seeds that failed to germinate at 72 hours. Using mass spectrometry, 128 unique proteins were identified. Comparison of each group using sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH–MS) revealed substantial differences in the abundance of specific proteins. Proteins that were abundant at the ‘before germination stage’ included proteins whose main role is in protecting the embryo during the dry desiccated period of seed storage such as late embryogenesis protein (LEA19A). The ‘after 24 hours germination’ group included abundant proteins involved in starch and protein breakdown such as beta-amylase (AMYB) which provide the young developing embryo with nutrition. Many proteins were not different in abundance in the samples that failed to germinate and to the samples that had been growing for 72 h. However, select proteins including H2A.6 were significantly different in abundance (p<0.05) between these groups. Together, our data suggests that seeds that fail to germinate are still metabolically active, but lack key regulatory events required for progression to germination.

3.1 Introduction

Barley (Hordeum vulgare subsp. vulgare L.) is an important cereal crop, ranking fourth in global cereal production after maize, wheat and rice (Schulte et al., 2009). The production of barley is high on a global basis because it is one of the main ingredients in beer making (Iimure and Sato, 2013). Its other uses include human food and animal feed. Barley is one of the most researched crops, it has been subjected to analyses including genomics and transcriptomics making it a model organism for the Triticeae tribe of plants (Iimure and Sato, 2013).

Germination refers to the process of emergence of the radicle from the seed following imbibition of water and takes into account all biochemical processes that take place within a seed until the emergence of the radicle (Nonogaki et al., 2010). Dormancy on the other hand is the failure of a seed to germinate even when presented with conditions favouring seed germination (Gubler et al., 2005; Hori et al., 2007; Romagosa et al., 1999). Dormancy is an important trait in plants because it offers the plant tolerance/resistance to pre-harvest sprouting (PHS), a condition whereby seeds germinate in the field while still on the mother plant if exposed to moist conditions. However, when the dormancy is too strong, the seed requires additional storage time to pre-condition the seeds to a germinable state, in order to attain the uniform germination desired in the malting process. This process in barley is highly controlled in order to attain uniformly germinated grains with a high germination percentage (Gubler et al., 2005; Hori et al., 2007; Finnie et al., 2009).The malting process involves three main steps which include: Steeping; involves exposing the grain to periods of water soaking and aeration in order to raise the grain moisture content to about 35-45%., followed by germination; is done over 4-6 days with controlled relative humidity of 100% and temperature (12-250C) in order to allow for the seedling growth and development and lastly kilning; involves the drying of the seeds in large kilns in order to reduce the moisture content to about 4-6% depending with the malts final use, (Burger and LaBerger, 1985)

Proteomics is a powerful tool which allows the acquisition of both qualitative and quantitative data on the proteins contained in a particular organism at the time of analysis (Finnie and Svensson, 2002). Proteomics has been used previously in studies to address many aspects of research, such as identifying the proteins contained in a tissue or sample (Finnie and Svensson, 2003; Bonsager, 2007; Daneri-castro et al., 2016), at a particular stage of development (Finnie et al., 2011, Irar et al., 2005). There has been an increased use of proteomics recently to study

29 the changes occurring in barley seeds during stages such as seed filling, grain maturation and during germination as reviewed in (Finnie and Svensson, 2009).

It is crucial to have high quality protein extract to be used in downstream proteomics applications and to enable acquisition of high quality data (Wang et al., 2005). In this dormancy study, we perform protein extraction adopting a protocol from (Peak et al., 2015) and followed with proteomic analysis using SWATH-MS in order to determine the proteome changes in the course of germination and in the seeds which failed to germinate. For physical germination visualization purposes, an x-ray technique was used. X-ray has been used previously in barley to induce mutations by long exposure (Stadler L, 1928), to predict the seedling morphology by embryo and endosperm visualization serving as a non-destructive method of seedling development predictions (Van der Burg et al., 1994) and in wheat to detect sprouted kernels in an effort to tackle the sticky dough problem (Neethirajan and White, 2007)

3.2 Materials and methods 3.2.1 Germination test

Seeds from one barley variety, Stirling, were used in this study. Prior to germination, five seeds were selected and placed in 1.5 mL protein LoBind tubes. The seeds were stored at -20℃ to be used later for protein extraction to represent the day zero samples. Five replicates of 100 seeds were then counted and surface sterilized with 1% hypochlorite. These were rinsed thoroughly, air-dried and placed in five 9mm petri dishes lined with two Whatman #1 filter papers. Double distilled water (4 mL) was added and the dishes covered and set aside in the dark for germination, at 22℃.

Seeds for proteomic analysis were selected on a single seed basis, where one seed was selected at random for each treatment category from each replicate dish. The seed sampling schedule was as documented in Table 1 below. After sampling, each seed was placed in a separate protein LoBind tube and stored in -20˚C freezer until extraction.

3.2.2 X-Ray visualisation

X-ray visualisation of randomly selected seeds was performed using a Faxitron X-Ray MX-20 before germination and then every 24 hours up to 72 hours during germination.

Table 3.1. Seed sampling schedule, five replicate seeds were sampled at each sampling period

Sample code Stage of germination sampled

C Before imbibition NG Seeds that germinated after 24 hours imbibition SG Seeds that germinated 72 hours after imbibition LS Seeds that germinated at 24 hours after imbibition and were sampled at 72 hours FG Seeds sampled at 72 hours that failed to germinate

3.2.3 Protein extraction

Individual grains were ground using a mortar and pestle to yield a homogenous powder, then placed in 2 mL Eppendorf tubes. Proteins were denatured and reduced/alkylated as previously reported (Peak et al., 2015) as follows; to each sample, 1 mL of 50 mM Tris HCl buffer (pH 8) and 6M guanidinium hydrochloride was added to the samples and then mixed thoroughly by vortexing. Cysteines were reduced by addition of dithiothreitol (DTT) to a final concentration of 10 mM with incubation at 37℃ for 30 min with constant shaking. Cysteines were alkylated by addition of acrylamide to a final concentration of 30 mM with incubation at 37℃ for 30 min with constant shaking. Excess acrylamide was quenched by addition of DTT to a final concentration of 5 mM. Samples were centrifuged at 18,000 rcf for 5 min and 10 µL of the supernatant transferred into a new protein LoBind tube. Protein was precipitated by addition of 100 µL 1:1 acetone: methanol and samples were stored at -20℃ for 2 h. Precipitated protein was pelleted by centrifugation at 18,000 rcf for 5 min, and the supernatant was discarded. The pellet was then air dried for 30 min. For protease digestion, the protein pellet was resuspended in 100 µL of 50 mM ammonium acetate with 0.5 µg trypsin and incubated at 37℃ for 16 h with constant shaking.

3.2.3.1 Mass spectrometry

Peptides were desalted using C18 ZipTips (Millipore) and proteins were measured by LC-ESI- MS/MS using a prominence nanoLC (Shimadzu) and Triple TOF 5600 instrument (SCIEX, Redwood City, USA) with a Nanospray III interface (SCIEX) as previously described (Bailey et al., 2012). Approximately 1 µg or 0.2 µg desalted peptides, as estimated by ZipTip binding capacity, were injected for data dependent acquisition (DDA) or data independent acquisition (DIA), respectively. LC parameters were identical for DDA and DIA, and LC-MS/MS was performed essentially as previously described (Peak et al., 2015). Peptides were separated with buffer A (1% acetonitrile and 0.1% formic acid) and buffer B (80% acetonitrile with 0.1% formic acid) with a gradient of 10–60% buffer B over 14 min, for a total run time of 24 min per sample. Gas and voltage setting were adjusted as required. For DDA analyses, an MS TOF scan from m/z of 350–1800 was performed for 0.5 s followed by DDA of MS/MS from m/z of 50-1800 with 26 m/z isolation windows and 1 m/z window overlap each for 0.1 s across an m/z range 400-1250 in high sensitivity mode. Identical LC conditions were used for DIA

SWATH, with an MS-TOF scan from an m/z of 350–1800 for 0.05 s followed by high- sensitivity DIA of MS/MS from m/z of 50-1800 with 26 m/z isolation windows with 1 m/z window overlap each for 0.1 s across an m/z range of 400–1250. Collision energy was automatically assigned by the Analyst software (SCIEX) based on the centre of each m/z window range. The normalization of fluctuation was done by the machine by calibrating every 5 samples.

The proteins identified by ProteinPilot 5.1 (SCIEX), searching against all high confidence proteins from transcripts of the barley genome (downloaded 28 April 2017; 248180 total entries), with settings; sample type, identification; cysteine alkylation, acrylamide; instrument, TripleT of 5600; spicies, none; id focus, biological modifications; enzyme, trypsin; search effort, through ID. These proteins identified were used to generate ion libraries for analysis of SWATH data. Protein abundances were measured with PeakView 2.1 (SCIEX) as described (Zacchi and Schulz, 2016). The setting for PeakView 2.1 (SCIEX) were as follows: shared peptides, allowed; peptide conference threshold, 99%; false discovery rate, 1%; XIC extraction window, 6 Min; XIC width, 75 ppm. Proteins identified were matched against UniProtKB (downloaded 2 December 2017: 555318 total entries). Protein relative abundances were compared based on protein intensities. The data is processing through both Protein Pilot and Peak View, was done to remove the noise and perform false discovery rate analysis and remove data that does not meet the stringent cut-off of 1. Analysis of variance was used to test for proteomic differences between the seeds sampled at each time period by assessing the variation amongst the entire protein set using their relative abundances. Analysis of variance was undertaken for each of 129 proteins using the R 2.14.0 statistical software package. Principal component analysis (PCA) was performed on the selected proteins using the samples as the individuals and the proteins as the attributes using R 2.14.0 software. Least significant difference testing (p=0.05) was conducted on individual selected proteins to test the mean differences. The graphs were plotted using GraphPad prism.

3.4 Results and discussion

A total of 15 proteins showed significant changes in abundance during the course of germination. The list of selected proteins which showed significant variation between germination stages are listed in Table 3.2. These were further analysed using principal components analysis to identify the pattern of clustering of the proteins in relation to the sample group as shown in Figure 3.1. The proteins were found to be clustered into three groups; (i) the proteins which were abundant at day zero before the seeds were germinated, (ii) the proteins that were abundant at the onset of germination and (iii) proteins which showed gradual increase over the course of germination.

The X-ray imaging revealed physical changes between the different time points before the germ began to grow and differences when it was grown and fully visible after 72 hours of germination, as shown below in Figure 3.2. Before germination, the seeds looked intact and the endosperm was darker than the other seed parts. At 24 hours after germination, the seeds appeared swollen and visible signs of germination were apparent, wherein the radicle could be seen emerging from the seed. Seventy-two hours after germination, the roots were very elongated and the plumule could be seen emerging from the seed. The plumule appeared darker than both the roots and the endosperm which, at this time, appeared lighter than they were at day 0 (before germination). The seeds which were classified in the group of failed to germinate at 72 hours showed no visible signs of germination by X-ray. While the seeds seemed to be swollen, the endosperm colour appeared unchanged. The germination percentage of seeds was an average of 82%, 93.5% and 97% for 24 hr, 48 hr and 72 hr after germination respectively.

3.4.1 Changes in the barley seed proteome over the course of germination

The protein data obtained revealed major changes in proteome during germination and also in the seeds which failed to germinate. A total of 129 proteins were identified, 74 which are important to barley as listed on Appendix 3.1. The abundance of proteins changed significantly depending on the period of sampling. Significant differences between the germinated seeds sampled at different times across the course of germination were detected for 15 proteins (p<0.05).

Table 3.2. List of selected proteins which showed significant variation (p=0.05) from 0-72 hours, their assigned group and their function/role. The accession number and role is based on their identification in the UniProt database

Accession Protein group Protein name Protein role number

DEF1 (Defensin like Inhibits protein translation in cell free protein) systems (antimicrobial) sp.P20230

Protects the cytoplasm during desiccation EM3 (EM protein) Proteins abundant period of the embryo at day 0 before sp.Q08000 germination LE19A (Late Osmoprotective protein for desiccation embryogenesis abundant tolerance protein) sp.Q05190 DHN4 (Dehydrin) sp.P12949 Stress response Alanine tRNA ligase Post translation modification (SYA-MOOTA) Q2RHZ3 Hydrolyses 1,4-glucosidic linkages of AMYB (β amylase) sp.P16098 starch PDI (Protein disulphide involved in folding of proteins containing Proteins with initial isomerase precursor) sp.P80284 disulphide bonds increase at day 1 germination HOG3 (Gamma hordein Transport and targeting of prolamins to the 3) vacuole of developing barley endosperm sp.P80198 ASPR (Phytepsin Storage protein breakdown precursor) sp.P42210 METE1 (Methyl Methionine formation transferase 1) sp.Q2QLY5 Transcription regulation, DNA repair, H2A6 (Protein H2A.6) DNA replication and chromosomal Proteins showing sp.Q43214 stability increase over time MDH (Malate Catalyses formation of oxaloacetate from from germination dehydrogenase malate to day 3 of seedling sp.Q9FSF0 development AMY6 (α amylase type B Catalyse hydrolysis of internal 1,4-alpha- isozyme precursor) sp.P04750 D-glucosidic bonds MHDC (Malate Catalyses formation of oxaloacetate from dehydrogenase, malate cytoplasmic) sp.Q7XDC8 HSP17 (Heat shock No group protein) sp.P19036 Desiccation tolerance

Figure 3.1. Protein abundance patterns from a PCA of 15 proteins measured on 25 seeds. The proteins clustered into three groups, which have been assigned as before germination abundant proteins, initial germination abundant proteins and proteins which increase in abundance over time. The 25 seeds can be grouped into 5 sets; C=before imbibition, N=seeds that germinated after 24 hours imbibition, S=seeds that germinated after 72 hours imbibition, L=seeds that germinated after 24 hours imbibition but sampled after 72 hours and F= failed to germinate after 72 hours imbibition. The protein list is found in Table 3.2.

Figure 3.2. X-ray image of seeds (A) before germination; (B) germinating after 24 hours imbibition. (C) Germinating from 24 hours imbibition after 72 hours imbibition (D) failing to germinate after 72 hours imbibition.

3.4.2 Proteins abundant before imbibition

The proteins which clustered in this group as the PCA figure 3.1 above included the proteins listed in Table 3.2. The late embryogenesis protein (LE19A) was of high significant abundance in the seeds assessed before imbibition (day zero seeds). The other proteins abundant at day zero were EM3, DEF1 and DNH4. During germination however, the abundance levels of LEA19A dropped significantly. The difference in abundance between 24 hours, 72 hours and the quiescent seed samples were however not significant. Protein dehydrin (DHN4) was the other protein abundant in non-germinated seeds and the abundance levels decreased significantly as the plants developed. The difference in abundance is however not significant at 24 hours, 72 hours and quiescent seed samples. EM3 and DEF1 showed similar trends where these proteins were highly abundant before germination and their abundance reduced significantly after germination. There is however no significant difference in abundance at 24 and 72 hours and also in the sample which failed to germinate.

The seeds which failed to germinate did not show any unique trend in this cluster. The analysis from the selected list of proteins showed that their level of abundance was significantly lower than the non-germinated group which indicates that there may be some biochemical activity initiated prior to germination, however, their abundance level was not significantly different from the 24 hours and 72 hours samples. It is apparent that is this measured level of biochemical activity is not sufficient to lead to germination.

Table 3.3. Mean relative abundance of proteins categorised as to have significant high abundance before germination. Superscripts of a, b and c indicate significantly different means at (p=0.05)

Condition DHN4 EM3 LE19A DEF1

Germinated after 24 hours imbibition 0.000010b 0.00008b 0.00058b 0.00104b

Germinated 24 hrs, sampled at 72 hrs 5.83E-06b 0.00004b 0.00060b 0.00088b

Germinated after 72 hours imbibition 7.87E-06b 0.00007b 0.00080b 0.00096b Failed to germinate 9.70E-06b 0.00005b 0.00014b 0.00095b Sampled before imbibition 0.00051a 0.00239a 0.00544a 0.00151a LSD (p=0.05) 0.00013829 0.00040166 0.000960294 0.0004251

The comparison of means using LSD tests revealed that there was a significant difference in means between the periods of sampling before germination and the rest of the sampling groups for the proteins shown in Table 3.3 above. All the proteins which clustered under the before germination group shows a similar trend as clearly shown in Figure 3.3 below with the day 0 samples being significantly different in abundance from the rest of the sampled periods.

P ro te in s a b u n d a n t b e fo re g e rm in a tio n

0 .0 1 0 C

0 .0 0 8 NG e

c FG

n 0 .0 0 6

a L S

d n

u 0 .0 0 4 S G

b A

0 .0 0 2

0 .0 0 0

A 1 4 A 3 9 F N Y M 1 E H S E E D | | L | |D 3 0 | 0 9 Z 0 0 3 4 0 9 2 H 8 1 0 9 R 2 2 0 5 2 1 Q 0 P P Q Q

Figure 3.3. Protein relative abundance changes over 72 hrs for late embryogenesis abundant protein, defensin-like protein, EM protein and protein dehydrin. Legend: C=before imbibition, N=seeds that germinated after 24 hours imbibition, S=seeds that germinated after 72 hours imbibition, L=seeds that germinated after 24 hours imbibition but sampled after 72 hours and F= failed to germinate after 72 hours imbibition.

The changes in proteome observed in seeds before and after germination included a change in abundance of proteins mainly involved with protection of the seed during the desiccation period. These proteins include the LE19A protein which belongs to the superfamily dehydrin and has been associated with desiccation tolerance during the periods of plant stress such as in dry seeds. The level of this protein during stress periods may rise to 4% of the total cellular proteins (Irar et al., 2006). Like LE19A, protein dehydrin belongs to superfamily dehydrin and its related genes are expressed during periods of low water content in the seed or when the seeds are exposed to environmental conditions favouring osmotic stress (Heyen et al., 2002).

Some of the other proteins which were significantly abundant before germination were also observed to have stress protective functions to the embryo. These proteins included Defensin- 39 like protein (DEF1) which has an antimicrobial role, preventing free cell systems such as bacteria and virus from translation of RNA to proteins, and EM protein (EM3) which have the role of desiccation tolerance in seeds.

3.4.3 Proteins that showed initial increase at 24 hrs germination

Protein Disulphide Isomerase (PDI) had high abundance in samples which germinated after 24 hours (Table 3.4). It is however not significantly different in abundance to samples which germinated after 24 hours and sampled at 72 hours imbibition. The protein is significantly lower in abundance in samples which were not germinated. Another protein, Gamma hordein 3 (HOG3), showed a similar trend showing high abundance at 24 hours germination. This was however not significantly different from the samples which failed to germinate, the samples which germinated after 72 hours and samples which germinated at 24 hours but sampled at 72 hours. The only significant difference observed was with the non-germinated samples. Beta amylase showed significantly higher abundance in samples germinating after 24 hours and this is different from the rest of the samples. The last two proteins, Phytepsin precursor (ASPR) and C Hordein fragment (HOR7) were also abundant at 24 hours germination and in the samples germinated after 24 hours but sampled at 72 hours.

Table 3.4. Mean relative abundance of proteins categorised to have an initial increase in abundance at 24 hours after germination. Superscripts of a, b and c indicate significantly different means at (p=0.05).

Condition PDI HOG3 ASPR AMYB

Germinated after 24 hours imbibition 0.0114a 0.0134a 0.0007a 0.0048a

Germinated 24 hrs, sampled at 72 hrs 0.0097ab 0.0102ab 0.0002b 0.0031b

Germinated after 72 hours imbibition 0.0096ab 0.0127a 0.0006ab 0.0030b

Failed to germinate 0.0094b 0.0126a 0.0004b 0.0027b Sampled before imbibition 0.0075c 0.0089b 0.0002b 0.0026b LSD (p=0.05) 0.0018 0.0033 0.0003 0.0013

The proteome changes observed after 24 hours included an increased abundance of proteins involved in protein folding and starch degradation. At the same time, there was a significant decrease in proteins observed before germination as shown in Figure 3.4, which were mainly the desiccation tolerance and water stress tolerance associated protein. This may be due to their degradation as the seeds move from the dry quiescent state to the imbibed state and to germination (Bonsager et al., 2007).

P r o te in s th a t s h o w e d in itia l in c r e a s e a t 2 4 h rs g e rm in a tio n

0 .0 8 C

N G 0 .0 6

F G

c n

a L S

d 0 .0 4

n u

b S G A

0 .0 2

0 .0 0

I 3 B D R Y P G P M | O S 4 H A |A 8 | | 3 2 8 0 9 0 9 1 9 8 1 2 2 P 0 2 8 8 4 P P P

Figure 3.4. Protein relative abundance changes over 72 hrs for Protein disulphide isomerase (PDI), Gamma hordein 3 (HOG3), Beta amylase (AMYB), C hordein fragment (HOR7) and Phytepsin precursor (ASPR). Legend: C=before imbibition, N=seeds that germinated after 24 hours imbibition, S=seeds that germinated after 72 hours imbibition, L=seeds that germinated after 24 hours imbibition but sampled after 72 hours and F= failed to germinate after 72 hours imbibition.

PDI has been detected in germinating barley seeds in previous studies by (Ostergaard et al., 2004; Bonsager et al., 2007). Its function has been described as a catalyst for the proper disulphide bonding during protein folding and also as a storage protein. It has been detected during early growth of plants. Proteins HOG3 and HOR7 have been identified as storage proteins whereas ASPR is involved with the breakdown of storage proteins. The protein increase and decrease trends is as illustrated on Figure 3.4.

3.4.4 Proteins abundant in the course of germination and during early radicle elongation

There were seven proteins that showed an increase in abundance over time (Figure 3.5). These included 5 methyltrahydropteroyltriglutamate/ homocysteine methyltranferase 1 (METE1) which had significantly high abundance in samples with 72 hours imbibition, it showed lowest abundance in non-germinated samples. In the rest of the samples, the abundance was intermediate with no significant difference as shown in Table 3.5.

Malate dehydrogenase, cytoplasmic (MDHC) was the other protein that was abundant in samples collected after 24 hours, 72 hours, samples which failed to germinate and the samples with extended radicle growth. It was however significantly lower in non-germinated samples. Malate dehydrogenase (MDH) protein showed significantly higher abundance in samples germinated after 24 hours, 72 hours, samples which failed to germinate and in samples with extended radicle growth for 72 hours. It was however significantly lower in abundance in non- germinated samples as shown in Table 3.4.

Protein H2A.6 (H2A6) showed significantly high abundance in samples which failed to germinate and significantly low abundance in samples which germinated after 72 hours. It showed intermediate abundance in the rest of the sample classes. This protein is the main constituent of the nucleosomes which function in wrapping and compacting of DNA into chromatin hence regulating its transcription, repair, replication and also chromosomal stability. This is achieved via post translational modifications of histones, also known as ‘histone code and nucleosome remodelling’ (Huh et al., 1995).

Table 3.5. Mean relative abundance of proteins categorised to have significant abundance in the course of germination with maximum level of abundance observed at 72 hours after imbibition. Superscripts of a, b and c indicate significantly different means at (p=0.05)

Condition AMY6 METE1 MDHC MDH H2A6 Germinated after 24 hours imbibition 0.0008b 0.0003bc 0.0075a 0.0020a 0.0021abc Germinated 24 hrs, sampled at 72 hrs 0.0018a 0.0006a 0.0077a 0.0022a 0.0023ab Germinated after 72 hours imbibition 0.0015ab 0.0004b 0.0068a 0.0021a 0.0019c Failed to germinate 0.0009b 0.0005ab 0.0068a 0.0020a 0.0025a Sampled before imbibition 0.0005b 0.0001c 0.0053b 0.0016b 0.0019bc LSD (p=0.05) 0.00082 0.00018 0.00138 0.00042 0.00043

Additional changes in proteome included Protein METE1 function which catalyses the transfer of methyl group from 5-methyltetrahydrofolate to homocysteine hence forming methionine, an amino acid. This is in the ‘L-methionine biosynthesis pathway’ which is part of the amino acid biosynthesis pathway. Protein MDHC on the other hand catalyses redox reactions within the cytoplasm for energy production e.g. in the tricarboxylic cycle where it catalyses the reversible dehydrogenation of malate to oxaloacetate by coupling with the reduction of NAD+ to NADH (Lin et al., 2004).

Protein MDH was the other protein which is similar to MDHC in function, catalysing the reduction of carbonyl group of oxaloacetic acid (Matsushima et al., 2008) to release energy.

P ro te in s th a t s h o w e d in c re a s e o v e r tim e

0 .0 1 0 C

0 .0 0 8 NG e

c FG

n 0 .0 0 6

a L S

d n

u 0 .0 0 4 S G

b A

0 .0 0 2

0 .0 0 0

6 6 H C A 1 Y D H E 2 T M |M D |H E A 0 M 4 | F | 1 |M 0 8 2 5 S C 5 7 F 3 Y 4 9 D 4 L 0 Q X Q Q P 7 2 Q Q

Figure 3.5. Protein relative abundance change for proteins Malate dehydrogenase, cytoplasmic (MDHC), Malate dehydrogenase (MDH), homocysteine methyltranferase 1 (METE1), Actin 1 (ACT1), protein H2A.6 (H2A6), 26 kDa endochitinase 2 (CH12) and Alpha amylase (AMY6). Legend: C=before imbibition, N=seeds that germinated after 24 hours imbibition, S=seeds that germinated after 72 hours imbibition, L=seeds that germinated after 24 hours imbibition but sampled after 72 hours and F= failed to germinate after 72 hours imbibition.

Beta amylase is normally present in the seed prior to germination. Its synthesis occurs during the grain filling stage. The enzyme is bound in the storage protein matrix (hordein) surrounding the starch granules and its release during germination is in response to the hormone GA, after this release its amount concentration in the cell is higher hence detection at the onset of germination (Finnie et al., 2011).

3.4.5 Proteins present in barley with little change during germination

Four proteins were identified in this category, including a group of proteins which are very abundant in beer and hence are an important component of beer quality determination. Non- specific lipid-transfer protein 1 (NLTP1) and Probable non-specific lipid-transfer protein (LTP2) are both important proteins in lipid transfer. The LTPs are the most common plant binding proteins. They were originally identified by their ability to catalyse lipid transfer between cell membranes (Steiner et al., 2011). They have been identified as one of the major beer proteins which affect the beer foaming properties (Iimure and Sato, 2013). Foam stability in beer is intensified by an interaction between LTPs with both low and high molecular weight polypeptides such as hordeins, resultant polypeptides and serpins (Iimure and Sato, 2013)

Both NLTP1 and NLTP2 showed no significant change in abundance during germination or lack thereof.

p ro te in s p re s e n t in b a rle y w ith little c h a n g e

0 .1 5 C

e 0 .1 0

c FG n

a L S

d n

u S G

b 0 .0 5 A

0 .0 0

1 4 2 7 P Z P Z T P T S S L | L |B |N 3 |N 2 7 9 5 9 9 2 4 4 5 6 1 3 7 0 0 4 0 P 2 Q P P

Figure 3.6. Protein relative abundance change for proteins Serpin-Z4, serpin-Z7, Non- specific lipid-transfer protein 1 (NLTP1) and Probable non-specific lipid-transfer protein (LTP2). Legend: C=before imbibition, N=seeds that germinated after 24 hours imbibition, S=seeds that germinated after 72 hours imbibition, L=seeds that germinated after 24 hours imbibition but sampled after 72 hours and F= failed to germinate after 72 hours imbibition.

Serpin-Z4 showed highest abundance levels in the dormant group of seed (FG). This increase in abundance was however non-significant at p=0.05 Serpin-Z7 on the other hand remained fairly constant in abundance in the groups sampled with little apparent change over the germination process. The Serpin family of proteins has three known isoforms which include Z4, Z7 and Zx, with first two isoforms commonly abundant in both barley and beer while the third is not so common (Iimure and Sato, 2012; Steiner et al., 2011). They are a major component of the albumin in the grain and act as storage proteins during grain filling. They also act as germination inhibitors through their serine-type endopeptidase inhibitor activity. They are common beer proteins and have a suggested role in beer stability (Steiner et al., 2011).

3.5 Conclusion

The germination process in the seed is initiated by water uptake. The water as is being taken up passes through the embryo and present in the embryo id the GA hormone which will be transported along with the water to the aleurone layer. Here, the initiation of degradation of storage proteins and activation of starch degrading enzymes occur. Beta-amylases are activated and Alpha-amylases are generated. The enzymes are then transported to the endosperm where the hydrolysis of starch to provide sugars for the growing embryo begins (Lee et al., 2011).

According to (Mikola, 1981), there are three stages in reserve protein mobilisation during the germination process of barley grain; the degradation of stored proteins to supply amino acids for novel synthesis of proteins, second degradation of endosperm proteins to provide amino acids for the new embryo growth and lastly the breakdown of other proteins such as structural proteins as a backup supply once all seed reserves have been mobilised. From our study, we identified three classes of protein expression associated to three stages of plant growth; before germination, the onset of germination and the growth and elongation of the root and shoot.

Studying proteome change during germination by varying the sampling periods over the course of germination gave us a clear view of the change in abundance as germination progressed. The proteins observed before germination were found to be mainly protective proteins which protect the embryo against the harsh desiccated conditions during the dry storage phase of the seed. However, at imbibition we saw a decrease in the desiccation tolerance proteins. Our findings were similar to (Finnie and Svenson, 2009), they observed the proteins related to desiccation-, osmotic-, oxidative-, and salt stress were the first spots to disappear within 4-12 hours post imbibition. These proteins have been suggested to protect the seed during the resting/storage periods, we concluded that the proteins were being degraded since the water and salinity stress within the seed is relieved with rehydration.

The proteins observed to be highly abundant when the seed germinated included mainly those responsible for starch degradation and protein folding. This ensures the young embryo gets sufficient nutrition as it starts the growth process. The changes included an increase in starch degrading enzymes alpha- and beta-amylases. Beta-amylase showed highest abundance 24 hours post imbibition at (P≤0.05). Starch degradation is initiated by alpha-amylases which cleave the α-1, 4 glucosidic linkages. Beta amylase and limit dextrinases then break down the

47 resulting products into simpler sugars. Amylases have been identified to increase in abundance in germinating seeds (Jin et al., 2013; Finnie et al., 2002). Beta amylases increase in abundance is due to its release from its bound form. Gamma hordein 3 (HOG 3) was another protein which increased in abundance with germination. It has been suggested to not only act as a storage protein but is involved in the transport of prolamins to the developing embryo in a complex and not well understood chain (Piston et al,. 2004).

Lastly the third group of proteins which were observed to increase over time from germination and onto early seedling growth included those responsible for cell metabolism, DNA replication and other important cell growth function. The group which failed to germinate clustered with the other seeds and showed no unique protein except for the protein H2A.6 which is responsible for folding of DNA into chromatin. The long term storage, handling, dehydration and rehydration processes which leads to oxidative stress can cause damage in DNA hence loss in variability in seeds. There were no other proteins which showed high significant abundance in this group or unique clustering implying the seeds were indeed alive and had initiated the process of germination. This highlights the need for further research in dormancy to identify the proteins that may be hindering germination.

4.0 Genomic and proteomic analysis of barley seeds to better understand dormancy

4.1 Introduction Barley (Hordeum vulgare) grain that goes into the malting process needs to have rapid and uniform germination. Very high dormancy is one of the main causes for non-uniform germination during malting, and this results in a reduction of malt quality which translates to losses to the brew house. Low or lack of dormancy however is not desirable as this would cause pre-harvest sprouting, a condition whereby the seeds germinate while still attached to the plant in the field due to exposure to wet conditions (Ullrich et al., 2009). It is therefore a pre-requisite for producing quality grain for malting to find a balance between the two varying physiological states.

Dormancy has been defined as an event whereby viable seed fails to germinate under conditions which would otherwise favour germination (Finkelstein et al., 2008). Seed dormancy is critical for plant survival and crop production because it determines the crop pressure by regulating the seed germinating each season hence ensuring seed is left in the soil for the next seasons cropping (Finkelstein et al., 2008, Nakamura et al., 2016). Despite a lot of research going into dormancy (Ullrich et al., 2009; Holdsworth et al., 2007; Finkelstein et al., 2008; Potikana et al., 2002; Gubler et al., 2005), the mechanisms regulating it still remain unclear (Nakamura et al., 2016). This lack of clarity could possibly be due to dormancy being regulated by multiple factors (Finkelstein et al., 2008).

Dormancy is further complicated by some seed requirement to break it such as stratification, scarification or the need for after-ripening (Finkelstein et al 2008). Many seeds will display varying levels of dormancy with the individual being anywhere between very dormant to non- dormant. It therefore still remains unclear as to the point at which the seed turns from dormant to non-dormant (Finkelstein et al 2008). Dormancy has been found to be regulated by Quantitative Traits Loci (QTLs) located on chromosome 4H, 5H and 6H (Zhang et al, 2005; Bonnardeaux et al, 2008; Ullrich et al, 2009). The two major QTLs, SD1 and SD2, being located on chromosome 5H (Nakamura et al, 2016).

Germination on the other hand is complex and requires the matched expression of a number of genes and tissues within the seed. The different seed tissues including the embryo, aleurone, scutellum, and starchy endosperm all need synchrony both spatially and temporally (Potikana et al., 2002). Germination occurs in three phases. Phase I includes water uptake and re-initiation of metabolic processes. Phase II is also known as the ‘lag phase’, as water uptake is paused for a short while. Lastly, Phase III is marked by further water uptake and finally radicle emergence (Holdsworth et al., 2007). During Phase I and II, a seed can prolong dormancy and enter into secondary dormancy. These seeds will not enter Phase III until dormancy is relieved, a process known as after-ripening.

The processes which resume upon imbibition include, but are not limited to, respiration, metabolic activities, enzymatic activities, and RNA and protein synthesis. These, as well as other important cellular activities, are all essential for growth and development of the young seedling (Potikana et al., 2002). While a lot of research is focused on enzymes associated with mobilization of food reserves, there is little knowledge of how the embryo mobilizes its internal reserves of carbohydrates, lipids and proteins during germination (Potikana et al., 2002). There has been some reports of stored messenger RNA (mRNA) in dry desiccated seeds and also reports on the need for novel RNA synthesis and translation for germination to commence (Potikana et al., 2002; Holdsworth et al., 2008).

The aim of this chapter was to study the proteome and genome changes of germinated and non- germinated/dormant barley seeds in storage for a 10-12 month period. Germination tests were carried out every two months during the storage period in order to monitor the point at which the germination percentages of the barley varieties used in the experiment would start dropping off, as this indicated a loss of germinability. The storage conditions were regulated to emulate the conditions used for storage of commercial malting barley.

4.2 Methodology 4.2.1 Germination tests Seeds from three varieties of barley were obtained from Hermitage Research Station, Warwick, Qld. These seeds were pure seed samples of varieties Schooner, Stirling and Gairdner. The seeds acquired were a small sample hence the need for seed increase. Seed increase was done in Warwick between June and December 2017. Growing was done in single plots, the plots were each harvested separately, threshed and stored in cold seed storage facilities at Leslie Research Centre for further processes. The temperature for storage was maintained at 210C which is the optimal storage temperature.

Seed surface sterilisation was completed before the germination tests were conducted. For seed sterilisation, seeds were soaked in 70% ethanol for 1 min with constant swirling. The ethanol was then discarded and immediately replaced with 1% NaOCl for another minute with constant swirling. The seeds were then rinsed off with double distilled water and patted dry. Counts of 100 seeds were placed in each of three x 9 mm petri dishes lined with two Whatman #1 filter papers. To each petri dish, 4 ml double distilled water was added and the dishes covered and set aside for three days in the dark, for germination at 22 ℃.

Germination was undertaken in three replicates for each variety tested, and this process was repeated every two months over a year. The whole experiment resulted in a test of 100 seeds in 3 petri dishes (3 replicates by 3 varieties) at each of 6 times of testing, resulting in germination counts on 54 petri dishes. Refer to the seed sampling schedule in Table 4.1. Samples taken included 2 germinated seeds and 2 non-germinated/dormant seeds at day three from each petri dish. The seed samples were then kept frozen at -80 0C until further processing.

Table 4.1. Grain sampling schedule over a period of 12 months and the germination percentages of barley varieties Schooner, Gairdner and Stirling. N.D. denoting T0 where the results are missing

SCHOONER GAIRDNER STIRLING

R1 R2 R3 R1 R2 R3 R1 R2 R3 T0 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. T1 53 60 56 66 53 46 4 6 4 (2MNTHS) T2 97 94 95 98 98 98 95 96 98 (4MNTHS) T3 93 91 96 99 98 100 97 98 99 (6MNTHS) T4 88 82 90 92 97 93 100 99 99 (8MNTHS) T5 86 87 90 93 93 95 100 99 95 (10MNTHS) T6 88 82 83 94 90 93 86 100 98 (12MNTHS)

4.2.2 Combined extraction methods of RNA and protein in germinated and non- germinated barley seeds Seed pulverisation was carried out using lysing matrix A, MP biomedicals. Single seeds were added to the tubes, then tubes placed in liquid nitrogen for 5-10 mins and transferred to pre- cooled TissueLyser adapter set 2 *24. This was then firmly placed on the Qiagen TissueLyser 85300 and samples ground for 2 min at 30 Hz with rotation after a minute. To each tube, 1 ml TRIzol was added and samples incubated at room temperature for 5 min. Subsequently, 0.2 ml chloroform was added and the samples mixed by up and downward suction using a micropipette. Samples were incubated at room temperature for 5 min, then centrifuged at 12,000 *g for 15 min at 4 0C. Phase separation is seen at this stage, with the top phase containing the RNA and DNA, and the mid and organic phase containing both protein and DNA. The top phase was transferred to a new RNAse free tube for further processing while the mid and lower phase go into protein processing.

RNA extraction was done using Zymo-research Direct-Zol RNA MiniPrep plus with slight modification on the protocol. To the upper phase, equal volumes of 100% ethanol was added and the sample mixed thoroughly using a vortex. Samples were then transferred to Zymo- spin™ IIC column inserted in a collection tube and then centrifuged at 16,000 *g for 30 sec. The flow through was then discarded. For DNA removal, 400 µl RNA wash buffer was added to the column, then centrifuge at 16, 000 *g for 30 sec then a mix of 5 µl DNAse I + 75 µl DNA digestion buffer added, samples were then incubated at room temperature for 15 min. 400 µl of Direct-zol RNA pre-wash buffer added to the column and centrifuge at 16, 000*g for 30 sec, the flow through was discarded and this pre-wash step repeated. 700 µl RNA Wash buffer was then added to the column then samples centrifuged at 16,000 *g for 2 min, the flow through was then discarded. A second spin was done on the empty column at 16,000*g for 30 sec to ensure all the wash buffer was discarded. The column was transferred to an RNAse free microfuge tube and 50 µl of DNAse and RNAse free water added directly to the column matrix. The samples were centrifuged at 16,000*g for 30 sec. RNA quality and quantity checks were carried out using Qubit 3.0 Fluorometer and bioanalyzer (Agilent). The RNA was immediately stored in -80 0C until further processing. Protein extraction was undertaken following the TRIzol protein extraction manufacturer’s instructions with modifications (Brockman et al., 2018). To the mid and lower phases, 750 µl of isopropanol was added and mixed by gentle inversion. This was followed by incubation at room temperature for 3 mins, then samples were centrifuged at 7,500 x g for 5 min at 4˚C. 95% ethanol was added to wash the samples and they were incubated for 5 minutes. Samples were then centrifuged at 7,500 x g for 5 min at 4 ˚C and the supernatant removed. This step was repeated three times. 10 µl 100% Ethanol was added to proteins pellet and incubated at -20 ˚C for 16 hrs. 500 µl 6M guanidine-HCl 50 mM Tris HCl buffer pH 8, was added then incubated for 20 min. Then DTT was added to a final concentration of 10 mM, incubated for 30 min, then added acrylamide to final concentration of 25 mM, incubated 30 min, and finally added DTT to final concentration 10mM and incubated for 30min. Proteins were then precipitated with 1 ml 1:1 methanol/acetone by inverting gently 15 times and incubated at -20 ˚C for 16 hours and then suspended in 100 µl of 50 mM Tris-HCl (pH 8) with 1 µg of trypsin and incubated for 16 hrs at 37°C. Tryptic-digested peptides were cleaned with C18 ZipTips (Millipore, Burlington, MA). The samples were stored at -20˚C until further processing. Following this result, a decision was made to observe the proteomic profile of Schooner noting the decline in germination percentage. Protein and RNA was extracted from Schooner samples.

RNA samples were stored at the University of Southern Queensland lab freezer whereas the protein samples were fragmented, cleaned and sent for further LC-MS/MS processing. Following the RNA extractions, quality checks were done using the 2100 Bioanalyzer, the quality (RIN) was found to be low hence it was not possible to advance the samples further. From the analysis, 278 proteins were identified from the 6 sampling periods, that is, samples taken immediately after harvest and every two months thereafter for 10 months storage. The samples included two replicates of germinated seed and dormant seed after three days of standard germination. MSstats (Zacchi and Schulz 2016) was done to identify proteins which are highly abundant at selected storage periods. This included comparison between; Dormant and germinated samples after harvest, dormant and germinated samples after 10 months storage, dormant samples at harvest and after 10 months storage. Proteins showing significant Log2FC >1 and a p value <0.005 were selected for every category compared using MSstats. The ID mapping, functions and sequence information retrieval was done from an online database Uniprot. Gene ontologies (GO) were then generated using a web tool EggNOG and run on another web tool, cateGOriser in order to classify the protein according to their function groups (Figure 4.2) Over 20% of the GOs were classified under biological process whereas the functional group with the lowest GO (<1%) was cytoskeleton function. 41 GOs were classified under dormancy initiation and maintenance (appendix 4.1).

4.3 Results Barley grain going into malt production is purchased after harvest and stored in optimum conditions over 12 months duration during which grains are used in batches for malting. The grain ideal for malting is dormant at harvest, and then breaks dormancy uniformly, with a germination percentage of 98% or higher. To monitor germination and track the breakdown of dormancy in different varieties, the change in barley seed germination percentage was measured over a twelve month time period. Seed sampling was done every two months with seeds germinated over three days. The seeds were then re-sampled in replicates for both dormant and germinated seeds on day three. These seeds were immediately transferred to - 800C for long term storage until further processing.

The germination percentage was low after harvest. This percentage increased to 95% and above for all the varieties after 4 months storage, with Gairdner reaching 98% for all the replicates. At 6 months storage, Gairdner showed the highest germination percentage of 100% followed by Stirling. Schooner on the other hand had started declining in germination percentage by month 4, Schooner maintained the slow decline of germination percentage with increased storage. At 12 months the lowest germination percentage recorded was from Schooner at 82%. The other two varieties showed small decline in germination percentage (Table 4.1; Figure 4.1). The extracted RNA showed low quality. No matter which method used or how the protocol was manipulated, the quality of RNA was not improved to the quality standard needed for sequencing. The extraction results from bioanalyzer (Agilent) are as shown in appendix 4.2- 4.4. Due to the quality issues experienced, we did not proceed with RNA work. The results showed are from protein analysis.

120

100

40 Germination percentage

0 T0 T1 (2Mnths) T2 (4Mnths) T3 (6Mnths) T4 (8Mnths) T5 (10Mnths) T6 (12Mnths) Time (MNTHS)

ScR1 ScR2 ScR3 GrR1 GrR2 GrR3 StR1 StR2 StR3

Figure 4.1. Germination percentage of barely varieties Gairdner (Gr), schooner (Sc) and Stirling (St) sampled over twelve months. R1, R2 and R3 representing replicate 1 to 3 for each variety. Line indicating T1 where observations began.

Go terms classification

generation of precursor metabolites and energy cell communication post-embryonic development response to external stimulus reproduction lipid metabolic process transferase activity response to abiotic stimulus hydrolase activity protein metabolic process carbohydrate metabolic process response to stress multicellular organismal development membrane transport catabolic process cellular component organization and biogenesis binding cytoplasm nucleobase, nucleoside, nucleotide and nucleic acid… biosynthetic process intracellular catalytic activity cell molecular_function metabolic process 0.00% 2.00% 4.00% 6.00% 8.00% 10.00% 12.00%

Figure 4.2. Classification of gene ontologies (GOs) according to function associated with it.

4.3.1 Protein comparison between germinated versus dormant grain at harvest

Sixteen proteins showed significant changes in abundance between the germinated and dormant seed sampled immediately after harvest (Figure 4.3). These proteins classified according to function fell into the following broad categories; plant defence, desiccation tolerance which included late embryogenesis abundant (LEA) proteins B19.1A, ABA- inducible protein PHV A1, LEA protein D-34, dehydrin DHN3, and LEA protein 1, protein related to binding activities of both metal and ion, proteins involved in translation, proteins showing inhibition activities which included alpha-amylase inhibitor (BDAI-1) and Bowman- Birk type trypsin inhibitor, and grain structure including Hordoindoline-A

Table 4.2. Proteins with significantly different abundance and large fold change (log2FC) in germinated versus dormant samples at harvest (G0vD0) P≤0.05

Protein Protein names log2FC P31110 Thaumatin-like protein -2.637 Q05190 Late embryogenesis abundant protein B19.1A -1.914 (B19.1) P14928 ABA-inducible protein PHV A1 -1.680 Q9M4E3 Hordoindoline-A -1.560 P09444 Late embryogenesis abundant protein D-34 -1.501 (LEA D-34) Q10464 Puroindoline-B -1.399 P13691 Alpha-amylase inhibitor BDAI-1 -1.221 P12948 Dehydrin DHN3 (B17) -1.134 Q96564 40S ribosomal protein S27 (Manganese -1.115 efficiency-related protein 1) A2XG55 Late embryogenesis abundant protein 1 -1.086 Q9M5M1 40S ribosomal protein S11 -1.070 P12940 Bowman-Birk type trypsin inhibitor -1.043 Q9ZRA9 Tubulin beta-4 chain (Beta-4-tubulin) 1.084 O04138 Chitinase 4 (EC 3.2.1.14) (OsChia2b) 1.225 (Pathogenesis related (PR)-3 chitinase 4) P25250 Cysteine proteinase EP-B 2 (EC 3.4.22.-) 1.339 Q339K6 Laccase-15 (EC 1.10.3.2) (Benzenediol:oxygen 1.427 oxidoreductase 15) (Diphenol oxidase 15) (Urishiol oxidase 15)

The proteins associated with desiccation tolerance were significantly higher (P≤0.05) in relative abundance amongst the dormant samples than in the germinated samples at harvest. These included four late embryogenesis abundant (LEA) proteins LEA D-34, LEA B19, LEA 1 and ABA-inducible protein PHV A1 and dehydrin. The LEA proteins showed increases and decreases in abundance during storage, the highest relative abundance recorded being after 6 months in storage and then they start to decrease steadily thereafter. Protein dehydrin (DHN3) on the other hand showed high relative abundance at harvest in dormant samples then reduces to near zero but steadily peaks at six months storage and finally starts on a steady decline (Figure 4.4).

Desiccation tolerance is an important trait in plants which confers its survival during the dry seed storage period where moisture content often drops to below 50%. Additionally, it is associated with increased salt accumulation within the seed, a condition which would otherwise kill the young embryo (Bartels., et al 1988, Irar et al., 2006). Several proteins have been associated with desiccation tolerance including the LEA proteins and dehydrin amongst others (Bonsager et al., 2003).

Dehydrins are a sub-family of group 2 LEA proteins which are expressed in desiccated conditions in seeds. These are associated with low moisture content and increased salinity (Calestani et al., 2015). Dehydrins were detected in an experiment by (Calestani et al., 2015) which was aimed at enhancing the germination of wild type Arabidopsis seeds by the incorporation of barley dehydrin gene aba2. The transgenic lines were able to show improved resistance to salinity and reduced moisture content and increase their germination percentage by 90-100%. The wild type counterparts’ seeds however gave low germination percentages of 15-45% when exposed to the same conditions. These desiccation tolerance proteins (LEA and Dehydrins) were among the proteins showing significant changes in abundance in previous chapter 3.

Late embryogenesis abundant protein D-34 Relative Relative abundance

0mnths 2mnths 4mnths 6mnths 8mnths 10mnths Time (mnths)

Germinated samples (G) Dormant samples (D)

ABA-inducible protein PHV A1 (LEA1) 0.012 0.01 0.008 0.006 0.004 0.002 0 Relative Relative abundance 0mnths 2mnths 4mnths 6mnths 8mnths 10mnths -0.002 -0.004 Time (mnths)

Germinated samples (G) Dormant samples (D)

Late embryogenesis abundant protein 1 0.006 0.005 0.004 0.003 0.002 0.001

Relative Relative abundance 0 0mnths 2mnths 4mnths 6mnths 8mnths 10mnths -0.001 Time (mnths)

Germinated samples (G) Dormant samples (D)

Figure 4.3. Protein abundance change over 10 months storage for LEA proteins; LEA protein D-34, ABA-inducible protein PHV A1, and LEA protein 1 sampled from Schooner.

Dehydrin DHN3 0.009 0.008 0.007 0.006 0.005 0.004 0.003

0.002 Relative Relative abundance 0.001 0 0mnths 2mnths 4mnths 6mnths 8mnths 10mnths -0.001 Time (mnths)

Germinated samples (G) Dormant samples (D)

Figure 4.4. Protein abundance change over 10 months storage for the protein dehydrin DHN3 sampled from Schooner.

4.3.2 Comparison of protein abundance between germinated and dormant grain after 10 months storage

Thirty proteins were included in the comparison category showing >1 Log2FC and lowest significant probability values. Of these, 16 showed negative Log2FC while 14 showed positive Log2FC. The largest negative fold change (G5vD5) was evident in the protein hordoindoline- B2, followed by histone H1. The highest positive fold change was from 60s ribosomal protein L18a (Table 4.3). Protein histone H1 is shown to be high in dormant samples at 10 months storage period. This is one of the proteins involved in folding DNA into higher structure such as chromosome (Marian et al., 2003). In the previous chapter (chapter 3) histone protein H2A was found to be significantly higher in dormant samples than all the other tested grain. Of particular interest was the proteases and protease inhibitors. These included serpin Z7 and serpin Z2a, serine carboxypeptidase and aspartic proteinase which will now be discussed in more detail.

Table 4.3. Proteins with significantly different abundance and large fold change (log2FC) in germinated and dormant grain after 10 months storage (G5vD5), P≤0.05.

Protein Protein names Protein function log2FC Q9LEH8 Hordoindoline-B2 (Puroindoline-B) defence/grain -3.116 texture/hardness P27806 Histone H1 Binding (DNA) -2.653 G1JSL4 Peroxygenase 1 (AsPXG1) (EC Binding (ions)/catalyst -2.247 1.11.2.3) Q9ST57 Serpin-Z2A (TriaeZ2a) (WSZ2a) Inhibitor -2.232 P32936 Alpha-amylase/trypsin inhibitor Inhibitor -2.224 CMb (Chloroform/methanol-soluble protein CMb) P08818 Serine carboxypeptidase 2 (EC Storage protein breakdown -1.993 3.4.16.6) (CP-MII P07596 Alpha-amylase/subtilisin inhibitor Inhibitor -1.700 (BASI) P01545 Alpha-hordothionin (Purothionin II) Defense -1.519 [Cleaved into: Alpha-hordothionin; Acidic protein] P13691 Alpha-amylase inhibitor BDAI-1 Inhibitor -1.513 C5X2M4 Thiamine thiazole synthase 2, Biosynthesis (thiamine) -1.487 chloroplastic (EC 2.4.2.60) (Thiazole biosynthetic enzyme 2) Q08062 Malate dehydrogenase, cytoplasmic Catalyst (central metabolism -1.471 (EC 1.1.1.37) and redox homeostasis) P09842 Granule-bound starch synthase 1, amylose synthesis -1.414 chloroplastic/amyloplastic (EC 2.4.1.242) (Granule-bound starch synthase I) (GBSS-I) Q42456 Aspartic proteinase oryzasin-1 (EC Storage protein breakdown -1.331 3.4.23.-) Q948T6 Lactoylglutathione lyase (EC 4.4.1.5 catalyst/ stress -1.143 response/detoxifiation Q43492 Serpin-Z7 (BSZ7) (HorvuZ7) Inhibition -1.042 P08488 Glutenin, high molecular weight Storage protein -1.003 subunit 12 P23345 Superoxide dismutase [Cu-Zn] 4A Destroys radicals which are 1.025 (EC 1.15.1.1) normally produced within the cells and which are toxic to biological systems

P22953 Probable mediator of RNA RNA mediator/defense 1.035 polymerase II transcription subunit response/chaperone 37e (Protein EARLY-RESPONSIVE TO DEHYDRATION 2) Q9SNX2 Phosphoglucomutase, cytoplasmic synthesis and breakdown of 1.044 (PGM) (EC 5.4.2.2) (Glucose glucose phosphomutase) Q8SVY9 Uncharacterized protein 1.113 ECU03_1610 Q01899 Heat shock 70 kDa protein, Binding 1.196 mitochondrial Q9FLF0 40S ribosomal protein S9-2 Binding (RNA) 1.318 P33679 Zeamatin Defense (Anti-fungal) 1.412 P41098 60S ribosomal protein L34 translation, ribosomal 1.512 constituent P81370 Thaumatin-like protein Defense (Anti-fungal) 1.568 P07597 Non-specific lipid-transfer protein 1 Lipid transport/ wax 1.730 (LTP 1) (Probable amylase/protease deposition inhibitor) P46259 Tubulin alpha-1 chain Binding (Ion)/ microtubule 1.735 constituent Q40082 Xylose isomerase (EC 5.3.1.5) Catalyst 1.756 Q7FAX1 Peroxygenase (EC 1.11.2.3) storage lipid 2.018 degradation/catalyst/defense response Q943F3 60S ribosomal protein L18a translation, ribosomal 3.294 constituent

At 10 months storage, the relative abundance of serpins Z7 and serpin Z2a was significantly higher in dormant samples than in germinated samples. Serpin Z7 and Z2a were both detected throughout the storage period. Serpin Z2a showed higher abundance in non-germinated (dormant) grain and reduced to almost zero after 10 months storage. Serpin Z7 on the other hand was high in germinated samples at harvest, then increased with storage to a peak at 4 months storage. It then decreased to levels below the non-germinated samples. Serine protease inhibitors (Serpins), also known as protein Z, have been identified as inhibitors of chymotrypsin-like enzymes, and have been suggested to inhibit cysteine proteinases (Ostergaard et al 2000). Its isoforms have been implicated for beer foam stabilization due to its high surface viscosity and elasticity (Iimure et al 2013). There are three isoforms in barley; Zx, Z7 and Z4, with the latter two being commonly identified in both barley grain, malt and beer. Serpin is one of the proteins which is identified for increasing during germination due to being release from its bound form by proteolysis (Ostergaard et al 2004).

Serpin-Z2A 0.0025 0.002 0.0015 0.001 0.0005

0 Relative Relative abundance -0.0005 0mnths 2mnths 4mnths 6mnths 8mnths 10mnths Time (mnths)

Germinated samples (G) Dormant samples (D)

Serpin-Z7 (BSZ7) 0.025 0.02 0.015 0.01 0.005

0 Relative Relative abundance -0.005 0mnths 2mnths 4mnths 6mnths 8mnths 10mnths Time (mnths)

Germinated grain (G) Non-germinated grain (D)

Figure 4.5. Change in protein abundance over 10 months storage period in dormant and germinated grain for proteins serpin Z2A and Z7 sampled from barley variety Schooner.

Proteinases are another important category of proteins. In this experiment, 2 proteinases were identified which showed significant log2FC among the G5vD5 group. These were aspartic proteinase and serine carboxypeptidase. During the 10 months storage, serine carboxypeptidase showed an increase in abundance in non-germinated grain while it showed a steady decrease in germinated grain. In aspartic proteinase however, there is little difference in the trends between the germinated and the non-germinated grain (Figure 4.6).

Aspartic proteinase oryzasin-1 0.015

0.01

0.005

0mnths 2mnths 4mnths 6mnths 8mnths 10mnths Relative Relative abundance Time (mnths)

Germinated samples (G) Dormant samples (D)

Serine carboxypeptidase 2 0.015

0.01

0.005

Relative Relative abundance 0mnths 2mnths 4mnths 6mnths 8mnths 10mnths -0.005 Time (mnths)

Germinated samples (G) Dormant samples (D)

Figure 4.6. Change in protein abundance over 10 months storage period in dormant and germinated grain for the proteinases aspartic and serine carboxypeptidase 2 sampled from Schooner.

4.3.3 Comparison of protein abundance between dormant grains at harvest versus dormant grains after 10 months storage (D5/D0)

When comparing proteins at harvest with proteins after 10 months of storage in dormant samples, 30 proteins were found to be significantly abundant at harvest, while 10 were found to be abundant after 10 months storage (Table 4.4). At harvest, protein showing the highest negative log2FC (D5vD0) value was 60s ribosomal protein L18a followed by late embryogenesis abundant protein B19.1A, with values of of -2.559 and -2.422 respectively. Other important storage proteins in this category included avenin-like a2, protein inhibitors such as alpha-amylase inhibitor BMA1, heat shock protein 1, and protein disulphide isomerase.

Table 4.4. Proteins with significantly different abundance and large fold change (log2FC) in dormant grains at harvest versus dormant grain after 10 months storage (D0vD5) P≤0.05.

Protein Id Protein name log2FC Q943F3 60S ribosomal protein L18a -2.559 Q05190 Late embryogenesis abundant protein B19.1A (B19.1) -2.422 Q9M4E3 Hordoindoline-A -2.412 P0CZ07 Avenin-like a2 -2.301 Q7X8H9 17kDa alpha-amylase/trypsin inhibitor 2 (allergen Ory s -2.150 17kD) P16968 Alpha-amylase inhibitor BMAI-1 (Alpha-amylase flour -2.083 inhibitor) (allergen Hor v 1) (Fragment) P01086 Trypsin inhibitor CMe (Alpha-amylase/trypsin inhibitor) -1.899 (BTI-CMe1) (BTI-CMe2.1) (BTI-CMe3.1) (Chloroform/methanol-soluble protein CMe) P12810 16.9 kDa class I heat shock protein 1 (HSP 16.9) (Heat -1.832 shock protein 16.9A) (Heat shock protein 17) (Low molecular weight heat shock protein) Q9FLF0 40S ribosomal protein S9-2 -1.540 P33679 Zeamatin -1.526 P11955 26 kDa endochitinase 1 (EC 3.2.1.14) -1.495 P34788 40S ribosomal protein S18 -1.452 Q10464 Puroindoline-B -1.432 P41098 60S ribosomal protein L34 -1.420 P13691 Alpha-amylase inhibitor BDAI-1 -1.409 P10387 Glutenin, high molecular weight subunit DY10 -1.409 P84516 Cationic peroxidase SPC4 (EC 1.11.1.7) -1.373 P49397 40S ribosomal protein S3a (CYC07 protein) -1.330 P26517 Glyceraldehyde-3-phosphate dehydrogenase 1, cytosolic -1.325 (EC 1.2.1.12) Q9ZUT9 40S ribosomal protein S5-1 (AtRPS5B) -1.314

Q7GCM7 Xylanase inhibitor protein 1 (XIP-I) (Class III chitinase -1.274 homolog a) (RIXI protein) P21569 Peptidyl-prolyl cis-trans isomerase (PPIase) (EC 5.2.1.8) -1.238 (Cyclophilin) (Cyclosporin A-binding protein) (Rotamase) Q9SCM3 40S ribosomal protein S2-4 -1.221 P12949 Dehydrin DHN4 (B18) -1.212 P09444 Late embryogenesis abundant protein D-34 (LEA D-34) -1.209 P31110 Thaumatin-like protein -1.206 A3BMZ5 Beta-glucosidase 26 (Os7bglu26) (EC 3.2.1.21) -1.165 P51414 60S ribosomal protein L26-1 -1.141 Q9M5Z9 40S ribosomal protein S23 -1.065 P80284 Protein disulfide-isomerase (PDI) (EC 5.3.4.1) (Endosperm -1.055 protein E-1) P01545 Alpha-hordothionin (Purothionin II) [Cleaved into: Alpha- -1.026 hordothionin; Acidic protein] F4IQK5 Vicilin-like seed storage protein At2g18540 (Globulin 1.005 At2g18540) B8AL97 Cupincin (EC 3.4.-.-) (52 kDa globulin-like protein) 1.097 (allergen Ory s NRA) O06005 Amino-acid permease AapA 1.182 P35793 Pathogenesis-related protein PRB1-3 (HV-8) (PR-1B) 1.224 Q8LB47 GrpE protein homolog 2, mitochondrial 1.361 Q08277 Heat shock protein 82 1.411 P08818 Serine carboxypeptidase 2 (EC 3.4.16.6) 1.422 Q9ZRA9 Tubulin beta-4 chain (Beta-4-tubulin) 1.470 Q07078 Heat shock protein 81-3 (HSP81-3) (Gravity-specific 1.528 protein GSC 381) P25250 Cysteine proteinase EP-B 2 (EC 3.4.22.-) 1.959

After 10 months storage, there were 10 proteins showing high positive log2FC in dormant grains as shown in table 4.4 above. The highest of these was cysteine proteinase (EP.B 2) and HSP 81-3 with values of 1.959 and 1.528 respectively. Others included serine carboxypeptidase 2 and pathogenesis related protein. Of interest was the desiccation tolerance and storage proteins. Late embryogenesis protein D-34 showed a decrease with storage in both dormant and germinated samples as shown in figure 4.6 below. This could be attributed to seed maturation in readiness to germinate. LEA proteins have been found to be high abundant as the seed enters the dry desiccated period. They have been identified to protect the seed and hence the embryo from the dry, moisture deficient and high salt concentration conditions hence ensuring survival (Bonsager et al., 2003).

Late embryogenesis abundant protein D-34 0.01

0.005

0 0mnths 2mnths 4mnths 6mnths 8mnths 10mnths

Time (mnths) Relative Relative abundance

Germinated seeds (G) Failed to germinate (F)

Figure 4.7. Relative protein abundance change over 10 months storage for the protein Late embryogenesis abundant protein D-34 in dormant and germinated samples which were sampled from Schooner.

Proteins avenin-like a2 and glutenin, high molecular weight subunit DY10 showed an increase in abundance trend with increase storage period in dormant grain (Figure 4.8). Both avenin and glutenin have been classified under prolamins which are storage proteins specialised in developing endosperms, (Shewry et al., 1984). Prolamins are composed mainly of prolines and glutamines. In wheat, high molecular weight glutenins and low molecular weight gliadins form gluten. It is readily soluble in alcohols including 60-70% ethanol, propan-1-ol and propan-2-ol at elevated temperatures (Shewry et al., 1984). Glutenins are an important proteins determining the quality of bread in wheat. It affects both elasticity and viscosity and these in turn reflect on the bread volume (Barak et al., 2014).

Avenin-like a2 0.0005 0.0004 0.0003 0.0002 0.0001

Relative Relative abundance 0 0mnths 2mnths 4mnths 6mnths 8mnths 10mnths Time (mnths)

Germinated seeds (G) Failed to germinate (F)

Glutenin, high molecular weight subunit DY10 0.006 0.004 0.002 0 0mnths 2mnths 4mnths 6mnths 8mnths 10mnths

Relative Relative abundance Time (mnths)

Germinated seeds (G) Failed to germinate (F)

The relative abundance of protein disulphide isomerase showed an increase-decrease trend through the storage period. In dormant samples, there is a significant decrease with the 10 month storage as shown in figure 4.8 below. PDI is an enzyme and has been implicated in protein folding in the endoplasmic reticulum and hence protein deposition (Johnson and Bhave 2004).

Protein disulfide-isomerase 0.035 0.03 0.025 0.02 0.015 0.01

0.005 Relative Relative abundance 0 0mnths 2mnths 4mnths 6mnths 8mnths 10mnths Time (mnths)

Germinated seeds (G) Failed to germinate (F)

Figure 4.9. Relative protein abundance change over 10 months storage for the protein disulphide isomerase, in dormant and germinated samples which were sampled from Schooner.

4.4 Conclusion From this study we have noted the following; there is a decrease in germination percentage with increased storage. The samples had a low germination percentages immediately after harvest, this increased and peaked at 4-6months storage period. The percentage then started on a slow decline.

The data has indicated that there is a change in protein composition as well as abundance during storage of barley seeds over a period of 10 months. This is evident in the proteins which showed significant changes over time. Some proteins seem to increase significantly with storage while others seem to decrease significantly over time. There were similarities in proteins identified from this study to proteins identified in chapter 3. These included protein groups such as, desiccation tolerance proteins, proteins involved in starch metabolism, storage proteins, serpins, LTPs and PDI. Unique proteins which showed significant change in abundance in this group which were not identified in the previous study included proteases and ribosomal proteins.

There was significant variation in relative protein abundance between the samples which germinated and those which failed to germinate. The dormant samples were however not lacking in proteins identified in germinating samples, an indication of them being alive. This can suggest that these samples are metabolising and performing all function related to live seeds but only lacking in particular aspect which hinders its germination. Maltsters have over time developed the art of predicting how and when barley grain germinates and have optimised the entire malting process (Daneri-Castro et al., 2016). But even with highly optimised methods, inaccuracies in predictions could potentially lead to heavy losses. Therefore we ask ourselves whether the development of a protein biomarker is feasible in malting barley. Our study’s goal was to identify a biomarker which can indicate the breaking point of dormancy in barley seed. A biomarker is a measurable indicator of a specific biological state. Examples include protein/blood presence in urine as an indicator for disease, high levels of blood protein e.g. troponin, c-reactive associated with the risk of stroke or heart disease (Baker, 2005). The process of biomarker discovery and use involves a comprehensive pipeline including the discovery of the protein, then qualification, followed by the verification and conducting research assays, then optimisation of the protein and lastly its validation (Rifai et al., 2006).

Our study was in the first step which was the identification of proteins signalling the break of dormancy. Candidate proteins from our study included LEA protein PER1, protein H2A.6 from chapter 3 and Serpins. These proteins have been identified to be involved in dormancy promotion and/or germination inhibition from previous studies. However, we were not able to come to conclusive results. The limitations to this study included not being able to fully accomplish the initial goal of the study which was to study both RNA and protein expression in barley over time during storage. The RNA extraction protocol was not optimisable to provide us with high quality RNA. We were however able to come up with a protocol for extraction of both RNA and protein from a single seed, the RNA quality can allow for lower technical research such as qPCR.

CHAPTER 5. Protein expression comparison of malt from different malting processes

Abstract Industrial malting relies heavily on rapid and uniform germination to yield maximum quantities of malt. Malting plants must optimise protocols for different barley cultivars, to attain the desired germination rate of 98% or higher, and so optimise malt yield and quality. This results in different malt quality which affects aspects of the end product, such as fermentation and flavour. Hence emphasis on improving the quality of malt, is just as desirable as malt yield. There was evidence to show that variations in malt quality may be due to the proteins that are expressed during various stages of the malting process, hence the use of proteomics in investigating the protein profiles of malting grains. This experiment compared the protein profiles of Buloke and Gairdner cultivars at six stages of the malting process. Samples were collected before steeping, and then on days one, two and three of germination, and after 12 hrs and 24 hrs of kilning. The samples were taken from two malting plants, which used different malting protocols. Samples were analysed using Sequential Window Acquisition of All Theoretical Mass Spectrometry (SWATH-MS) which showed protein profiles differed during the malting process.

5.1 Introduction Malt is the main raw material for beer and whisky production, and the malt which is most commonly used is barley malt (Lastovickova and Bobolava, 2012). Annually, about 22 million tons of barley malt is produced globally, with the European Union being the highest producers (Lastovickova and Bobolava, 2012). Malting is the germination of non-dormant barley seeds under controlled environment with the aim of maximising endosperm degradation but minimising radicle and rootlet growth. The process involves synthesizing hydrolytic enzymes which aid in the breakdown of starchy endosperm, cell wall and proteins therefore making the grain friable and releasing the starch granules from their embedded endosperm matrix.

Malting conditions are highly controlled to ensure uniform germination with high germination percentages, as ungerminated seedlings in the malt batch lead to reduced malt extract. The malting conditions ensure minimised rootlet growth while constant mixing during growth reducing rootlet matting which would pose a problem with infiltration during brewing (Briggs, 1998; Gupta et al., 2010). There are three stages in malting; steeping, germination or seedling growth and lastly, kilning. Steeping involves mixing the desiccated seed with water to allow for water uptake. This is done for a day during which air breaks are introduced so that the seeds do not die (Briggs 1998; Gupta et al., 2010). The moisture content is increased from 14% to 45% at the end of the steep period. The seeds are then transferred to the germination tanks to allow for controlled germination. Germination involves embryo growth, enzyme activation and some starch degradation. This is achieved in three to four days, depending on the malting

76 conditions of the malt plant. The grain is then moved to Kiln tanks where hot air is used to dry the seeds, thereby stopping the growth process and hence ensuring a stable product (Bonsager et al., 2007, Gupta et al., 2010).

Protein and starch content are important factors for malting grain and need to be balanced. A higher protein content decreases the grain starch content which in turn negatively impacts the brewing process (Fox et al., 2002). The total starch contained in barley grain ranges from 65% to 68% whereas the total protein content ranges from 8% to 15% (Gupta et al., 2010). The protein contained in barley grain is broken down to amino acids and peptides which affect the quality of malt. This in turn affects the quality of wort and hence beer (Steiner et al., 2011). Extensive research has been done on barley grain protein as whole seed, and on partitions into individual parts including the aleurone layer, endosperm and embryo (Finnie and Svensson, 2003). The degradation of proteins is done by peptidases and around 40 endopeptidases have been identified in malt. These have been classified into the following groups; metallo-, cysteine-, aspartic- and serine proteases (Jones, 2005) and exopeptidases including carboxypeptidases and aminopeptidases (Gupta et al., 2010)

The grain protein can be partitioned into albumins, prolamins, glutelins and globulins (Osborne, 2004). Proteins in barley malt contribute to the beer proteins, and these determine the beer quality aspects such as flavour, the mouth feel, colour, which is also described as the hazing property, the foaming properties and colloidal stability. Three aspects which have been studied widely include beer foam, and taste properties (Osman et al., 2003; Iimure and Sato, 2012; Bamforth, 1985; Evans et al., 1998; Evans and Hejgaards, 1998, Steiner et al., 2011).

Foam is an important property in beer and its creaminess, quantity, stability, density, adhesion and strength are all very important factors used by consumers to judge the quality of beer (Evans et al., 1998; Bamforth, 1985). Foam properties are determined by proteins found in beer, including water soluble proteins. These proteins resist the proteolysis and the brewing process heat. Therefore 20% of the total barley proteins ending up in beer belong to this group. This percentage increases to 40% during malting as a result of the breakdown of hordein, thereby releasing the latent proteins (Osman et al., 2003). Foam positive proteins include high molecular weight proteins which enhance foam stability such as serpins, LTP and hordein fragments, (Evans et al., 1998; Osman et al., 2003).

Beer haze is a negative quality aspect in beer during storage and might indicate spoilage or contamination. Haze results from interactions between polyphenols and proline residues during storage. Proteins such as hordein particles or segments are high in proline and glutamine and have been implicated in the hazing property, (Evans et al., 2003, Iimure et al., 2008). Types of haze include chill haze and permanent haze, which form during storage, and particulate haze, which forms as a result of shaking during transportation of beer. Haze formation is minimized by addition of silica papain or tannic acid which removes the haze active protein during brewing and also by minimizing oxygen exposure during the brewing process. Polyvinylpyrrolidone (PVPP) is also added to minimize the level of polyphenols (Evans et al., 2003).

In this chapter, we focus on proteins found in malt which affect beer properties and particularly look at the change in the proteins during the malting process.

5.2 Materials and methods 5.2.1 Plant material sampling and sample collection The malt samples were collected from two commercial scale, modern pneumatic Australian malting plants; plant 1 (JP) and plant 2 (BP). Both malting plants use the typical Australian malting program of 24 hrs steeping, 88 hrs germination and 24 hrs kilning to temperatures of 800C. The difference in design of the malting plant is that JP is a conventional malting plant whereas BP is an older malt house with dual steep and kiln steps. Samples were collected every twelve hours from barley grains to the last step, which is kilned malt. After sample collection, the samples were kept at -20 0C then freeze dried to reduce the moisture to below 10%. They were then bagged in zip lock bags until further processing. The variety Buloke was used. The sampling schedule followed is as documented in Table 5.1 below. Samples were collected in replicates then packed and sent to The University of Queensland for further analysis.

5.2.2 Protein isolation, LC-MS/MS and MS data analysis Once received at the laboratory, an incomplete block design was prepared for the protein extraction and purification processes with 2 replicate samples prepared for the MS process. This experimental design resulted in a total of 88 malt samples arising from a factorial combination of 2 MS replicates by 2 malt plants (MP) by 2 MP replicates by malt stages (10 for MP 1 and 12 for MP 2). A single seed was selected randomly from each sample batch and ground to powder using pestle and mortar. This was added to 1 ml extraction buffer (50mM Tris hydrochloride pH 8, 6 M guanidine hydrochloride), mixed properly then 10uL dithiothreitol (DTT) added and incubated for 30 min with constant shaking. Cysteine alkylation was done by addition of 25mM acrylamide followed by a 30 minute incubation. DTT was then added to a final concentration of 10 mM then samples centrifuged at 18000 g for 5 min. 10uL of the supernatant was added to 100 uL 1:1 methanol:acetone mixture. This was then stored for 16hrs in -200C freezer. The samples were centrifuged at 18000 g for 5 min to pelletise the protein and air dried for 30 min. Protein digestion was done using 0.5 ug trypsin in 50 mM ammonium acetate incubated at 37 0C for 16 hrs. Tryptic-digested peptides were cleaned with C18 ZipTips (Millipore, Burlington, MA). SWATH-MS analysis was performed as described previously in Chapter 3.

Table 5.1. Malt sample collection over a 12 hour period from two malt plants covering the stages of steeping, germination and kilning. Note that malt plant 2 had a reduced number of samples, due to a 3-day germination process. Sample Plant one Malting (BP) Plant two Malting (JP) No. 1 Barley (steep in around 2pm) Barley (steep in between 12-5pm) 2 Steep 6.30am (cast germination 12- Steep 5.30pm 2pm) 3 Day 0 Germination 5.30pm Steep 6.30am (steep transfer ~10am, 3 hrs.) 4 Day 1 Germination 6.30am Day 1 Germination 5.30pm 5 Day 1 Germination 5.30pm Day 1 Germination 6.30am 6 Day 2 Germination 6.30am Day 2 Germination 5.30pm 7 Day 2 Germination 5.30pm Day 2 Germination 6.30am 8 Day 3 Germination 6.30am Day 3 Germination 5.30pm 9 Day 3 Germination 5.30pm Day 3 Germination 6.30am 10 Day 4 Germination 6.30am (in to Kiln ~7.30am) 11 Kiln 7.30am (at bottom floor before start of 2nd Days kilning) 12 Kilned malt. Kilned malt.

5.2.3 Data processing Proteins were identified from the 10 and 12 malting stages as detailed in table 5.1 above. These proteins were from 2 malt replicates * 2 laboratory replicates. Using their relative abundances, proteins were subjected to analysis of variance (ANOVA), treatments used being the sampling time and the plant. A set of proteins were identified to vary significantly with varying sampling time, plant or both (sampling time*plant).

For all the proteins identified, ID mapping and sequence information were retrieved from UniProt (an online protein database). This sequence information was then used to generate gene ontologies (GO) using the online web tool EggNOG database. The GO were used for singular enrichment analysis tool in the database for agricultural community, AGRIGO in order to identify GO terms which include biological, molecular and cellular compartment categories that are enriched among the malting proteins. KeGG mapping was done and the starch and sucrose pathway plotted through the online web tool KeGG mapper (Mahalingam, R. 2017; Mahalingam, R. 2020).

5.2.3.1 Hierarchical clustering Proteins found to vary significantly in abundance from one malting step to another were further analysed to identify patterns of change within the malting process. The relative abundance data was normalised and used to generate clusters. An online web-based tool, HeatMapper, was used to generate heat maps with average linkage clustering based on a euclidean distance measure. This was done to identify the clustering patterns over time in the malting process. Using HClust, (Murtagh et al., 2014) in the R statistical package (R Core team, 2019), hierarchical clustering algorithm was run using Euclidean metric and Ward’s minimum variance. The generated dendogram was cut into 4 clusters. The Normalised relative abundance values were used to plot graphs in Excel to show variability within the clusters. The average for each cluster being plotted with individual relative abundance data for each sample (Mahalingam, R. 2017; Mahalingam, R. 2020).

5.2.3.2 Select proteins of importance to malting and brewing industry Proteins were selected which have been discussed in previous malting and brewing articles as of importance in the industry. These were then plotted in a single graph for both malt plants JP and BP. This was to show trends variability. Normalised relative abundance data was then averaged into three main malting stages namely barley seed, green malt and kilned malt. This was also plotted side by side for the malting plants JP and BP.

5.3 Results and discussion The commercial malting variety, Buloke, was used in this experiment. The samples were collected from two commercial scale, modern pneumatic Australian malting plants Plant 1 (JP) and plant 2 (BP). Samples were collected every 12 hrs from barley seeds to kilned malt as detailed in the table 5.1 above. Protein extraction, reduction and alkylation, desalting and digestion to peptides was then done. The peptides were detected using LC-MS/MS analysis, they were then identified by searching the UniProt database against all protein sequences from all species using the MS/MS fragmentation data. 618 unique proteins were identified from the four replicates, 2 technical*2 malt replicates and 10 JP and 12 BP malting stages. A small subset of proteins perceived to be important in malt and beer quality were then selected for further investigation. These included the Chloroform Methanol soluble proteins, hordeins, amylases, serpins, lipid-transfer protein and proteinases. The relative abundance of the proteins showed differences from one sampling point to the next, and also some differences were noted from one malting plant to another in some proteins.

A total number of 618 proteins were identified in the SWATH-MS data, with nine of these being uncharacterised proteins. Using UniProt ID mapping tool from the online database UniProt, information including protein sequence information, protein names, protein organism origin, and gene name among others were retrieved for 548 proteins. Using EggNog, an online database, gene ontologies and KeGG ontologies for the 548 proteins was retrieved. The number of GO retrieved totalled 16, 574. Using the webtool cateGorizer 5258 GOs were mapped against 103 Plant_Go slim classes. The method for mapping used was consolidated single occurance count method to avoid repetition. Of these, 1138 GOs were mapped against biological processes, 368 were mapped against molecular function and 203 mapped against cellular component. The unique GO terms identified were 1710 belonging to 87 plant_Go slim cases. Using REVIGO, the biological terms GO were reduced and visualised, and 38 protein functions were identified, corresponding to the dormancy process. Using analysis of variance, 277 proteins were found to have a significant change in abundance from one malting step to another. 13 were found to have significant difference among the two malting plants. Using the webtool HeatMapper, a heat map was generated to show the clustering of proteins over time. Then using HClust package in R, a dendogram was plotted showing the protein grouping according to similarities in abundance change over the malting period. The data was cut into 4 clusters and plotted in excel using standardised data.

5.3.1 Selected proteins of importance to malting and brewing Amylases and their inhibitors The Chloroform Methanol soluble (CM) proteins identified from this study included CMa, CMb, CMc, CMd and CMe. The relative abundance of the CM proteins showed somewhat similar increase and decrease trends from sampling point 1 (barley seed) to sampling point 10 (kilned malt) for the two malting plants (Figure 5.2). The differences in relative abundance amongst the two malting plants was however not significant by SWATH-MS comparison. From the results, CMa was initially high before malting, the abundance levels however dropped in the green malt and kilned malt (Figure 5.3). Both malting plants JP and BP showed similar trends in abundance. The abundance level changes were however not significant. CMb on the other hand appeared more abundant in barley seeds and green malt than in kilned malt in plant JP (Figure 5.3). The plant BP showed a different trend with small decrease and an increase in the final product.

Barley CM proteins include the chloroform methanol extractable proteins CMa-e. These proteins have been grouped to be in a homologous family based on their amino acid composition and their weak “immunochemical cross-reactivity” (Barber et al., 1985). During the malting and brewing process, proteinases work to degrade storage proteins partially or fully and they therefore cause modifications which do affect the end product, beer by affecting the hazing properties (Robinson et al., 2004). Proteases have inhibitors which affect the protein modification process. The CM proteins are one of such inhibitors and belong to the trypsin/alpha-amylase inhibitor family. Their action involve inhibition of exogenous alpha- amylase, trypsin and serine proteases (Robinson et al., 2004).

Trypsin inhibitor CMc showed a decrease in abundance through the malting process at plant JP, but at plant BP there was a slight increase noted with the kilned malt (Figure 5.3). The trypsin inhibitor CMd showed varied abundance increase and decrease pattern in the two malting plants with JP having high abundance in green malt whereas BP had high malt protein abundance levels (Figure 5.3).

Trypsin inhibitor CMe had a decrease pattern on both plants JP and BP with kilned malt having the lowest abundance of the protein. Cme has been suggested to be a have positive protein. It has low molecular weight (13,626 SE +ve in some varieties and 13,840SE –ve in others), has hydrophobic properties and a low proline content of about 8% (Robinson et al., 2004). Beer haze is affected by the proteins which survive the malting and brewing process and end up in beer. These would include proteins such as hordein in varying molecular weight fragment, high in proline and hydrophobic in nature.

0.05

0.045

0.04 CMa

0.035 CMb CMc 0.03 CMd 0.025 CMe 0.02 Cma*

Relative Relative abundance 0.015 CMb*

0.01 CMc *

0.005 CMd* CMe* 0 1 2 3 4 5 6 7 8 9 10 11 12 Sampling time points

Figure 5.3. The effect of malting and kilning on protein Alpha-amylase/trypsin inhibitors chloroform methanol (a-e). The first three bars from malting plant JP and the last three from plant BP.

Another important group of proteins include the amylases. Two amylases were identified by the study, Alpha-amylase type A and B isozymes and Beta-amylase (Figure 5.4). The abundance of α-amylase isozyme A and B varied such that, type A showed low abundance in barley seeds, an increase in green malt and finally highest abundance in kilned malt. The trend in increasing abundance was similar in both malting plants JP and BP. Isozyme B however showed highest abundance in barley seeds, with lowest abundance in kilned malt. The trend in abundance decrease for isozyme B was similar in both malting plants JP and BP, however, the level of abundance differs.

Alpha-amylase is one of the starch degrading enzymes found in plants. It cleaves the α-1, 4 glucosidic bonds in glucose, maltose, amylose, amylopectin and polysaccharide from starch resulting in oligosaccharides Muralikrishna and Nirmana, 2005). Alpha and Beta-amylases are

86 important enzymes in the brewing industry because they are among the enzymes which determine diastatic power in malt, that is, the ability of malt to hydrolyse starch into fermentable sugars (Georg-Kraemer et al., 2000). The activity of α-amylase has been found to be low during the first days of germination, then increase gradually in the later days. This corresponds with our findings for isozyme type A, but differs from isozyme type B. This response is hypothesised to arise from the inhibitor BASI whose primary purpose is to prevent premature germination in seed. The levels of α-amylase increase due to de-novo synthesis in the aleurone layer during germination (Georg-Kraemer et al., 2000).

Beta-amylase identified in the study in malt plant JP showed a slight decrease in abundance from barley seeds, green malt and lastly to kilned malt. In malt plant BP however, barley seeds showed low abundance, followed by a slight increase in green malt and finally a slight drop in levels of abundance in kilned malt.

Barley beta-amylase is synthesised during grain development hence accumulates during this period. It is stored in grain in two forms; insoluble protein complex outside of the starch granule and soluble free form within the grain complex. This therefore means that no de-novo synthesis of beta-amylase occur during germination (Georg-Kraemer et al., 2000). Low levels of β- amylase detected on day one germination followed by a steep increase in its activity as the enzyme is released from its bound to free form by the reduction of disulphide bonds hence the activation of the enzymes active sites, an event that corresponds to our study findings. Beta- amylase is considered the principal diastatic power enzyme. It catalyses the removal of beta- maltose from the non-reducing chain ends of gelatinised starch and related compounds. Its release and activation has been suggested to be due to cysteine protease activity (Koehler & Ho, 1990). Heat treatment at normal pH has been found to inactivate amylases at 700C, however, alpha- amylase has been found to be more heat stable than beta-amylase.

Figure 5.4. The effect of malting and kilning on protein Alpha-amylase type A and B isozymes and Beta-amylase. The first three bars from malting plant JP and the last three from plant BP.

Protein Z (Serpins) Protein Z was another one of the selected barley proteins, they fall under serine protease inhibitor (serpin) group of proteins. The isoforms serpin Z4 and Z7 were detected in this study. From the data, a slight increase in abundance after germination is followed by a decrease, then

88 a sharp increase with slight decrease towards the end of germination and finally a decrease was noted with kilning. Serpin Z7 was lowest in abundance for both the malting plants JP and BP (figure 5.5).

0.03

0.025

Serpin 0.02

Serpin-Z4

0.015 Serpin-Z7

Serpin* Relative Relative abundance 0.01 Serpin- Z4*

Serpin-Z7 0.005 *

0 1 2 3 4 5 6 7 8 9 10 11 12 Sampling points

From the study, Serpin-Z7 increased in abundance as the seed germinates i.e. green malt and decreases during malting for both plants JP and BP (Figure 5.6). A study conducted previously (Evans and Hejgaard, 1999) revealed similar pattern in Serpin-Z7. They attributed the increase of abundance in green malt to the release of bound protein by cleaving, hence allowing for more free protein molecules. They noted an increase of up to 70% of protein amounts. Protein

Z is present in seed in bound thiol form and is released upon germination. Its synthesis and deposition on the seed is respondent to soil nitrogen availability (Steiner et al., 2011).

Protein Serpin-Z4 showed an increase in abundance in the green malt and this was followed by a decrease in the kilned malt for plant JP (Figure 5.6). Plant BP however showed a decrease in abundance but at very low levels through the malting process. (Evans and Hejgaard, 1999) observed an increase of between 44-47% in the green malt from an initial 19-33%. They noted that malting reduced the abundance to 4-27%.

Protein Z is one of the major beer proteins and has been suggested to have foam stabilizing property. This is because of its high surface viscosity and elasticity (Iimure and Sato, 2013). It belongs to the family Serpin and has three known isoforms Z4, Z7 and Zx, it is high in leucine (20 lysine and 2 cysteine units per monomer) and has hydrophobic properties. It is classified as an albumin and accounts for 5-7% seed albumin fraction, they have been implicated for the inhibition of serine proteases including chemotrypsin and trypsin, (Steiner et al., 2011).

Protein Z has been suggested to be a form positive protein, it survives the malting and brewing while retaining both its molecular weight and immunochemical properties hence ending up in beer (Evans and Hejgaard, 1999).

Figure 5.6. The effect of malting and kilning on Serpin-Z7, the first three bars from malting plant JP and the last three from plant BP.

Lipid-transfer protein (LTP1) and putative linoleate 9s-lipoxiganase From this study, the isoforms of LTP which were detected included non-specific LTP 1, 2 and 4 (NSLTP) and probable non-specific LTP (Figure 5.7). NSLTP1 was generally higher in abundance than the other variants. LTP was one of the proteins of interest in this study and findings from the study showed in plant JP that barley seeds contain higher levels of the proteins, as the malting proceeds, the levels of LTP1 drops and rises again in the kilned malt, (Figure 5.7). In plant BP however, the starting amount of LTP1 is lower than that in the kilned 91 malt. (Evans and Hejgaard, 1999) revealed small increases from the seed to green malt and kilned malt of 10% and 7-37% respectively. LTP1 is a plant protein commonly found in beer. It consists of approximately 1% total beer protein and has been suggested by various studies to be a form positive protein (Iimure and Sato, 2013; Colgrave et al., 2013; Steiner et al., 2011; Evans and Hejgaard, 1999).

Figure 5.7. The effect of malting and kilning on Non-specific lipid-transfer protein 1, the first three bars from malting plant JP and the last three from plant BP.

LTP affects beer quality in two ways, by foam stabilization which is brought about by its interaction with other low molecular weight polypeptides and Serpins. The other one is causing beer gushing (over foaming). This is especially so when the grain used was infected by fungi which causes overexpression of LTP (Heppeli and Hetch, 2009; Iimure and Sato, 2013).

Putative linoleate 9s-lipoxypenase 3 (LOX) relative abundance was found to differ significantly from one malting plant to the other at 99% confidence by SWATH-MS data comparison analysis. LOX showed a slow increasing trend over the malting period (Figure 5.8). (Walker et al., 1996) measured the amounts of hydroperoxy fatty acids 9 and 11 released as a result of LOX oxidation and noted and increase in amounts of the hydroperoxy fatty acids during germination, peak at day 3 then reduced quantity after day three. The hydroperoxy fatty acids were not detected in kilned malts but the increases were even higher during mashing than in malting. They suggested a conversion of hydroperoxy fatty acids to volatile substances during kilning hence remain in the malt and will later on cause the formation of stale beer. The amounts of hydroperoxy fatty acids released correspond to amounts of LOX in the malt.

LOX are catalysts in nature, they catalyse the reactions between oxygen and poly-unsaturated fatty acids hence resulting in stale beer. Beers going stale occurs during storage in cans or bottles. This is a negative trait in beer. LOX is available in barley grain (Walker et al, 1996).

Q 7 G 7 9 4 P u ta tiv e lin o le a te 9 S -lip o x y g e n a s e 3

0 .0 0 4

0 .0 0 3

n a

d 0 .0 0 2

b A 0 .0 0 1

0 .0 0 0

t t lt l lt l d a d a e a e a m m e m e m s n d s n d y e e y e e le e n le e n r r il r r il a G K a G K B B

Figure 5.8. The effect of malting and kilning on putative linoleate 9s-lipoxygenase protein (LOX), the first three bars from malting plant JP and the last three from plant BP.

0.007

Non-specific lipid- transfer protein 1 0.006 Non-specific lipid- transfer protein 4

0.005 Non-specific lipid- transfer protein 2

Probable non-specific lipid-transfer protein 0.004 Putative linoleate 9S- lipoxygenase 3

Non-specific lipid-

0.003 transfer protein 1* Relative Relative abundance Non-specific lipid- transfer protein 4*

0.002 Non-specific lipid-transfer protein 2* Probable non- 0.001 specific lipid- transfer protein* Putative linoleate 9S-lipoxygenase 3 * 0 1 2 3 4 5 6 7 8 9 10 11 12 Sampling points

Aspartic proteinase and Cysteine Proteases The analysis identified three proteinases; two variations of the cysteine proteinase EP-B1 and EP-B2 and aspartic proteinase (Figure 5.10). Cysteine proteinase EP-B1 was generally in relative abundance and showed little decreases and increases throughout the malting process. Cysteine proteinase EP-B2 however showed significant changes, noted by a significant increase in abundance during the germination period and a drop during malting. The trend is however similar in both malting plants with only slight variations. Aspartic proteinase on the other hand showed only slight changes throughout the malting process with noted low abundance in kilned malt.

Hydrolysis of proteins during malting is a very important step. Hordeins and other proteins get broken down into peptides and amino acids. This process is regulated by proteases (Jones 2005). There are four classes of proteases; cysteine, aspartic, metallo and serine. This study

95 identified two proteases; cysteine and aspartic. The total endoproteolitic activity has been found to increase during germination from the first to the fifth day (Zhang and Jones, 1995).

A study by (Wrobel and Jones, 1992) found that the general endoprotease activity shows a decrease with steeping of about 47%, then an increase of 26 fold with maximum activity levels being detected at day two germination. The hordein degrading enzymes however appear in germination, then increase in number and activity greater than 20 fold that of dormant grain with more than 90% of the endoproteases surviving the kilning process and ending up in mash (wort) (Osman et al., 2002). This finding corresponds to our findings on cysteine proteinase, which showed a steady increase in abundance from the seed. It however varies with the aspartic proteinase findings, which shows a steady decrease in abundance over the course of germination and onto malting (Figure 5.11).

Previous studies by (Zhang and Jones, 1995) classified cysteine and aspartic proteinase as “housekeeping” enzymes, meaning they are always present in the grain and/or plant tissue and are involved in everyday proteolytic activities. Cysteine proteinase however has also been grouped as one of the most important proteinases in hordein degradation hence the germination process and has also been found to be relatively heat stable (Osman et al., 2002).

0.006

0.005 Cysteine proteinase EP-B 1 0.004 Cysteine proteinase EP-B 2 Aspartic 0.003 proteinase

Relative Relative abundance 0.002

0.001

0 1 2 3 4 5 6 7 8 9 10 11 12 Sampling time points

Figure 5.11. The relative abundance of the proteinases cysteine and aspartic proteinase and cysteine proteinase inhibitor. Data in continuous lines denoting samples from the first plant JP and the dotted lines from the second malting plant BP. The sampling time points correspond to the table 5.1 above.

Hordeins and hordoindolines Various forms of hordeins and hordoindolines were able to be identified in this study. These included B1-, B3-, C-, γ-, and γ3- hordeins and hodoindoline-A, -B1 and –B2. There is a general decrease in relative abundance trend both with the hordeins and hordoindolines (Figure 5.12). Previous studies on beer and wort have been able to identify B-, C-, D- and γ-Hordeins (Asano et al., 1982; Evans et al., 2003; Silva et al., 2008; Colgrave et al., 2013; Schulz et al., 2018). A study by (Fladvora et al., 2012) noted a decrease in C-hordein of up to 35% from Malting to Kilning. They was also able to detect B1- and B3-Hordeins reporting only slight decreases in their intensities during the malting process. The B3-hordein was bit of a contrast with our data, we noted 83% decrease from malting to kilning. B-1 and C-hordein had only slight decreases in relative abundance. Studies by (Fladrova et al., 2012) attributed the slight loss in abundance to hordeins’ high resistance to the heating and proteolysis process which take place during malting and kilning.

0.06

0.05

B1-hordein B3-hordein 0.04 C-hordein Gamma-hordein Gamma-hordein-3 Hordoindoline-A Hordoindoline-B1 0.03 Hordoindoline-B2 B1-hordein*

Relative Relative abundance B3-hordein* C-hordein* 0.02 Gamma-hordein* Gamma-hordein-3* Hordoindoline-A* Hordoindoline-B1* Hordoindoline-B2* 0.01

0 1 2 3 4 5 6 7 8 9 10 11 12 Sampling time points

Figure 5.12. The relative abundance of the hordein proteins. Data in continuous lines denoting samples from the first plant JP and the dotted lines from the second malting plant BP. The sampling time points correspond to the table 5.1 above.

Hordeins are barley grain’s major storage proteins (Iimure and Sato, 2013) and is classified into B-, C-, D- and γ- hordein based on their electrophoretic mobility. During the malting

98 process hordeins are degraded by proteolysis process to amino acids and low molecular weight polypeptides. Hordeins from 30-50% of total grain composition. They form a matrix surrounding the starch molecules located in the endosperm. During malting, the most important physical and chemical change is the degradation of the hordein matrix to allow access to starch by the starch enzyme and hence partial hydrolysis. D-hordein has been reported to degrade rapidly compared to B- and C- hordeins which are a bit more resistant to degradation (Garcia –villalba et al., 2006). Malt defined as well modified should, after kilning, contain less than half its starting hordein fraction (Celus et al., 2006).

5.3.2. Employing different databases to better understand malting data Some of the important GO functions related to malting included carbohydrate metabolic process which had 60 proteins involved in it, protein metabolic process with 42 proteins, and lipid metabolic process with 34 proteins as shown in percentages (Figure 5.13). All the GO functions are important processes in germination leading to the breakdown of starch during the brewing process.

transporter response to lipid metabolic activity endogenous process 1% stimulus 1% post- 1% cell communication embryonic respons 1% protein modification nucleic growth protein metabolic anatomicaldevelopme structure e to process acid morphogenesisnt biotic 1% process cell differentiation 1% binding 1%1% stimulus 1% 1% reproduction 1% 1%plastid 1% 1% response to response to abiotic external stimulus stimulus 1% 1%

response to stress cellular process 2% 19% hydrolase activity 2% transferase carbohydrateactivity metabolic2% process 2% membrane 2% catabolic process metabolic process 2% 17%

multicellular organismal development binding 2% 3% cytoplasm 3% catalytic activity cellular 7% component organization cell and intracellular 5% biogenesis 5% 2% transport biosynthetic process 2% 5% nucleobase, nucleoside, nucleotide and nucleic acid metabolic process 4%

Figure 5.13. Classification of proteins according to their gene ontology terms. The biological process had the number of gene ontologies allocated to it.

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Post embryonic development with 31 proteins, cell differentiation with 20 proteins, cellular organisation and biogenesis with 17 proteins, cell growth with 10 proteins, ribosome activity with 7 proteins, translation with 5 proteins, translation factor activity with 1 protein, nucleic acid binding with 2 proteins and DNA metabolic process with 1 protein which are important processes towards the growth of the young embryo. The processes leading to germination ,initiation and the growth and development of the young embryo. Majority of the proteins were involved in cellular processes and metabolic functions; 19% and 17% respectively. Other important category were proteins involved in stress response which included 56 proteins. These proteins may have been activated during the seed stress periods and included proteins such as ribonuclease TUDOR 1, heat shock protein 82, and thiamine thiazole synthase 2, chloroplastic amongst others.

Of interest was the biological process GO. Using ReViGo online database, the biological process were reduced to 284 (Figure 5.14). The highest partitioning for this category was the GOs grouped as response to redox state. This had 85 GO terms assigned to it. The GO terms included were such as; hyperosmotic salinity response regulated by proteins such as Delta-1- pyrroline-5-carboxylate synthase (Zhang et al., 2015), cellular response to abscisic acid stimulus regulated by guanine nucleotide-binding protein subunit beta-like protein A (Nakashima et al., 2009). Redox state reactions together with increased production of reactive oxygen species (ROS) signal response to plant biotic and abiotic stress. An example of such reactions is the chloroplast and mitochondrial electron transport chains and has been suggested to interact leading to important plant responses such as cell death and or stress tolerance (Queval et al., 2007).

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RNA secondary structure unwinding oxazole or thiazole metabolism ethylene biosynthesis oxazole or thiazole biosynthesis primary amino compound metabolism mucilage metabolism olefin metabolism protein unfolding stomatal closure succinyl-CoA metabolism protein folding in endoplasmic reticulum phytoalexin metabolism stomatal movement protein autoprocessing choline metabolism auxin homeostasis starch metabolism double fertilization forming a zygote and endosperm establishment or maintenance of transmembrane… amylopectin biosynthesis jasmonic acid metabolism glyoxysome organization dormancy process response to redox state 0 10 20 30 40 50 60 70 80 90 GO's

Figure 5.14. Gene ontology term biological process partitioning. Highest number of GO’s proteins were classified under response to redox state, followed by dormancy process with 85 and 39 respectively.

Figure 5.15. Partitioning of biological process gene ontology showing the different function groups that the proteins are part of dormancy

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The GO terms associated with dormancy process were one of the most important categories to the study (Figure 5.14). This is because the study was aimed looking at proteins associated with dormancy in malting barley. The dormancy process had 39 GO terms categorised under it (Figure 5.15). It included GO terms such as; maintenance of dormancy regulated by proteins such as 1-cys perodoxin PER1. PER 1 has been described as an uncharacteristic LEA gene. They have been implicated to protect the embryo against respiration by-products which may lead to lipid peroxidation and phospholipid de-esterification. The by-products include ROS such as superoxide dimutases and catalases (Stacy et al., 1999). It has been reported in a previous study by (Stacy et al., 1999) that PER 1 levels increase enormously up to 11 days after imbibition in seeds which were dormant and was found to disappear soon after 24 hours in seeds which germinated. PER 1 has therefore been implicated for the maintenance of dormancy and its level increase in dormant seed is an indication of the proteins stability.

Acquisition of desiccation tolerance in seed was another important GO term related to the dormancy process. Proteins LEA 19 and LEA EMB564 were linked to the GO term. LEA proteins are desiccation tolerance proteins which are produced when the seed goes into the desiccated period right after maturation. This is an adaptation of seed to ensure survival during storage and tolerance to harsh conditions (Leprince et al., 1993).

Starch metabolism was the other important category of GO terms related to malting. This is important because it involves the breakdown of starch into simple sugars, a very important component in beer manufacturing. It also encompasses starch biosynthesis hence available within the seed during the malting process.14 GO terms were linked to starch metabolism. A few of these included; sucrose metabolic process which is regulated by alpha-amylase type B isozyme. This enzyme catalyses the breakdown of internal alpha-glucosidic linkages in starch, oligosaccharides and polysaccharides (Kadziola et al., 1994). Glucose catabolic process which involves both the biosynthesis and breakdown of glucose and is regulated by protein triosephosphate isomerase, cytosolic (TPI). TPI is a well-known catalyst which catalyses the interconversion of dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (GAP) (Dorion et al., 2005).

Hierarchical clustering Hierarchical clustering was done in order to identify patterns of change in the protein profile of malting barley sampled every 12 hours of malting from seed to kilned malt. The results

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(Figure 5.16) show the period sampled with highest abundance of proteins to be the last day of germination (marked G6) just before green malt was transferred for kilning. Barley seeds prior to steeping had the lowest abundance of proteins. The abundance then increases in sort of a uniform manner to reach its peak at the last day of germination. This is as denoted by the colour changes in Figure 5.16. The abundance then reduces with kilning, the intensity of the yellow colour reduces and in some protein starts turning back to blue to indicate reduced abundance.

Figure 5.16. Heat map cluster analysis of proteins showing statistical significant change in abundance over the 10 stages of mating. Samples were taken every 12 hrs. G1-G6 indicating the sampling periods during germination process.

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From this clustering, the 277 proteins which showed significant changes in abundance at different malting stages were able to be grouped to four clusters using HClust R statistical package as shown in (Figure 5.16). The generated dendogram was cut into the four cluster: cluster 1; 78 proteins, cluster 2; 83 proteins, cluster 3; 60 proteins and cluster 4; 56 proteins.

Figure 5.17. Dendrogram cluster analysis of proteins showing statistically significant change in abundance during malting. The groups indicate the different patterns of change in abundance that the clusters fall into

Cluster 1 proteins showed increased abundance after 24 hours (imbibition) and a slight drop in abundance after 72hrs (Day 2 germination). Cluster 2 proteins showed a steady increase in abundance with a slight drop after 72hrs. Cluster 3 proteins were a bit similar to cluster 1 proteins in the abundance pattern profile, however, the loss of abundance after 132hrs (kilning) was slightly higher. Lastly cluster 4 proteins showed loss in abundance at 24 hrs and the highest loss of abundance after 132 hrs (kilning) compared to all the other clusters (Figure 5.17).

Cluster 1 had the highest number of ribosomal proteins which is 16, it also had, 4 LEA proteins, 2 serine carbopeptidase proteinases, Protein disulphide-isomerase, Linoleate 9S-lipoxygenase 1 (LOX), 2-Cys peroxiredoxin BAS1 chloroplastic and serpin Z7 amongst others. Cluster 2 on

105 the other hand had 13 ribosomal proteins, 4 LEA proteins, and 3 hordein class proteins, 2 types of alpha-amylase inhibitors, protein disulphide-isomerase, glutelin and NS lipid transfer protein amongst others.

Cluster 3 had 12 ribosomal proteins, 2 types of alpha-amylase inhibitors, 2 different hordein class proteins, aspartic proteinase and 2 types of NS lipid transfer protein among others. Cluster 4 had the lowest number of ribosomal proteins being 9, 4 LEA protein types, 3 uncharacterised proteins, vicilin-like storage protein, alpha-amylase type A isozyme and Linoleate 9S- lipoxygenase 1 (LOX).

Figure 5.18. The four clusters from the analysis of proteins which showed statistical significant difference in abundance within the different malting steps. Cluster 1, 2, 3 and 4 each have 78, 83, 60 and 56 proteins respectively. Normalised predictions average data was used.

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5.4 Conclusion This study revealed the change in proteome during malting as shown by the change in abundance from one malting step to another. There were variations in protein expression from one malting plant to another although these were not dominant. The proteins abundance is shown to increase as the seeds germinate, then increase over time, the proteins then decrease significantly with malting. Qualitative and quantitative analysis identified key DP enzymes as well as other important malt enzymes (proteases) and important beer quality proteins (LOX and LTP) Some proteins were selected and their profiles closely examined as they were seen to be important in the malting process. Their importance is summarised as follows:

i. Barley chloroform methanol (CM) proteins: these are low molecular weight proteins which show alpha-amylase inhibitory activity and are suggested to affect beer haze property ii. Serpins: Act as storage protein during grain filling, suggested germination inhibition and are major beer protein, suggested foam stabilizing activities. Identified in both chapter 3 and 4. iii. Aspartic and Cysteine proteinases: Protease class, completely or partially hydrolyse hordeins and modify other proteins. Proteases solubilize proteins hence affecting beer quality especially clarity. Cysteine has been implicated in beta amylase activation. This was also identified in chapter 4 of this study. iv. LOX: Initiates fatty acid production during wort production i.e. lipid metabolism, oxylipin pathway. Suggested for the ‘bench taste’ in stored beer. Role in growth and development, insect resistance and senescence v. LTP: Is a major beer protein, binds foam destabilizing lipids hence promoting foam stability. Overexpressed in cases of grain infection by fungi hence if grain is used in malting results to ’gushing’ (Iimure and Sato 2012; L.H. Robinson et., 2006; www.uniprot.org; Schmitt and Marinac, 2007; Mechelen et., al. 1999).

This study opens up the possibility for more research in the malting barley proteomics.

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6. Summary of the thesis 6.1 General discussion In this projects, methodical research has been done to explore the protein expression in malting barley in the dormant, resting and germinative phase, overtime in storage, and during the malting period. This thesis research was conducted with the aim of addressing the following goals;

1. Chapter 3 of the research was aimed at studying the change in proteome of barley over the course of germination. This included looking at the proteome of barley at its resting, dormant and germinative phase. 2. Chapter 4 of the research was aimed at studying the genome and proteome expression of germinating vs dormant barley. Germination and dormancy was monitored over a year in storage 3. Chapter 5 of the research was aimed at studying the proteome change of malt acquired from two malting plants and investigating the site (environmental) effect on the proteome of malted barley

6.2 Summary of conclusions 6.2.1 Differences in protein expression in dormant and non-dormant barley An aspect of importance in the malting industry is protein. The main source of protein in beer is from barley grain therefore the amount of protein in the grain is closely monitored. Barley varieties chosen to go into malting should have the right amount of protein in order not to affect the quality of the final product negatively.

In this research, we characterised protein expressed from barley in five categories; before germination (resting phase), early germinating (germinated on day 1), late germinating (germinated after 3 days), dormant (failed to germinate) and in the course of germination (early germinating, but left to grow radicle for three days). We concluded that the barley proteome changes from one phase of growth to another as was indicated by the significant changes in relative abundance within the proteins which were identified. The protein abundant before germination were mainly proteins whose role/function is protective, that is, desiccation tolerance. The abundance of these proteins dropped significantly with the onset of germination. The relative abundance of starch degrading enzymes however showed an increase with germination onset, an indication of the onset of starch breakdown. Proteins found to be

108 significantly abundant in the course of germination and radicle elongation included those whose function encapsulates promotion of growth and the development of the young seedling.

6.2.2 Genomic and proteomic analysis of barley seeds to better understand dormancy

The normal practice in the malting industry involves buying barley grain in large amounts and storing it in large silos over a year, monitoring temperature, humidity and other environmental aspects closely. This will be sampled and tested for germination percentage and malted over the entire season. When the germination percentage drops below the desired 98%, the maltsters face heavy losses in terms of increased storage costs of the “carry over stock” from the previous season and lowered malt quality (Briggs, 1994).

In this research, we investigated the change in barley proteome of seed that was stored for a year after harvest. Sampling and germination testing was done every two months followed by SWATH-Ms analysis. The germination percentage found to be low immediately after harvest, increasing to 99% around 4-6 months and then decreasing slowly in some varieties. Comparing the dormant and non-dormant barley, samples showed differences in protein relative abundance. Gene ontology analysis identified 40 GOs classified to be directly involved with dormancy initiation and regulation, the proteins identified included those directly involved with plant growth processes. The genomic aspect of this study was however not realised.

6.2.3. Protein expression comparison of malt from different malting processes Malting involves controlled germination of barley seeds aiming at the activation of starch degrading enzymes hence breakdown of starch/sugars and the subsequent formation of alcohol. This process is highly controlled to ensure high quality malt with brewers defined quality aspects such as the distatic power, wort viscosity, malt extract among others. This research was aimed at investigating the differences in malt proteome sampled from two different malting plants and the differences in the protein expression within the different malting stages. The study revealed differences in proteome expression from one malting stage to the next as indicated by the significant change in protein relative abundances. There were small variations in protein expression from one malting plant to the other. From the hierarchical clustering, we noted the abundance of proteins was low at day 1 malting, then increases and peaks at day 6 then decreases to lower levels that day 1 during kilning. The increase was related with the release of bound protein whereas the decrease during kilning was related to heat

109 degradation of proteins. Of interest were the hordein proteins which showed a decreasing slope during the malting process, an indication of breakdown to release starch.

6.3 Future research plans A lot of research has gone towards dormancy (Oberthur et al., 1995; Hans et al., 1996; takeda 1996; Romagosa et al., 1999, Zhang et al., 2005, Romagosa et al., 1999) and protein exression during germination of malting barley (Finnie and Svensson, 2003, Bonsager, 2007, Finnie et al., 2009, Iimure and Sato, 2013, Daneri-castro et al., 2016). While the study of proteins to understand plant processes is very important, the study of genomics is also of similar value. Monitoring dormancy using proteins has been done repeatedly, however, no ideal dormancy break indicator exists to date. Little research is directed towards gene expression in dormancy. Studying the genomics of dormancy mechanisms can lead to better understanding of dormancy and finally to the much desired dormancy break indicator. Genes are transcribed to mRNA and translated to amino acids. However, there are modifications within the process as the final product, protein, is generated. I speculate that the modifications could hold the key to whether the seed gets a go ahead signal to germinate or remain dormant. It would be interesting to direct research towards this area in future. Another much desired discovery is the non-invasive seed sampling and hence allowing us to monitor the seed beyond dormancy into germination. This would be ideal for maltsters to monitor the state of dormancy or germination readiness of their entire stock by scanning and monitoring, hence no destructive and repeated germination percentage testing and also regulation of grain purchase.

The above research breakthrough will however be dependent on the development of efficient single seed RNA extraction protocols with high yields and quality. The current barley RNA extraction protocol goes for seed bulking of 5-15 seeds, limiting collaborative study (relating gene and protein from the same seed). In our research we were able to isolate high quantity RNA from a single seed, however, an aspect which we struggled to attain in this thesis due to unavoidable circumstances was high quality which can be used for Next Gen sequencing.

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Appendices

3.1 List of barley proteins from the data set

N Accession Name Species Peptides (95%) 1 P09842 Granule-bound starch synthase 1 HORVU 21 2 P06293 Serpin-Z4 HORVU 42 3 P80284 Protein disulfide-isomerase HORVU 21 4 P06471 B3-hordein (Fragment) HORVU 24 5 Q43492 Serpin-Z7 HORVU 21 Glyceraldehyde-3-phosphate dehydrogenase 2, 6 P08477 cytosolic (Fragment) HORVU 14 7 P31922 Sucrose synthase 1 HORVU 10 8 P07596 Alpha-amylase/subtilisin inhibitor HORVU 13 9 P17990 Gamma-hordein-1 HORVU 23 10 P11643 Alpha-amylase/trypsin inhibitor CMd HORVU 33 11 P04399 Protein synthesis inhibitor II HORVU 10 12 P28041 Alpha-amylase/trypsin inhibitor CMa HORVU 12 13 P16968 Alpha-amylase inhibitor BMAI-1 (Fragment) HORVU 14 14 P34937 Triosephosphate isomerase, cytosolic HORVU 10 15 P31923 Sucrose synthase 2 HORVU 9 16 P80198 Gamma-hordein-3 HORVU 20 17 P52572 1-Cys peroxiredoxin PER1 HORVU 6 18 P52894 Alanine aminotransferase 2 HORVU 5 19 P07597 Non-specific lipid-transfer protein 1 HORVU 24 20 Q9LEH8 Hordoindoline-B2 HORVU 6 21 P16062 Subtilisin-chymotrypsin inhibitor CI-1A HORVU 6 22 Q43772 UTP--glucose-1-phosphate uridylyltransferase HORVU 5 23 P13691 Alpha-amylase inhibitor BDAI-1 HORVU 8 24 P01086 Trypsin inhibitor Cme HORVU 8 25 P32936 Alpha-amylase/trypsin inhibitor CMb HORVU 12 26 Q9ZRI8 Formate dehydrogenase, mitochondrial HORVU 5 27 P23951 26 kDa endochitinase 2 HORVU 8 28 P28814 Barwin HORVU 4 29 Q9M4E3 Hordoindoline-A HORVU 5 Glucose-1-phosphate adenylyltransferase large subunit 30 P30524 1, chloroplastic/amyloplastic HORVU 4 31 Q9FSI9 Hordoindoline-B1 HORVU 6 32 P16063 Subtilisin-chymotrypsin inhibitor CI-1B HORVU 5 33 P34951 Trypsin inhibitor CMc HORVU 8 34 P06470 B1-hordein HORVU 22 Glucose-1-phosphate adenylyltransferase small 35 P55238 subunit, chloroplastic/amyloplastic HORVU 5 36 P20145 Probable non-specific lipid-transfer protein HORVU 4 Glyceraldehyde-3-phosphate dehydrogenase 1, 37 P26517 cytosolic HORVU 8 38 P11955 26 kDa endochitinase 1 HORVU 5

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39 P01545 Alpha-hordothionin HORVU 8 40 P16098 Beta-amylase HORVU 35 41 Q43470 14-3-3-like protein B HORVU 2 42 P23901 Aldose reductase HORVU 2 43 Q43472 Glycine-rich RNA-binding protein blt801 HORVU 2 44 Q05968 Pathogenesis-related protein 1 HORVU 2 45 P35793 Pathogenesis-related protein PRB1-3 HORVU 2 46 P35792 Pathogenesis-related protein PRB1-2 HORVU 2 47 Q05190 Late embryogenesis abundant protein B19.1A HORVU 2 48 P46532 Late embryogenesis abundant protein B19.1B HORVU 2 49 P14928 ABA-inducible protein PHV A1 HORVU 3 50 Q9ZRR5 Tubulin alpha-3 chain HORVU 2 51 Q96460 Tubulin alpha-2 chain HORVU 2 52 P20230 Defensin-like protein 1 HORVU 3 53 P04750 Alpha-amylase type B isozyme HORVU 1 54 P04063 Alpha-amylase type B isozyme HORVU 1 55 P22244 Protein synthesis inhibitor I HORVU 9 56 Q43470 14-3-3-like protein B HORVU 2 57 P29305 14-3-3-like protein A HORVU 2 58 P17991 C-hordein (Fragment) HORVU 4 59 P06472 C-hordein (Fragment) HORVU 2 60 Q9M5G3 Translationally-controlled tumor protein homolog HORVU 1 61 P27337 Peroxidase 1 HORVU 1 62 P25250 Cysteine proteinase EP-B 2 HORVU 1 63 P25249 Cysteine proteinase EP-B 1 HORVU 1 64 P55307 Catalase isozyme 1 HORVU 1 65 P46532 Late embryogenesis abundant protein B19.1B HORVU 2 66 P33044 Antifungal protein R (Fragment) HORVU 1 67 P29114 Linoleate 9S-lipoxygenase 1 HORVU 1 68 P12949 Dehydrin DHN4 HORVU 2 69 P42210 Phytepsin HORVU 4 Pyrophosphate-energized vacuolar membrane proton 70 Q06572 pump O HORVU 1 71 P36183 Endoplasmin homolog HORVU 1 72 Q05191 Late embryogenesis abundant protein B19.4 HORVU 0 73 Q02400 Late embryogenesis abundant protein B19.3 HORVU 0 74 P31923 Sucrose synthase 2 HORVU 9

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4.1 GO’s analysis classified 41 GOs within the dormancy promoting role as shown in table – below. These are associated with plant growth aspects which have been related to dormancy promotion in one way or another. These however need to be studied more deeply to be able to find the link with dormancy. dormancy process GO:0022611 LE19A_HORVU negative regulation of seed germination GO:0010187 MD37E_ARATH plant organ development GO:0099402 PFKA7_ARATH; meristem development GO:0048507 Y8359_ORYSI vegetative to reproductive phase GO:0010228 Y8359_ORYSI transition of meristem seed coat development GO:0010214 SBT17_ARATH plant epidermis development GO:0090558 PFKA7_ARATH polar nucleus fusion GO:0010197 SEBP1_ARATH developmental maturation GO:0021700 GLUD1_ORYSJ SUS1_HORVU SSG1_AVESA root system development GO:0022622 PFKA7_ARATH GBLPA_ORYSJ Y8359_ORYSI negative regulation of post-embryonic GO:0048581 MD37E_ARATH development megagametogenesis GO:0009561 EDA2_ARATH SEBP1_ARATH regulation of post-embryonic GO:0048580 MD37E_ARATH development seed dormancy process GO:0010162 LE19A_HORVU leaf senescence GO:0010150 AATC_ORYSJ fruit development GO:0010154 GLUD1_ORYSJ PXG_ORYSJ PXG1_AVESA OLE16_BROSE mucilage extrusion from seed coat GO:0080001 SBT17_ARATH embryo sac development GO:0009553 EDA2_ARATH SEBP1_ARATH acquisition of desiccation tolerance GO:0097439 LE19A_HORVU anatomical structure maturation GO:0071695 GLUD1_ORYSJ SSG1_AVESA LE19A_HORVU SUS1_HORVU meristem maintenance GO:0010073 ENPL_HORVU stamen development GO:0048443 GRP2_NICSY seed maturation GO:0010431 SUS1_HORVU root meristem growth GO:0010449 Y8359_ORYSI plant organ morphogenesis GO:1905392 PFKA7_ARATH seedling development GO:0090351 OLE16_BROSE GBLPA_ORYSJ

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RBG4_ARATH TSN1_ARATH aging GO:0007568 AATC_ORYSJ shoot system development GO:0048367 GBLPA_ORYSJ AATC_ORYSJ CAPP2_SORBI GRP2_NICSY leaf development GO:0048366 AATC_ORYSJ CAPP2_SORBI root development GO:0048364 PFKA7_ARATH GBLPA_ORYSJ Y8359_ORYSI mucilage metabolic process involved in GO:0048359 SBT17_ARATH seed coat development seed development GO:0048316 GLUD1_ORYSJ SSG1_AVESA LE19A_HORVU LE193_HORVU meristem growth GO:0035266 Y8359_ORYSI seed oilbody biogenesis GO:0010344 OLE16_BROSE OLEO1_ORYSJ OBP1A_MAIZE plant organ senescence GO:0090693 AATC_ORYSJ seed germination GO:0009845 OLE16_BROSE GBLPA_ORYSJ RBG4_ARATH TSN1_ARATH endosperm development GO:0009960 PDI_HORVU acquisition of desiccation tolerance in GO:0048700 LE19A_HORVU seed gametophyte development GO:0048229 EDA2_ARATH ORYB_ORYSJ SEBP1_ARATH regulation of meristem structural GO:0009934 ENPL_HORVU organization plant epidermal cell differentiation GO:0090627 PFKA7_ARATH

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4.2 Single seed RNA quality check for 24 samples. The quality of RNA is very poor due to high degradation. Sterile water pr epared manualy in the lab was used, this impacted the quality negatively. RIN lower than 3.

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4.3 RNA quality check of the trizol extraction protocol. It was noticed that the sample was precipitating out before addition to the filter column hence little or no RNA detected. The RNA quantity is between 800 – 1112 ng/µl, the RNA integrity number (RIN) was however low-between 4- 5.

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4.4 Single seed modified TRIzol RNA extraction. RIN all samples but 6 and 12 were 6 and above. The RNA concentration was however low (41- 232 ng/µl) due to increased RNA purification steps.

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