MANIPULATING RAB GTPASE ACTIVITY IN WHEAT TO ALTER GLUTEN QUALITY FOR BREADMAKING ADAM MICHAEL TYLER, BSc. (Hons) Thesis submitted to the University of Nottingham for the degree of Doctor of Philosophy MAY 2012 Adam M Tyler i University of Nottingham I. Abstract In the developing endosperm of bread wheat (Triticum aestivum), seed storage proteins are produced on the rough endoplasmic reticulum (ER) and transported to protein bodies, specialised vacuoles for the storage of protein. The important gluten proteins of wheat are transported to the protein bodies they are stored in by two distinct routes. One route consists of vesicles that bud directly off the ER, while the other involves transport through the Golgi (Arcalis et al, 2004). In plants, the RabD clade mediates ER to Golgi vesicle transport (Batoko et al, 2000). Available sequence information for Rab GTPases in Arabidopsis, rice, Brachypodium and bread wheat was compiled and compared in phenetic trees. Partial genetic sequences were assembled using the first draft of the Chinese Spring wheat genome. A suitable candidate gene from the RabD clade (TaRabD2a) was chosen for down-regulation by RNA interference (RNAi) and an RNAi construct was used to transform wheat plants. Using real time PCR, all four available RabD genes were shown to be knocked down in the developing endosperm of transgenic wheat. The transgenic grain was found to produce flour with significantly altered processing properties when measured by farinograph and extensograph. SE-HPLC found that a smaller proportion of HMW-GS and large LMW-GS are incorporated into the glutenin macropolymer in the transgenic dough. Lower protein content but a similar protein profile on SDS-PAGE was seen in the transgenic grain. Adam M Tyler ii University of Nottingham II. Acknowledgements The glasshouse work at Rothamsted Research, including potting up and harvest of T0 plants and sowing, potting up and harvesting of T1 and T2 plants, was done by Mrs Fiona Gilzean, Mr Anthony Griffin and Mrs Melloney St Leger. The biolistic transformation work was performed by Amanda Riley at Rothamsted Research and the screening of the T0 lines was performed by Caroline Sparks. A sample of pHMW-Adh-Nos plasmid vector was provided by Huw Jones. Agilent RNA gels were performed at Sutton Bonington by Linda Staniforth and Ruth Cornock. Doug Smith at Campden BRI performed the 2D-PAGE and SE-HPLC of T3 flour and assisted with the SDS-PAGE of T1 seeds. Milling of T3 grain was performed by Leigh Wyatt at Campden BRI. Rheology of T3 dough and Dumas protein content measurement was carried out by the Cereals and Milling Department at Campden BRI. I would also like to thank my supervisors Grantley Lycett, Chungui Lu, Huw Jones, Johnathan Napier and Mervin Poole for their considerable help and input throughout the project. Finally I would like to thank the BBSRC and Campden BRI for funding through the Industrial CASE Partnership Award. Adam M Tyler iii University of Nottingham III. List of Contents I. Abstract ............................................................................. ii II. Acknowledgements ............................................................. iii III. List of Contents ................................................................. iv IV. List of Figures .................................................................... xi V. List of abbreviations ...........................................................xv 1 General Introduction ........................................................... 1 1.1.1 Functions of the Rab GTPase family............................ 1 1.1.2 Development of wheat and cereal seeds ..................... 2 1.1.3 Storage molecules of cereal and wheat seeds .............. 5 1.1.4 The role of gluten proteins in baking performance ........ 6 1.1.5 Transport of seed storage proteins during wheat endosperm development ...................................................... 7 1.1.6 Potential for manipulation of Rab GTPase activity in the developing endosperm of bread wheat................................... 9 2 General Materials and Methods ........................................... 10 2.1.1 Laboratory chemicals and reagents .......................... 10 2.1.2 Primers & DNA ...................................................... 10 2.1.3 Genomic DNA extraction from wheat leaf .................. 10 2.1.4 Polymerase Chain Reaction (PCR) ............................ 11 2.1.5 Agarose gel ........................................................... 12 2.1.6 DNA precipitation from PCR product ......................... 12 Adam M Tyler iv University of Nottingham 2.1.7 Gel purification of DNA fragments ............................ 13 2.1.8 Plasmid miniprep ................................................... 14 2.1.9 Escherichia coli glycerol stock .................................. 14 2.1.10 E. coli transformation and culture ......................... 14 2.1.11 Spectrophotometric analysis of nucleic acids .......... 15 2.1.12 DNA sequencing ................................................. 15 2.1.13 Cloning of PCR products for sequencing ................. 15 2.1.14 Total RNA extraction from developing wheat endosperm ....................................................................... 16 2.1.15 cDNA synthesis using random hexamer primers ...... 17 2.1.16 Quantitative reverse transcriptase polymerase chain reaction (qPCR) ................................................................ 18 3 Bioinformatics of wheat Rab GTPases .................................. 20 3.1 Introduction ............................................................... 20 3.1.1 The protein life-cycle of Rab GTPase ........................ 20 3.1.2 Membrane fusion of vesicle and target compartments 24 3.1.3 Rab GTPases in the plant endomembrane system ...... 25 3.1.4 The RabD/Rab1 clade of Rab GTPases ...................... 26 3.2 Materials and Methods ................................................. 28 3.2.1 Sequence data and analysis .................................... 28 3.2.2 Gene expression profiling ........................................ 28 3.2.3 Phenetic analysis ................................................... 29 Adam M Tyler v University of Nottingham 3.3 Results....................................................................... 30 3.3.1 Sequence collection and analysis ............................. 30 3.3.2 Phenetic analysis of the Rab GTPase family ............... 31 3.3.3 Construction of RabD gene sequences using PCR and GSS database ................................................................... 38 3.3.4 Expression of RabD genes in developing wheat endosperm ....................................................................... 42 3.4 Discussion .................................................................. 46 3.4.1 Rab gene sequences .............................................. 46 3.4.2 Rab genes in the wheat genome .............................. 47 4 Cloning of vector construct, transformation and screening of transgenic wheat .................................................................... 49 4.1 Introduction ............................................................... 49 4.1.1 The RNA interference technique ............................... 49 4.2 Materials and Methods ................................................. 53 4.2.1 cDNA synthesis using specific primers ...................... 53 4.2.2 Reverse transcriptase polymerase chain reaction (RT- PCR)..... .......................................................................... 53 4.2.3 Cloning of RNAi construct ....................................... 53 4.2.4 Wild-type wheat growth and seed collection .............. 55 4.2.5 Preparation of wheat embryos and gold particles for transformation.................................................................. 56 4.2.6 Biolistic transformation of wheat embryos ................. 58 Adam M Tyler vi University of Nottingham 4.2.7 Regeneration of T0 transgenic wheat plants .............. 59 4.2.8 PCR screening of T0 plants for transgene .................. 61 4.2.9 96-well plate DNA extraction ................................... 62 4.2.10 96-well plate to 1.5ml tube DNA extraction ............ 62 4.2.11 Transgene screening PCR to eliminate null and hemizygous T2 lines .......................................................... 63 4.3 Results....................................................................... 65 4.3.1 Single target Rab strategy ...................................... 65 4.3.2 RabD consensus strategy ........................................ 65 4.3.3 RNAi target region selection and PCR primer design ... 68 4.3.4 PCR to amplify RabD2a target region........................ 68 4.3.5 Cloning of RNAi vector construct .............................. 74 4.3.6 Screening of T0 plants for transgene ........................ 77 4.3.7 Transgene screening PCR to eliminate null and hemizygous lines from T1 plants ......................................... 78 5 Analysis of transgenic wheat .............................................. 82 5.1 Introduction ............................................................... 82 5.1.1 Gluten protein structure and function - High molecular weight glutenin subunits .................................................... 82 5.1.2 Gluten protein structure and function - Low molecular weight glutenin subunits .................................................... 84 5.1.3 Gluten protein structure and function - Gliadins ......... 86 5.1.4 Gluten protein genetics .......................................... 87 Adam M Tyler vii University of Nottingham 5.1.5 Wheat processing quality ........................................ 88 5.2 Materials and Methods ................................................. 92 5.2.1 Sodium dodecyl sulphate polyacrylamide gel electrophoresis of T1 seeds ................................................ 92 5.2.2 Sowing and tagging of T1 wheat, harvesting during development and harvesting when mature ........................... 94 5.2.3 Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of T2 seeds .............................. 95 5.2.4 Grain size and shape analysis .................................. 97 5.2.5 Milling of T2 grain .................................................. 99 5.2.6 Determination of flour moisture content ..................100 5.2.7 Measurement of flour protein content ......................100 5.2.8 Small-scale breadmaking quality test - Reomixer ......101 5.2.9 Sowing, transport and potting up of T2 wheat ..........103 5.2.10 Tagging and harvesting of developing T3 seeds and harvest of mature seeds ...................................................104 5.2.11 Milling of T3 grain ..............................................104 5.2.12 Size exclusion high-performance liquid chromatography (SE-HPLC) ...............................................105 5.2.13 Statistical analysis .............................................108 5.2.14 Two-dimensional polyacrylamide gel electrophoresis (2D PAGE) ......................................................................108 5.2.15 Brabender farinograph ........................................108 Adam M Tyler viii University of Nottingham 5.2.16 Brabender extensograph .....................................110 5.3 Results......................................................................114 5.3.1 Protein analysis of grain from T0 plants ...................114 5.3.2 T2 grain size and shape analysis .............................114 5.3.3 T2 grain protein analysis........................................121 5.3.4 T2 dough rheology ................................................125 5.3.5 Expression of RabD genes in T3 RabD knockdown line....... .........................................................................127 5.3.6 T3 grain size and shape analysis .............................131 5.3.7 Yield of T3 grain harvest ........................................133 5.3.8 Analysis of T3 grain protein content ........................133 5.3.9 Two-dimensional gel analysis of T3 lines ..................135 5.3.10 Analysis of glutenin macropolymer composition in T3 dough...... ......................................................................135 5.3.11 T3 dough rheology .............................................143 5.4 Discussion .................................................................145 5.4.1 Protein content of transgenic wheat ........................145 5.4.2 Gluten bonding strength ........................................146 5.4.3 Potential post-translation modification differences in storage proteins of transgenic wheat ..................................147 6 General Discussion ...........................................................150 6.1.1 Expression of RabD genes in developing wheat endosperm of wild type and transgenic Cadenza wheat ........150 Adam M Tyler ix University of Nottingham 6.1.2 Effect of RNAi on Rab gene expression ....................152 6.1.3 Trafficking of gluten proteins ..................................154 6.1.4 Gluten quality in transgenic wheat ..........................155 6.1.5 Future prospects ...................................................157 7 References ......................................................................161 8 Appendix ........................................................................179 8.1 Alignment of wheat RabD nucleotide sequences .............179 8.2 SDS-PAGE gels ..........................................................184 Adam M Tyler x University of Nottingham