Rht-1 Semi-Dwarfing Alleles Increase the Abundance of High Molecular Weight Glutenin Subunits Emma Jobson, Jae-Bom Ohm, John Martin, Mike Giroux Jobson, E. M., Ohm, J. B., Martin, J. M., & Giroux, M. J. (2021). Rht‐1 semi‐dwarfing alleles increase the abundance of high molecular weight glutenin subunits. Cereal Chemistry, 98(2), 337-345. 10.1002/cche.10371 This is the peer reviewed version of the following article: [Rht‐1 semi‐dwarfing alleles increase the abundance of high molecular weight glutenin subunits. Cereal Chemistry (2020)], which has been published in final form at https://doi.org/10.1002/cche.10371. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self- Archived Versions: https://authorservices.wiley.com/author-resources/Journal-Authors/licensing/ self-archiving.html#3. Made available through Montana State University’s ScholarWorks scholarworks.montana.edu 1 2 DR. MICHAEL J. GIROUX (Orcid ID : 0000-0001-7343-6719) 3 4 5 Article type : Research 6 7 8 Rht-1 Semi-Dwarfing Alleles Increase the Abundance of High Molecular 9 Weight Glutenin Subunits 10 Emma Jobson1, Jae-Bom Ohm2, John Martin1, Mike Giroux1,3 11 12 1Department of Plant Sciences and Plant Pathology, 119 Plant Bioscience Building, Montana 13 State University, Bozeman, MT 59717-3150, USA 14 2USDA‐ARS, Edward T. Schafer Agricultural Research Center, Cereal Crops Research Unit, 15 Hard Spring and Durum Wheat Quality Lab., Fargo, ND 58108 16 3Corresponding author. Email: mgiroux@montana.edu Phone: (406) 994-7877 17 18 Funding Information 19 This project was supported by the USDA National Institute of Food and Agriculture awards 20 2017- 67014-26190, 2019-67014-29199, by the Montana Wheat and Barley Committee, and the 21 Montana Agricultural Experiment Station. 22 ABSTRACT 23 Background and Objectives: Grain protein and starch abundance and composition are 24 quantitative traits that play key roles in wheat quality. The semi-dwarfing alleles of the Reduced This is the author manuscript accepted for publication and has undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/cche.10371 This article is protected by copyright. All rights reserved 25 height (Rht-1) gene increase tillers and yield but also reduce seed size and protein content. 26 Despite their negative impact on grain protein content the semi-dwarfing alleles increase dough 27 mixing time and tolerance. This study used near isogenic lines that were either tall or semi-dwarf 28 lines that carried Rht-B1b, Rht-D1b, or Rht-8 to investigate how each semi-dwarfing allele 29 impacts gluten composition and flour pasting properties. . 30 Findings: None of the semi-dwarfing alleles impacted starch properties. Each reduced flour 31 protein content compared to the tall variety with the largest decreases in Rht-B1b (1.8%) and 32 Rht-D1b (1.5%). The semi-dwarfing lines increased the gluten index (21.5%) compared to Rht- 33 1a. Using SE-HPLC we determined that the semi-dwarfing lines had an increased relative 34 abundance of high molecular weight glutenins compared to the tall variety. 35 Conclusions: This study indicates that the Rht-1 semi-dwarfing alleles increase dough mixing 36 time and tolerance by increasing the relative abundance of high molecular weight glutenins 37 yielding stronger dough. 38 Significance and Novelty: The semi-dwarfing alleles developed primarily for agronomic 39 purposes have significant impacts on gluten index and starch swelling power. 40 KEYWORDS: gluten, Rht-1, semi-dwarf, starch, wheat 41 1. INTRODUCTION 42 Wheat (Triticum aestivum) is one of the most important crops for human consumption. Its 43 popularity is driven by its ability to grow well within a wide range of environments, as well as 44 the products derived from wheat dough. Specifically, the extensibility and elasticity unique to 45 wheat dough which allows gas bubbles to be trapped during the baking process has contributed 46 to the global success of wheat. Both extensibility and elasticity play important roles in dough 47 development. Breadmaking doughs are typically stronger and more elastic compared to pastry 48 doughs which have lower protein content and greater extensibility. Together, extensibility and 49 elasticity are referred to as dough viscoelasticity and grain storage proteins are the major 50 determinant of the viscoelasticity of dough (Shewry, Halford, Belton, & Tatham, 2002). Wheat 51 grain protein accounts for approximately 8 - 18% of grain dry weight (Shewry et al., 2009). The 52 major class of storage protein in wheat grain is gluten (Shewry, et al., 2002). Gluten is composed 53 of hundreds of different protein subunits which form a complex matrix in dough (Wrigley & 54 Bietz, 1988: reviewed in Wieser, 2007). Gluten can be separated into its two most prevalent 55 fractions based on their solubility; the alcohol soluble gliadins, and the alcohol insoluble This article is protected by copyright. All rights reserved 56 glutenins. Gliadins are considered less elastic than glutenins and are primarily responsible for the 57 extensibility of the dough. The glutenins are responsible for dough elasticity and cohesion which 58 determines dough strength (Weiser, 2007). Furthermore, insoluble polymeric proteins are 59 positively correlated with bake mixing time, whereas soluble polymeric proteins are negatively 60 correlated with bake mixing time (Park, Bean, Chung, & Seib, 2006). 61 Glutenins are further characterized by their molecular weight. Low molecular weight 62 glutenins account for approximately 20% of gluten proteins (Weiser & Kieffer 2001). High 63 molecular weight glutenins account for 10% of gluten proteins and have a molecular weight 64 ranging from 67 – 83 kDa (Wieser, 2007). Of gluten component proteins, high molecular weight 65 glutenins have the greatest impact on dough strength (Wrigley, Bekes, & Bushnuk, 2006; Weiser 66 & Kieffer 2001). High molecular weight glutenins are largely controlled by loci found on the 67 group 1 chromosomes, Glu-A1, Glu-B1, and Glu-D1 (Payne, 1987). Recent studies have shown 68 that these genes, as well as those associated with gliadins, are regulated by both cis and trans loci 69 (Plessis, Ravel, Bordes, Balfourier, & Martre, 2013). These transcription factors have been 70 shown to impact the quantity and composition of glutenins and gliadins (Plessis et al., 2013). 71 In addition to grain protein, starch is also important in dough rheology. Starch accounts 72 for 65-73% of the dry weight of flour (Pomeranz, 1988). It plays a key role in water absorption 73 and interacts directly with the gluten matrix (Sandstedt, 1961, 1955; Petrofsky & Hoseney, 74 1995). Furthermore, the rate of starch gelatinization directly impacts dough expansion during 75 baking (Kusunose, Fugii, & Matsumoto, 1999). 76 Starch is composed of large A-type and small B-type granules. The A-type granules have 77 a diameter greater than 10 µm and are more disk shaped than spherical; B-type granules have a 78 diameter smaller than 10 µm and are spherical (Soulaka & Morrison, 1985; Vermeylen, Goderis, 79 Reynaers & Delcour, 2005; Kim & Huber, 2008). Although A-type granules account for greater 80 than 70% of total starch weight, 90% of granules are B-type (Bechtel, Zayas, Kaleikau, & 81 Pomeranz, 1990; Raeker, Gaines, Finney, & Donelson, 1998; Peng, Gao, Abdel-Aal, Hucl, & 82 Chibbar, 1999). The surface of these granules has a direct impact on dough rheology (Sipes, 83 1993) and the ratio of A- and B-type granules impacts bread making (Park, Chung, & Seib, 84 2005). 85 Grain protein and starch content are quantitative traits controlled by many genes and 86 influenced by the environment. One gene that has large impacts on protein content is Reduced This article is protected by copyright. All rights reserved 87 Height (Rht-1). There is a Rht-1 gene on each of the group 4 chromosomes (Gale, Law, & 88 Worland, 1975; Gale & Marshall, 1975, 1976; McVittie, Gale, Marshall, & Westcott, 1978; 89 Sourdille et al., 1998). Dominant acting mutant forms of Rht-1 reduce plant height and increase 90 yield. The two most prevalent semi-dwarfing mutations are found in the B and D genome, Rht- 91 B1b and Rht-D1b, respectively. Both alleles contain a premature stop codon near the RHT 92 protein N terminus (Peng, Richards, & Hartley, 1999). The resultant truncated protein partially 93 inhibits the plant’s ability to respond to gibberellic acid (GA) (Allan, Vogel, & Craddock, 1959). 94 The agronomic result is a 20% height reduction, increased productive tillers, and a 10% increase 95 in grain yield (Flintham, Börner, Worland, & Gale, 1997). Rht-B1b and Rht-D1b are also 96 associated with decreased kernel size and grain protein content (Flintham et al., 1997; Gooding, 97 Cannon, Thompson, & Davies, 1999; Lanning et al., 2012; Mann et al., 2009). Rht-8 is another 98 gene which reduces plant height but does not interfere with the plant’s ability to perceive 99 gibberellic acid (Korzun, Röder, Ganal, Worland & Law, 1998; Rebetzke & Richards, 2000). 100 Rht-8 reduces plant height approximately 6.5% by impacting the plant’s ability to respond to 101 brassinosteroids (Lanning et al., 2012; Gasperini, Greenland, Hedden, Dreos, Harwood, & 102 Griffiths, 2012). Rht-8 was included as part of this study to ensure that the impact of Rht- 103 B1b/Rht-D1b was due to the Rht-1 mutations, and not semi-dwarfed plant architecture. 104 Limited work has been done to investigate the impact of the Rht-1 semi-dwarfing alleles 105 on bread making and end use quality. Sherman et al. (2014) associated Rht-D1b with decreased 106 flour yield, protein, and loaf volume, but increased mixing tolerance and bake mixing time. We 107 used near isogenic lines carrying either Rht-B1b, Rht-D1b, or no semi-dwarfing mutation to 108 evaluate the impact of the semi-dwarfing alleles on end use quality (Jobson, Martin, Schneider, 109 & Giroux, 2018). We also observed a decrease in flour protein content (1.8% Rht-B1b, 1.5% Rht- 110 D1b), but an increase in mixograph mixing time (1.8 minutes) and tolerance when compared to 111 Rht-1a. 112 The purpose of this study was to investigate the impact of the Rht-1 semi-dwarfing alleles 113 on dough strength and grain composition to better understand how they increase dough strength 114 despite reducing flour protein content. For this study we used near isogenic lines developed in a 115 tall hard red spring wheat cultivar. Lines either carried Rht-B1b, Rht-D1b, no semi-dwarfing 116 gene (Rht-1a), or Rht-8. This article is protected by copyright. All rights reserved 117 The Rht-1 semi-dwarfing alleles have been incorporated into most modern wheat 118 cultivars. Therefore, it is important to not only understand their agronomic impact, but also their 119 impact on end use quality and bread making. This study provides new insight into the impact of 120 the semi-dwarfing alleles on grain protein and starch composition in relation to bread making 121 quality. The semi-dwarfing alleles are some of the most broadly used genes in wheat breeding 122 programs. Although there has been extensive research regarding their impact on plant growth 123 and development, there is very limited research regarding their impact on bread making and end 124 use quality. This study provides a comprehensive analysis of the impact of the semi-dwarfing 125 alleles on starch and protein in relation to bread making; and illustrates how genes which 126 significantly impact agronomic traits also influence product quality. 127 2. MATERIALS AND METHODS 128 2.1. Plant Material 129 This study used near isogenic lines (NILs) which carried either no semi-dwarfing alleles, Rht- 130 B1b, Rht-D1b, or Rht-8. The NILs were developed in the standard height, hard red spring 131 wheat, “Fortuna” (CI 13596) as described by Lanning et al. (2012). “Hi-Line” (PI 132 549275) was the donor parent for the Rht-B1b allele, “McNeal” (PI 574642) served as the 133 donor of the Rht-D1b allele, and “Mara” (PI 244854) was the donor of the Rht-8 allele. 134 All lines were backcrossed to Fortuna as the recurrent parent to the BC4 generation. The 135 genotype of each line was confirmed in the BC4 generation using the markers described 136 by Ellis, Spielmeyer, Gale, Rebetzke, & Richrds (2002) and Ellis, Rebetzke, Azanza, 137 Richards, & Spielmeyer (2005). 138 139 2.2. Field Design and Conditions 140 Trials for this study were grown as described in Jobson et al., (2018) in 2017 at the 141 Arthur H. Post Field Research Center near Bozeman, MT (latitude 45.6 N, longitude 111.00 W, 142 elevation 1,455 m, soil type: Amsterdam silt loam). Plants were grown under both irrigated and 143 rainfed conditions. The trials received 13.8 cm of precipitation throughout the growing season 144 with the irrigated field receiving an additional 10.2 cm of water with half supplied one week 145 prior to and half one-week post heading. This article is protected by copyright. All rights reserved 146 The NILs (Rht-B1b, Rht-D1b, Rht-8, and Rht-1a) were grown in a randomized complete 147 block design with five replications in both the irrigated and rainfed trials. The plots were 3 m 148 long and 4 rows wide, with 30 cm between rows. Seeds were planted at a rate of 3.3 g per m of 149 row. 150 2.3. Milling 151 Milling and quality analysis were done according to AACC approved methods (American 152 Association of Cereal Chemists, 2000). Samples were cleaned using a Forster Cyclone Grain 153 Scourer, 1930, size 6 (Forster Manufacturing CO, Wichita, KS 154 ). Dockages were removed using a Dockage Test Machine XT7 2014 (Carter International, 155 Minneapolis, MN). Samples were tempered to 14.5 % moisture (AACC Method 26-10.02) and 156 milled into straight grade white flour and bran fractions using a Quadrumat Jr II Mill (C.W. 157 Brabender Instruments Inc., Hackensack, NJ). The milled samples were further cleaned using a 158 149 µm USA Standard Testing Sieve (Seedburo Equipment CO., Chicago, IL) and a Ro-Tap 159 RX-29 shaker (W.S. Tyler, Mentor, OH). 160 2.4. Flour Properties 161 Flour protein and moisture content were measured using a Foss Infratec 1241 Machine 162 (Foss Analytics, Eden Prairie, MN; AACC Method 39-11.01). Whole wheat flour for flour 163 swelling power analysis and starch swelling power was milled using a Laboratory Mill 3303 164 (Perten, Springfield, IL). Starch was extracted and purified from 300 mg of whole wheat flour as 165 described by Hogg et al. (2013). Flour swelling power was measured according to AACC 166 Method 56-21.01. Gluten Index was measured using the Glutomatic System (Perten, Springfield, 167 IL; AACC Method 38-12.02). The pasting property of flour was measured using a Perten Rapid 168 Visco Analyser 4500 (Perten; AACC Method 76-21.02). 169 2.5. Starch Granule Visualization 170 Measurements of starch granules were done using images captured using a Zeiss Supra 171 55 VP field emission gun-scanning electron microscope, (Carl Zeiss Microscopy, Peabody, MA). 172 Three starch samples purified from flour from each genotype grown under irrigated conditions 173 were imaged. Each sample was imaged 5 times. Measurements of A- and B-type granules were 174 determined by measuring the maximal diameter of three A- and three B-type granules from each 175 image; totaling 15 measurements for each of the three biological replicates. 176 2.6. Protein molecular weight distribution This article is protected by copyright. All rights reserved 177 Flour from 3 replications of each genotype grown under irrigated conditions was 178 analyzed at the USDA-ARS Hard Spring and Durum Wheat Quality Laboratory at Fargo, North 179 Dakota for protein molecular weight distribution analysis. Protein molecular weight distribution 180 (MWD) parameters were measured using size exclusion high performance liquid 181 chromatography (SE-HPLC) as described by Gupta, Khan, & MacRitchie (1993) and Ohm, 182 Hareland, Simsek, & Seabourn (2009). The extractable and unextractable protein fractions were 183 obtained from 10mg (14% moisture) of flour using a sodium phosphate/ sodium dodecyl sulfate 184 (SDS) solution (0.5% SDS and 0.5 M sodium phosphate, pH 6.9). The SDS extractable protein 185 fraction was then solubilized in 1 ml of a buffer solution, and vortexed for 5 minutes at 2,000 186 rpm using a vortex mixer (Pulsing Vortex Mixer; Fisher Scientific, Hampton NH). The 187 extractable protein fraction was then separated by centrifugation at 20,000 g (Eppendorf 188 Centrifuge 5424, Hamburg, Germany) and filtered through a 0.45 μm polyvinylidene difluoride 189 syringe filter. The unextractable protein fraction was solubilized by sonicating the residue in 1 190 mL of the buffer solution for 30 sec (Sonic Dismembrator 100; Fisher Scientific). The 191 unextractable protein fraction was then separated using centrifugation and filtration as described 192 for the extractable fraction. Immediately after filtration both the SDS extractable and 193 unextractable protein fractions were heated at 80 °C for 2 min to prevent protein hydrolysis. Ten 194 μL of each fraction was then injected individually for SE-HPLC fractionation. 195 Size exclusion HPLC was done using a liquid chromatograph (Agilent 1100; Agilent 196 Technologies, Santa Clara, CA) loaded with a size exclusion narrow bore column (300 × 4.6 197 mm, Yarra 3 um SEC SEC-4000; Phenomenex, Torrance, CA) and a guard cartridge (BIOSEP 198 SEC S4000; Phenomenex). The SE-HPLC system was run at a flow rate of 0.5 mL/min using an 199 isocratic mobile phase of 50% acetonitrile and 0.1% (v/v) trifluoroacetic acid aqueous solution. 200 Absorbance data were attained at 214 nm by a photodiode array detector (Agilent 1200; Agilent 201 Technologies, Santa Clara, CA). UV absorbance data were analyzed by in-house programs coded 202 using MATLAB software (MathWorks, Natick, MA) as described by Ohm, Hareland, Simsek, & 203 Seabourn et al. (2009). Size exclusion HPLC profiles were divided into four fractions (F) as 204 follows, F1: 3.5– 4.7 min, F2: 4.7–5.2 min, F3: 5.2-5.8 and F4: 5.8-7.4 min. Size exclusion 205 HPLC fractions (F1–4) were reported to be composed primarily of polymeric proteins for F1 and 206 F2, gliadins for F3, and albumin and globulins for F4 (Larroque, Gianibelli, Batey, & This article is protected by copyright. All rights reserved 207 MacRitchie, 1997; Malalgoda, Ohm, Meinhardt, & Simsek, 2018). The protein molecular weight 208 distribution parameters were derived from UV absorbance data from the four fractions. 209 2.7. RNA Sequencing 210 Grain used for expression analysis was collected and immediately frozen using liquid 211 nitrogen at 21 days past anthesis. The frozen grain was ground using a mortar and pestle, and 212 total RNA was extracted using a RNeasy Plant Mini Kit (Qiagen, Valencia, CA) (Oiestad et al., 213 2016). The extracted RNA was quantified using an Agilent 2100 Bioanalyzer (Agilent 214 Technologies, Santa Clara, CA). ArrayStar (DNASTAR, Madison, WI) was used to analyze the 215 sequence data. The parameters were match setting at 90% for a minimum of 100 bp. The data are 216 reported as reads per kilobase of transcript for million mapped reads (RPKM) (Mortazavi, 217 Williams, McCue, Schaeffer, & Wold, 2008). The data was initially analyzed globally using the 218 most recent wheat genome sequence (Appels, et al., 2018). We then performed a targeted 219 expression analysis focused on genes previously identified to be involved in grain starch (Cao, 220 Hu, & Wang, 2012) and protein synthesis (Kawaura, Mochida, & Ogihara, 2005). The data were 221 normalized to Act-2. Act-2 encodes an actin-like protein and has been previously shown to be a 222 reliably expressed gene for RNAseq dataset normalization (Tenea, Peres, Cordeiro, & Maquet, 223 2011). 224 2.8. Statistical Analysis 225 Response variables where data were obtained on all replications were analyzed using an 226 analysis of variance model for a randomized complete block design which combined both 227 rainfed and irrigated environments. All factors were considered fixed. A model for a completely 228 randomized design was used for protein molecular weight distribution and starch granule size 229 variables where data were collected on a subset of the replications. The Least Significant 230 Difference (LSD) value to compare differences between genotypes was calculated following a 231 significant F ratio (P<0.05) using the “Agricolae” R v1.3.3 package (De Mendiburu & Simon, 232 2015). The P value presented for the expression analysis was calculated using a two-tailed, 233 independent sample t-test. This value represents any variance in expression between the wildtype 234 and Rht-B1b lines. 235 3. RESULTS 236 3.1. Flour and Starch Pasting Properties This article is protected by copyright. All rights reserved 237 We observed the expected reduction in flour protein in the semi-dwarf lines compared to 238 the tall Rht-1a. Rht-B1b and Rht-D1b decreased flour protein content by 1.8% and 1.5% 239 compared to Rht-1a (P<0.05; Table 1). Rht-B1b and Rht-D1b increased gluten index 21.5% 240 compared to Rht-1a (95 % vs 74 %) (P<0.05; Table 1). Rht-8 had intermediate flour protein 241 content and gluten index between the semi-dwarf and tall varieties. Rht-B1b and Rht-D1b had no 242 measurable impact compared to Rht-1a on starch swelling power (Table 1), flour swelling 243 power, or starch granule size (data not shown). 244 The semi-dwarfing alleles had an impact on the pasting properties of the flour measured 245 using RVA. The peak viscosity value was increased 20% in the semi-dwarfing lines compared to 246 Rht-1a (data not shown) and time to peak was increased slightly. The total setback value was 247 also increased in Rht-B1b (26%) and Rht-D1b (22%) compared to Rht-1a (data not shown) 248 3.2. Protein Molecular Weight Distribution 249 SE-HPLC results are divided by those proteins which were SDS-Extractable, and those 250 which were not. The first two parameters in each group (UP1, UP2, EP1, EP2) represent the 251 proportion of polymeric glutenin proteins in total proteins. Specifically, UP1 and UP2 are 252 associated with the large molecular weight glutenins. We observed an increase in UP1 and UP2 253 in Rht-B1b (UP1: 14.92%, UP2: 4.39%) and Rht-D1b (UP1: 14.65%, UP2: 4.33%) compared to 254 Rht-1a (UP1: 12.73%, UP2: 3.80%) (P<0.05; Table 2). The third fraction is associated with the 255 unextractable and extractable gliadin subunits (UP3 and EP3). The EP3 value was decreased in 256 the semi-dwarfing lines (Rht-B1b:32.7%, Rht-D1b:33.42%) compared to the tall variety 257 (35.84%) (P<0.05; Table 2). There was no measurable difference between genotypes for the 258 unextractable gliadins. The fourth fraction (UP4 and EP4) is associated with the albumins and 259 globulins, we did not detect any significant differences between genotypes for this fraction. 260 3.3. RNA Sequencing 261 RNA sequencing data was initially analyzed globally. We also performed a targeted 262 analysis focused on genes associated with seed storage proteins (Table 3). We found no 263 statistically significant differences in storage protein gene expression between Rht-B1b and Rht- 264 1a. 265 DISCUSSION 266 Rht-B1b and Rht-D1b have a significant impact on many aspects of wheat plant growth 267 and development. Because they reliably decrease plant height and increase yield, they are now This article is protected by copyright. All rights reserved 268 present in most modern wheat varieties. However, despite their agronomic importance, there is 269 limited research regarding their impact on protein and starch in relation to bread making. Since 270 the ability of wheat flour to be baked into bread is one of the primary reasons for the global 271 success of wheat, it is important to understand how yield genes, such as Rht-1, impact end use 272 quality and bread making. 273 A previous study (Jobson et al., 2018) showed that Rht-B1b and Rht-D1b increase dough 274 mixing time (4.6 minutes) while reducing flour protein. In this study we evaluated the impact of 275 Rht-B1b and Rht-D1b on dough gluten index, starch properties, and storage protein composition 276 to understand how Rht-B1b and Rht-D1b impact dough mixing properties. Rht-B1b and Rht-D1b 277 did not impact flour or starch swelling power, or starch granule size. Our previous study also 278 showed Rht-B1b and Rht-D1b had no impact on alpha amylase activity (Jobson et al., 2018). 279 Based on these results, it is unlikely that the semi-dwarfing alleles significantly impact starch 280 content or composition. 281 There were measurable differences in pasting viscosity between Rht-B1b/Rht-D1b and 282 Rht-1a. Semi-dwarf NILs flour had increased final viscosity as well as peak time compared to 283 the tall NIL flour. This may be explained by previous findings which describe an inverse 284 relationship between flour protein content and final viscosity and total setback (Lee, 2016; 285 Katyal et al., 2018). 286 Despite having a lower flour protein, Rht-B1b and Rht-D1b increased gluten index 21.5% 287 compared to Rht-1a (95% vs 74%). This agrees with our previous study which illustrated that 288 despite decreasing total protein content, the Rht-B1b and Rht-D1b semi-dwarfing alleles increase 289 dough strength (Jobson et al., 2018). This may be explained by a difference in the abundance of 290 major storage proteins. Barak, Mudgil, and Khatkar (2014) and Dhaka and Khatkar (2015) found 291 that an increased ratio of gliadins to glutenins decreased gluten index and dough stability. Barak 292 et al. (2014) added fractionated glutenins and gliadins to fortified flour in increments of 2, 4, 6, 293 8, and 10%. The addition of gliadins decreased the stability and mixing time of the dough, while 294 the addition of glutenins increased the dough stability and mixing time. A 2% addition of 295 glutenins resulted in a 100% increase in the dough stability. Based on SE-HPLC data, Rht- 296 B1b/Rht-D1b have a positive impact on glutenin percentage, specifically the high molecular 297 weight SDS unextractable polymers. Previous SE-HPLC studies have shown that these high 298 molecular weight SDS unextractable protein polymers have a significant impact on increasing This article is protected by copyright. All rights reserved 299 dough strength, which may partially explain the increased dough strength associated with the 300 semi-dwarfing alleles (Tsilo, Mudgil, & Khatkar, 2010; Dachkevitch & Autran 1989; Singh, 301 Donovan, & MacRitchie, 1990; Bangur, Batey, McKenzie, & MacRitchie, 1997; Park et al., 302 2006). 303 For almost all traits, Rht-8 was intermediary between Rht-B1b/Rht-D1b and Rht-1a. This 304 may be due to its intermediate grain protein content. However, further research is needed to 305 understand the mechanism behind the impact of Rht-8 on grain composition and end use quality. 306 CONCLUSIONS 307 This study provides a comprehensive analysis of the impact of the Rht-B1b and Rht-D1b 308 semi-dwarfing alleles on flour protein composition, starch and flour pasting properties. We 309 found that despite decreasing total flour protein content, Rht-B1b and Rht-D1b increase dough 310 strength compared to the tall NIL by altering the composition of gluten component storage 311 proteins. We observed that Rht-B1b and Rht-D1b increase the relative abundance of glutenins 312 compared to gliadins, which has previously been shown to increase dough strength. Further 313 studies will be needed to determine how the semi-dwarfing alleles alter gluten storage protein 314 composition. 315 316 ACKNOWLEDGEMENTS 317 We would like to acknowledge Luther Talbert and Nancy Blake for the use of their 318 isolines in this study; Douglas Engle and Craig Morris for completing the RVA; and Deanna 319 Nash and Harvey TeSlaa for their assistance with milling. 320 321 REFERENCES 322 Allan, R. E., Vogel, O. A., & Craddock, J. C. (1959). Comparative response to gibberellic acid of 323 dwarf, semi-dwarf and standard short and tall winter wheat varieties. Agronomy 324 Journal, 51, 737–740. 325 326 AACC International. (2000). Approved Methods of Analysis (11th Ed). AACC International: St. 327 Paul, MN. 328 This article is protected by copyright. All rights reserved 329 Appels, R., et al. (2018). Shifting the limits in wheat research and breeding using a fully 330 annotated reference genome. Science, 361, 661. 331 332 Bangur, R., Batey, I.L., McKenzie, E., & MacRitchie, F. (1997). Dependence of extensograph 333 parameters on wheat protein composition measured by SE-HPLC. Journal of Cereal 334 Science, 25, 237-241. 335 336 Barak, S., Mudgil, D., & Khatkar, B.S. (2014). Influence of gliadin and glutenin fractions on 337 rheological, pasting, and textural properties of dough. International Journal of Food 338 Properties, 17, 1428-1438. 339 340 Bechtel, D. B., Zayas, I., Kaleikau, L., & Pomeranz, Y. (1990). Size-distribution of wheat starch 341 granules during endosperm development. Cereal Chemistry, 67, 59-63. 342 343 Cao, Y., Hu, H.G., & Wang, C.S. (2012). Expression profiles of genes involved in starch 344 synthesis in non-waxy and waxy wheat. Russian Journal of Plant Physiology, 59, 632- 345 639. 346 347 Dachkevitch, T. & Autran J.C. (1989). Prediction of baking quality of bread wheats in breeding 348 programs by size-exclusion high-performance liquid chromatography. Cereal Chemistry, 349 66, 448-456. 350 351 De Mendiburu, F., & Simon, R. (2015). Agricolae: Ten years of an open source statistical tool 352 for experiments in breeding, agriculture and biology. PeerJ PrePrints, 3, e1404v1. 353 354 Dhaka, V. & Khatkar, B. 2015. Effects of gliadin/glutenin and HMW‐GS/LMW‐GS ratio on 355 d ological properties and bread‐making potential of wheat varieties. Journal of 356 Food Quality. 38:71-82. 357 This article is protected by copyright. All rights reserved 358 Ellis, H., Spielm‐ eyer, W., Gale, K., Rebetzke, G., & Richards, R. (2002). “Perfect” markers for 359 the Rht B1b and RhtD1b dwarfing genes in wheat. Theoretical and Applied 360 Genetics, 105, 1038–1042. 361 362 Ellis, M. H., Rebetzke, G. J., Azanza, F., Richards, R. A., & Spielmeyer, W. (2005). Molecular 363 mapping of gibberellin responsive dwarfing genes in bread wheat. Theoretical and 364 Applied Genetics, 111, 423– 430. 365 366 Flintham, J. E., Börner, A., Worland, A.J., & Gale, M.D. (1997). Optimizing wheat grain yield: 367 effects of Rht (gibberellin-insensitive) dwarfing genes. The Journal of Agricultural 368 Science, 128, 11-25. 369 370 371 Gale, M. D., Law, C.N., & Worland, A.J. (1975). The chromosomal location of a major dwarfing 372 gene from Norin 10 in new British semi-dwarf wheats. Heredity, 35, 417-421. 373 374 Gale, M.D. & Marshall G.A. (1976). The chromosomal location of Gai 1 and Rht 1, genes for 375 gibberellin insensitivity and semi-dwarfism, in a derivative of Norin 10 wheat. Heredity, 376 37, 283-289. 377 378 Gale, M.D. & Marshall, G.A. (1975). Nature and genetic-control of gibberellin insensitivity in 379 dwarf wheat grain. Heredity, 35, 55-65. 380 381 Gasperini, D., Greenland, A., Hedden, P., Dreos, R., Harwood, W., Griffiths, S. (2012). Genetic 382 and physiological analysis of Rht8 in bread wheat: an alternative source of semi- 383 dwarfism with a reduced sensitivity to brassinosteroids. Journal of Experimental Botany, 384 63, 4419-4436. 385 386 Gooding, M. J., Cannon, N.D., Thompson, A.J., & Davies, W.P. (1999). Quality and value of 387 organic grain from contrasting breadmaking wheat varieties and near isogenic lines 388 differing in dwarfing genes. Biological Agriculture & Horticulture, 16, 335-350. This article is protected by copyright. All rights reserved 389 390 Gupta, R., Khan, K., & MacRitchie, F. (1993). Biochemical basis of flour properties in bread 391 wheats. I. Effects of variation in the quantity and size distribution of polymeric protein. 392 Journal of Cereal Science, 18, 23-41. 393 394 Hogg, A.C., Gauss, K., Hofer, P., Martin, J.M., Graybosch, R.A., Hansen, L.E., & Giroux, M.J. 395 (2013). Creation of a high-amylose durum wheat through mutagenesis of starch synthase 396 II (SSIIa). Journal of Cereal Science, 57, 377-383. 397 398 Jobson, E. M‐ ., Martin‐, J.M., Schnei 399 Rht B1b, Rht D1b, and Rht‐der, T.M., & Giroux, M.J. (2018). The impact of the 8 wheat semi‐dwarfing genes on flour milling, baking, and 400 micronutrients. Cereal Chemistry, 95, 770-778. 401 402 Katyal, M., Virdi, A.S., Singh, N., Kaur, A., Rana, J.C., & Kumari, J. (2018). Diversity in 403 protein profiling, pasting, empirical and dynamic dough rheological properties of meal 404 from different durum wheat accessions. Journal of Food Science and Technology, 55, 405 1256-1269. 406 407 Kawaura, K., Mochida, K., & Ogihara, Y. (2005). Expression profile of two storage-protein gene 408 families in hexaploid wheat revealed by large-scale analysis of expressed sequence tags. 409 Plant Physiology, 139, 1870-1880. 410 411 Kim, H.S., & K. C. Huber. (2008). Channels within soft wheat starch A-and B-type granules. 412 Journal of Cereal Science, 48, 159-172. 413 414 Korzun, V., Röder, M.S., Ganal, M.W., Worland, A.J., & Law, C.N. (1998). Genetic analysis of 415 the dwarfing gene (Rht8) in wheat. Part I. Molecular mapping of Rht8 on the short arm of 416 chromosome 2D of bread wheat (Triticum aestivum L.). Theoretical and Applied 417 Genetics, 96, 1104-1109. 418 This article is protected by copyright. All rights reserved 419 Kusunose, C., Fujii, T., & Matsumoto, H. (1999). Role of starch granules in controlling 420 expansion of dough during baking. Cereal Chemistry, 76, 920-924. 421 422 Lanning, S. P., Martin, J.M., Stougaard, R.N., Guillen-Portal, F.R., Blake, N.K., Sherman, J.D., 423 Robbins, A.M., Kephart, K.D., Lamb, P., Carlson, G.R., Pumphrey, M., & Talbert, L.E. 424 (2012). Evaluation of near-isogenic lines for three height-reducing genes in hard red 425 spring wheat. Crop Science, 52, 1145-1152. 426 Larroque, O. R., Gianibelli, M.C., Batey, I.L., & MacRitchie, F. (1997). Electrophoretic 427 char‐acterisation of f‐ractions collected from gluten protein extracts subjected to 428 size exclusion high performance liquid chromatography. Electrophoresis, 18, 1064-1067. 429 430 Lee, N.Y. (2016). Effects of blends of low-protein winter wheat flour and barley byproducts on 431 quality changes in noodles. Preventive Nutrition and Food Science, 21, 1870-1880. 432 433 Malalgoda, M., Ohm, J.B., Meinhardt, S., & Simsek, S. (2018). Association between gluten 434 protein composition and breadmaking quality characteristics in historical and modern 435 spring wheat. Cereal Chemistry, 95, 226-238. 436 437 Mann, G., Diffey, S., Cullis, B., Azanza, F., Martin, D., Kelly, A., McIntyre, L., Schmidt, A., 438 Ma, W., Nath, Z., Kutty, I., Leyne, P.E., Rampling, L., Quail, K.J., & Morell, M.K. 439 (2009). Genetic control of wheat quality: interactions between chromosomal regions 440 determining protein content and composition, dough rheology, and sponge and dough 441 baking properties. Theoretical and Applied Genetics, 118, 1519-1537. 442 443 McVittie, J. A., Gale, M.D., Marshall, G.A., & Westcott, B. 1978. The intra-chromosomal 444 mapping of the Norin 10 and Tom Thumb dwarfing genes. Heredity. 40:67-70. 445 446 Mortazavi, A., Williams, B. A., McCue, K., Schaeffer, L., & Wold, B. (2008). Mapping and 447 quantifying mammalian transcriptomes by RNA-Seq. National Methods, 5, 621–628. 448 This article is protected by copyright. All rights reserved 449 Ohm, J. B., Hareland, ‐G., Simsek, S., & Seabourn, B. (2009). Size‐exclusion HPLC of protein 450 using a narrow bore column for evaluation of breadmaking quality of hard spring wheat 451 flours. Cereal Chemistry, 86, 463-469. 452 453 Oiestad, A. J., Martin, J. M., & Giroux, M. J. (2016). Overexpression of ADP-glucose 454 pyrophosphorylase in both leaf and seed tissue synergistically increase biomass and seed 455 number in rice Oryza sativa ssp. japonica. Functional Plant Biology, 43, 1194–1204. 456 457 Park, S. H., Bean, S.R., Chung, O.K., & Seib, P.A. (2006). Levels of protein and protein 458 composition in hard winter wheat flours and the relationship to breadmaking. Cereal 459 Chemistry, 83, 418-423. 460 461 Park, S. H., Chung, O.K., & Seib, P.A. (2005). Effects of varying weight ratios of large and 462 small wheat starch granules on experimental straight‐dough bread. Cereal Chemistry, 82, 463 166-172. 464 465 Payne, P. I. (1987). Genetics of wheat storage proteins and the effect of allelic variation on 466 bread-making quality. Annual Review of Plant Physiology, 38, 141-153. 467 468 Peng, J., Richards, D., & Hartley, N. (1999). ‘Green revolution’ genes encode mutant gibberellin 469 response modulators. Nature, 400, 256-261. 470 471 Peng, M., Gao, M., Abdel-A‐al, E.-S‐.M., Hucl, P., & Chibbar, R.N. (1999). Separation and 472 characterization of A and B type starch granules in wheat endosperm. Cereal Chemistry, 473 76, 375-379. 474 475 Petrofsky, K., & Hoseney, R. (1995). Rheological properties of dough made with starch and 476 gluten from several cereal sources. Cereal Chemistry, 72, 53-57. 477 478 Plessis, A., Ravel, C., Bordes, J., Balfourier, F., & Martre, P. (2013). Association study of wheat 479 grain protein composition reveals that gliadin and glutenin composition are trans- This article is protected by copyright. All rights reserved 480 regulated by different chromosome regions. Journal of Experimental Botany, 64, 3627- 481 3644. 482 483 Pomeranz, Y. (1988). Chemical composition of kernel structures. In: Pomeranz, Y. (Ed.) Wheat 484 Chemistry and Technology, col. 1. AACC International, St. Paul, MN, 97-158. 485 486 Raeker, M., Gaines, C.S., Finney, P.L., & Donelson, T. (1998). Granule size distribution and 487 chemical composition of starches from 12 soft wheat cultivars. Cereal Chemistry, 75, 488 721-728. 489 490 Rebetzke, G. & Richards, R. (2000). Gibberellic acid-sensitive dwarfing genes reduce plant 491 height to increase kernel number and grain yield of wheat. Crop and Pasture Science, 51, 492 235-246. 493 Sandstedt, R.M. (1955). Photomicrographic studies of wheat starch. III. Enzymatic digestion 494 and granule structure. Cereal Chemistry, 32, 17-47. 495 Sandstedt, R.M. (1961). The function of starch in the baking of bread. Bakers Digest, 35, 36-44. 496 497 Sherman, J. D., Nash, D., Lanning, S.P., Martin, J.M., Blake, N.K., Morris, C.F., & Talbert, L. 498 (2014). Genetics of end-use quality differences between a modern and historical spring 499 wheat. Crop Science, 54, 1972-1980. 500 501 Shewry, P. R., D’Ovidio, R., Lafiandra, D., Jenkins, J. A., Mills, E. N. C., & Bekes, F. (2009). 502 Wheat grain proteins. In K. Khan, & P. R. Shewry (Eds.). Wheat: Chemistry and 503 Technology (pp. 223–298). (4th ed.). St. Paul, M.N., U.S.A.: A.A.C.C. 504 505 Shewry, P., Halford, N., Belton, P., & Tatham, A. (2002). The structure and properties of gluten: 506 an elastic protein from wheat grain. Philosophical Transactions of the Royal Society of 507 London. Series B, Biological sciences, 357, 133-142. 508 509 Singh, N., Donovan, R., & MacRitchie, F. (1990). Use of sonication and size-exclusion high- 510 performance liquid chromatography in the study of wheat flour proteins. II. Relative This article is protected by copyright. All rights reserved 511 quantity of glutenin as a measure of breadmaking quality. Cereal Chemistry, 67, 16l- 512 11.70. 513 514 Sipes, K. K. (1993). Factors affecting protein and starch interaction. MS thesis. Kansas State 515 University, Manhattan, KS. 516 517 Soulaka, A. B., & Morrison, W.R. (1985). The amylose and lipid contents, dimensions, and 518 gelatinisation characteristics of some wheat starches and their A‐and B‐granule fractions. 519 Journal of the Science of Food and Agriculture, 36, 709-718. 520 521 Sourdille, P., Charmet, G., Trottet, M., Tixier, M.H., Boef, C., Negre, S., Barloy, D., & Bernard, 522 M. (1998). Linkage between RFLP molecular markers and the dwarfing genes Rht-B1 523 and Rht-D1 in wheat. Hereditas, 128, 41-46. 524 525 Tenea, G. N., Peres, A. B., Cordeiro, F. R., & Maquet, A. (2011). Reference genes for gene 526 expression studies in wheat flag leaves grown under different farming conditions. BMC 527 Research Notes 4:373. 528 529 Tsilo, T. J., Mudgil, D., & Khatkar, B.S. (2010). Association of size‐exclusion HPLC of 530 endosperm proteins with dough mixing and breadmaking characteristics in a recombinant 531 inbred population of hard red spring wheat. Cereal Chemistry, 87, 104-111. 532 533 Vermeylen, R., Goderis, B., Reynaers, H., & Delcour, J.A. (2005). Gelatinisation related 534 structural aspects of small and large wheat starch granules. Carbohydrate Polymers, 62, 535 170-181. 536 537 Wieser, H. (2007). Chemistry of gluten proteins. Food Microbiology, 24, 115-119. 538 539 Wieser, H., & Kieffer, R. (2001). Correlations of the amount of gluten protein types to the 540 technological properties of wheat flours determined on a micro-scale. Journal of Cereal 541 Science, 34, 19-27. This article is protected by copyright. All rights reserved 542 543 Wrigley, C., Bekes, F., & Bushnuk, W. (2006). Gluten: a balance of gliadin and glutenin. In: 544 Wrigley, C. W., Bekes, F., & Bushuk, W., Eds.; Gliadin and glutenin. The unique balance 545 of wheat quality. AACC International Press, St Paul, MN. pp 2-32. 546 547 Wrigley, C.W., & Bietz, J.A. (1988). Proteins and Amino Acids. In: Y. Pomeranz, Ed. Wheat 548 Chemistry and Technology. AACC, St. Paul, MN. pp 159–275. 549 550 Tables: 551 Table I: Impact of Rht semi-dwarfing alleles on gluten index, starch swelling, and flour 552 viscosity. 553 Flour Protein Gluten Starch Swelling RVAb Final RVA Peak (%) Index (%) Power (g/g)a Viscosityc Time (min) Rht-1a 15.0 ± 0.13a 74 ± 0.03c 23.4 ± 3.31a 159.5 ± 6.8b 15.8 ± 0.09c Rht-8 14.6 ± 0.19b 82 ± 0.03b 20.28 ± 2.42a 168.1 ± 11.2b 15.9 ± 0.13bc Rht-B1b 13.2 ± 0.05d 96 ± 0.03a 21.3 ± 5.42a 199.1 ± 35.7a 16.0 ± 0.09ab Rht-D1b 13.5 ± 0.11c 95 ± 0.02a 21.9 ± 2.94a 195.2 ± 13.7a 16.03 ± 0.19a LSD (0.05) 0.16 2.36 N.S. 16.9 0.08 554 555 a Starch swelling power reported as grams of water absorbed/grams of starch. 556 b RVA: Rapid Visco Analyser 557 c Final Viscosity measured in Rapid Visco Units. 558 N.S. no significant difference between groups. 559 Values represent the mean of combined rainfed and irrigated plots ± standard error. 560 Means followed by different letters within the same column are statistically different (P ≤ 0.05). 561 n = 10, where n represents one plot in either rainfed or irrigated conditions. 562 Flour protein previously reported in Jobson et al. (2018). 563 564 This article is protected by copyright. All rights reserved 565 566 567 568 Table II: Impact of Rht-1 semi-dwarfing alleles upon wheat flour storage protein distribution. SDS Extractable Mean % Area EP1 Rht-1a 15.63 ± 0.08a Rht-8 14.96 ± 0.26b Rht-B1b 15.13 ± 0.34ab Rht-D1b 15.18 ± 0.38ab LSD 0.54 EP2 Rht-1a 7.60 ± 0.25a Rht-8 7.46 ± 0.17a Rht-B1b 7.49 ± 0.21a Rht-D1b 7.45 ± 0.17a LSD 0.38 EP3 Rht-1a 35.84 ± 0.35a Rht-8 35.18 ± 0.12b Rht-B1b 32.70 ± 0.18c Rht-D1b 33.42 ± 0.20d LSD 0.42 EP4 Rht-1a 16.34 ± 0.65a Rht-8 16.86 ± 0.68a Rht-B1b 17.04 ± 0.48a Rht-D1b 17.00 ± 0.54a LSD 1.12 SDS Unextractable UP1 Rht-1a 12.73 ± 0.64c Rht-8 13.54 ± 0.68bc Rht-B1b 14.92 ± 0.48a Rht-D1b 14.65 ± 0.59ab This article is protected by copyright. All rights reserved 569 LSD 1.14 UP2 Rht-1a 3.80 ± 0.07c Rht-8 4.06 ± 0.06b Rht-B1b 4.39 ± 0.14a Rht-D1b 4.33 ± 0.07a LSD 0.17 UP3 Rht-1a 5.21 ± 0.57a Rht-8 5.04 ± 0.11a Rht-B1b 5.26 ± 0.32a Rht-D1b 5.02 ± 0.10a LSD 0.42 UP4 Rht-1a 3.07 ± 0.04b Rht-8 2.95 ± 0.09ab Rht-B1b 2.91 ± 0.11a Rht-D1b 2.84 ± 0.13ab LSD 0.19 570 571 Values represent the mean of irrigated plots ± standard deviation, n=3. 572 Means followed by different letters within the same column are statistically different (P ≤ 0.05). 573 EP: SDS Extractable proteins, higher content of low molecular weight subunits. 574 UP: SDS Unextractable proteins, higher content of high molecular weight subunits. 575 EP1, EP2, UP1, and UP2 primarily represent the polymeric proteins, EP3/UP3 represent the 576 gliadins, and EP4/UP4 represent albumins and globulins (Larroque et al., 1997; Malalgoda et al., 577 2018). This article is protected by copyright. All rights reserved Table III: Expression of wheat seed storage protein genes in developing grains 21 days past anthesis. Data is reported as the average reads per kilobase million (RPKM), n=3 individual plants grown under irrigated conditions, P-value represents a two tailed independent t test, all expression values were normalized to actin (Tenea et al., 2011). Rht-B1b Average Rht-1a Average Rht-B1b/ Protein Type Accession # P-value RPKM RPKM Rht-1a alpha gliadin U51306 12353 22366 0.28 0.55 alpha/beta gliadin M11075 52764 58102 0.54 0.91 Gliadins gamma gliadin M16064 79750 69377 0.68 1.15 omega gliadin AF280605 6708 9290 0.36 0.72 HMW x-type Bx7 DQ119142 16179 15119 0.82 1.07 Glutenins HMW x-type 1Dx5 X12928 9380 8377 0.70 1.12 HMW y-type 1Dy X03041 9250 10509 0.17 0.88 Control Actin AB181991 353 353 This article is protected by copyright. All rights reserved