bioRxiv preprint doi: https://doi.org/10.1101/2021.11.08.467769; this version posted November 8, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 Dosage of duplicated and antifunctionalized homeobox proteins influences spikelet development in barley Venkatasubbu Thirulogachandar1,5,*, , Geetha Govind1,a, Götz Hensel1,b, Sandip M. Kale1, Markus Kuhlmann1,5, Lennart Eschen-Lippold2,c, Twan Rutten1, Ravi Koppolu1, Jeyaraman Rajaraman1, Sudhakar Reddy Palakolanu1,f, Christiane Seiler1, Shun Sakuma3,d, Murukarthick Jayakodi1, Justin Lee2, Jochen Kumlehn1, Takao Komatsuda3,g, Thorsten Schnurbusch1,4,*, and Nese Sreenivasulu1,5,e,* 1 Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstr. 3, OT Gatersleben,D-06466 Stadt Seeland, Germany 2 Leibniz Institute of Plant Biochemistry (IPB), Weinberg 3, D-06120 Halle, Germany 3 National Institute of Agrobiological Sciences (NIAS), Plant Genome Research Unit, Tsukuba 3058602, Japan 4 Institute of Agricultural and Nutritional Sciences, Faculty of Natural Sciences III, Martin Luther University Halle-Wittenberg, 06120 Halle, Germany 5 Research Group Abiotic Stress Genomics, Interdisciplinary Center for Crop Plant Research (IZN), Hoher Weg 8, 06120 Halle (Saale), Germany Present address a Department of Crop Physiology, College of Agriculture, Hassan, 573225, Karnataka, India b Institute of Plant Biochemistry, Heinrich Heine University, 40225 Düsseldorf, Germany d Faculty of Agriculture, Tottori University, Tottori 680-8550, Japan c Department of Crop Physiology, Institute of Agricultural and Nutritional Sciences, Martin Luther University, 06120 Halle-Wittenberg, Germany e International Rice Research Institute (IRRI), Grain Quality and Nutrition Center, DAPO Box 7777, Metro Manila, Philippines f Cell, Molecular Biology and Trait Engineering Cluster, International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Hyderabad, 502 324, Telangana, India g Crop Research Institute, Shandong Academy of Agricultural Sciences (SAAS), Jinan, Shandong 250100, China * Corresponding authors Sreenivasulu, N. (n.sreenivasulu@irri.org) Schnurbusch, T. (schnurbusch@ipk-gatersleben.de) Thirulogachandar, V. (venkatasubbu@ipk-gatersleben.de) 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.11.08.467769; this version posted November 8, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 Lead contact Thirulogachandar, V (venkatasubbu@ipk-gatersleben.de)  Short title: HD-ZIP I TFs influence spikelet development The authors responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (https://academic.oup.com/plcell/pages/General-Instructions) are: Sreenivasulu, N. (n.sreenivasulu@irri.org); Schnurbusch, T. (schnurbusch@ipk-gatersleben.de); Thirulogachandar, V. (venkatasubbu@ipk-gatersleben.de) ORCiD Venkatasubbu Thirulogachandar - 0000-0002-7814-5475 Geetha Govind - 0000-0003-0450-0814 Sandip M. Kale - 0000-0003-0665-509 Markus Kuhlmann - 0000-0003-3104-0825 Lennart Eschen-Lippold - 0000-0001-8907-6922 Götz Hensel - 0000-0002-5539-3097 Twan Rutten - 0000-0001-5891-6503 Ravi Koppolu - 0000-0001-8566-9501 Jeyaraman Rajaraman - 0000-0003-0946-0508 Sudhakar Reddy Palakolanu – 0000-0002-5341-187X Christiane Seiler - 0000-0001-7181-9855 Shun Sakuma - 0000-0003-1622-5346 Murukarthick Jayakodi - 0000-0003-2951-0541 Justin Lee - 0000-0001-8269-7494 Dierk Scheel - 0000-0002-2105-6711 Jochen Kumlehn - 0000-0001-7080-7983 Takao Komatsuda Thorsten Schnurbusch - 0000-0002-5267-0677 Nese Sreenivasulu - 0000-0002-3998-038X 86 87 88 89 90 91 92 93 94 95 2 bioRxiv preprint doi: https://doi.org/10.1101/2021.11.08.467769; this version posted November 8, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 96 Abstract 97 Illuminating the mechanisms of inflorescence architecture of grain crops that feed our 98 world may strengthen the goal towards sustainable agriculture. Lateral spikelet 99 development of barley (Hordeum vulgare L.) is such an example of a floral architectural 100 trait regulated by VRS1 (Vulgare Row-type Spike 1 or Six-rowed Spike 1, syn. 101 HvHOX1). Its lateral spikelet-specific expression and the quantitative nature of 102 suppressing spikelet development were previously shown in barley. However, the 103 mechanistic function of this gene and its paralog HvHOX2 on spikelet development is 104 still fragmentary.Here, we show that these duplicated transcription factors (TFs) have 105 contrasting nucleotide diversity in various barley genotypes and several Hordeum 106 species. Despite this difference, both proteins retain their basic properties of the 107 homeodomain leucine zipper class I family of TFs. During spikelet development, these 108 genes exhibit similar spatiotemporal expression patterns yet with anticyclic expression 109 levels. A gene co-expression network analysis suggested that both have an ancestral 110 relationship but their functions appear antagonistic to each other, i.e., HvHOX1 111 suppresses whereas HvHOX2 rather promotes spikelet development. Our transgenic 112 promoter-swap analysis showed that HvHOX2 can restore suppressed lateral spikelets 113 when expression levels are increased; however, at its low endogenous expression 114 level, HvHOX2 appears dispensable for spikelet development. Collectively, this study 115 proposes that the dosage of the two antagonistic TFs, HvHOX1 and HvHOX2, 116 influence spikelet development in barley. 117 118 Keywords 119 Inflorescence architecture, lateral spikelet, HD-ZIP class I transcription factors, 120 duplication, antagonistic transcription factors, antifunctionalization, 121 transcription factors, RNA-guided Cas9 endonuclease, site-directed mutagenesis, 122 nucleotide diversity, dosage of expression. 123 124 125 126 127 3 homeobox bioRxiv preprint doi: https://doi.org/10.1101/2021.11.08.467769; this version posted November 8, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 128 Introduction: 129 Cereals such as maize (Zea mays L.), rice (Oryza sativa L.), wheat (Triticum spp.), and 130 barley (Hordeum vulgare L.) are major grass species that feed most of the population 131 on earth. Understanding the genetic regulation of inflorescence (flower-bearing 132 structure) architecture in these cereal crops may shed light on the basic developmental 133 patterning of floral meristems and reveal potential pathways to improve their yield. 134 Barley, along with other major cereal crops (wheat, rye, and triticale) belonging to the 135 Triticeae tribe, possesses a branchless inflorescence known as ‘spike’ (Ullrich, 2011; 136 Koppolu and Schnurbusch, 2019). In general, a barley spike forms three spikelets on 137 its rachis (inflorescence axis) nodes – one central and two lateral spikelets in an 138 alternating, opposite arrangement (distichous) (Bonnett, 1935; Koppolu and 139 Schnurbusch, 2019; Zwirek et al., 2019). The spikelet, a small/condensed spike, is 140 considered the basic unit of the grass inflorescence (Clifford et al., 1987; Kellogg et al., 141 2013). A barley spikelet forms a single floret that is subtended by a pair of glumes. 142 Typically, a barley floret consists of one lemma, one palea, two lodicules, three 143 stamens, and a monocarpellary pistil (i.e., single carpel) (Waddington et al., 1983; 144 Forster et al., 2007). Based on the fertility of the lateral spikelets/florets, barley is 145 classified into two- and six-rowed spike types. In two-rowed types, the lateral spikelets 146 are smaller (compared to the central spikelets), awnless (extension of the lemma is 147 absent), and sterile, while the central spikelets are bigger, awned, and fertile. In six- 148 rowed types, both the lateral and central spikelets are awned and fertile. 149 The major gene responsible for the lateral spikelet fertility was found to be a 150 homeodomain leucine zipper class I (HD-ZIP I) transcription factor, known as VRS1 151 (Vulgare Row-type Spike1 or Six-rowed Spike 1, syn HvHOX1) (Komatsuda et al., 152 2007). Transcripts and proteins of HvHOX1 had previously been found in barley spikes, 153 predominantly in the lateral florets and most strongly in the carpels, corroborating a 154 role of HvHOX1 as negative regulator of lateral floret development and fertility 155 (Komatsuda et al., 2007; Sakuma et al., 2010; Sakuma et al., 2013). Recently, a very 156 similar function has also been identified for its orthologous wheat gene during apical 157 floret abortion(Sakuma et al., 2019). In recent years, HvHOX1 was shown to be also 158 expressed in other organs, such as leaves, where in analogy to its effects on lateral 159 spikelet development, it negatively affects the size of leaf primordia and results in 160 narrower leaves in two-rowed barleys (Thirulogachandar et al., 2017). Further 161 supporting its suppressive function, one specific allele of HvHOX1 is responsible for 4 bioRxiv preprint doi: https://doi.org/10.1101/2021.11.08.467769; this version posted November 8, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 162 the extremely reduced lateral spikelet size in deficiens barley (Sakuma et al., 2017). 163 Interestingly, HvHOX2, the paralog of HvHOX1, was also identified in barley. Although 164 HvHOX2 is expressed in a wide variety of organs including leaves, coleoptile, root, and 165 spike; tissue-wise, it is mainly found in vascular regions particularly those at the base 166 of lateral spikelets (pedicel) and rachis, thus suggesting a role in the promotion of 167 development (Sakuma et al., 2010; Sakuma et al., 2011; Sakuma et al., 2013). In 168 addition to HvHOX1, four other genes, VRS2, VRS3, VRS4, and VRS5 or INT-C 169 (intermedium-spike c), were reported to be involved in the suppression of lateral 170 spikelet fertility (Ramsay et al., 2011; Koppolu et al., 2013; Bull et al., 2017; van Esse 171 et al., 2017; Youssef et al., 2017). Notably, VRS4, the ortholog of maize RAMOSA2 172 (RA2) appeared to be functionally upstream of HvHOX1 but not of HvHOX2 (Koppolu 173 et al., 2013; Sakuma et al., 2013). Later, VRS3 was also identified as an upstream 174 regulator of HvHOX1, and in certain stages also of HvHOX2 (Bull et al., 2017; van 175 Esse et al., 2017). 176 Despite the detailed studies on HvHOX1’s expression pattern and mutants, the 177 mechanistic role of HvHOX1 on barley spikelet development is still unclear. The same 178 holds true for HvHOX2 while its suggested role in barley development has yet to be 179 validated(Sakuma et al., 2010; Sakuma et al., 2013). In this study, we show that 180 HvHOX1 and HvHOX2 proteins are functional HD-ZIP class I transcription factors. Our 181 transcript expression studies suggest that both have similar spatiotemporal expression 182 patterns; however, with a contrasting dosage of transcripts in central and lateral 183 spikelets during spikelet development. Based on our combined results, we conclude 184 that both genes are ancestrally related but act antagonistically to each other, i.e., 185 HvHOX1 suppresses whereas HvHOX2 rather promotes spikelet development. Our 186 transgenic promoter-swap analysis shows that HvHOX2 can restore suppressed 187 lateral spikelets when transcript levels are increased, most likely, by modulating the 188 adverse effects caused by HvHOX1. At low endogenous transcript levels, however, 189 HvHOX2 appears dispensable for spikelet development. Collectively, our findings 190 recommend that HvHOX1 and HvHOX2 act antagonistic to each other, and that the 191 dosage of their transcripts influences barley spikelet development. 192 193 5 bioRxiv preprint doi: https://doi.org/10.1101/2021.11.08.467769; this version posted November 8, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 194 Results: 195 HvHOX2 nucleotide diversity is highly conserved compared to its paralog 196 HvHOX1 197 The eight different natural alleles for HvHOX1 known so far are grouped into two-rowed 198 (Vrs1.b2, Vrs1.b3, Vrs1.b5, & Vrs1.t1) and six-rowed alleles (vrs1.a1, vrs1.a2, vrs1.a3, 199 & vrs1.a4) (Komatsuda et al., 2007; Sakuma et al., 2017; Casas et al., 2018). In 200 contrast, the nucleotide diversity of HvHOX2 is largely unknown. To fill this gap, we 201 sequenced the HvHOX2 promoter (one kb) and gene (including 5’ and 3’ untranslated 202 region) in 83 diverse spring barleys (44 two-rowed and 39 six-rowed). Surprisingly, we 203 found only four single nucleotide polymorphisms (SNPs), restricted to the promoter 204 (two SNPs), 5’UTR (one SNP), and intron-2 (one SNP). At the same time, the coding 205 sequence (CDS) was identical and highly conserved in all these accessions 206 (Supplementary Table 1). We further expanded our nucleotide diversity study by 207 sequencing the HvHOX1 and HvHOX2 in 24 Hordeum spp. (Supplementary Table 2), 208 which showed that the non-synonymous (Ka) and synonymous (Ks) substitution values 209 of HvHOX1 (Ka = 0.028, Ks = 0.049) and HvHOX2 (Ka = 0.008, Ks = 0.051). The 210 higher Ka value of HvHOX1 than that of HvHOX2 indicates that the evolutionary speed 211 of HvHOX1 is much faster than that of HvHOX2, otherwise, HvHOX2 has been well 212 conserved among the Hordeum species (Supplementary Table 3). A subsequent 213 comparison of the nucleotide diversity (π) of these two genes (HvHOX2, Chr.2H: 214 139932435-139953386; 215 domesticated barleys(Jayakodi et al., 2020) confirmed the lower nucleotide diversity 216 (π) of HvHOX2 compared to HvHOX1 (Supplementary Fig. 1A). The study also 217 revealed two major haplotypes for the HvHOX2 genic region, whereas HvHOX1 218 possesses multiple haplotypes that span the whole region analyzed (Supplementary 219 Fig. 1B). This difference in diversity might be due to their physical location, wherein 220 HvHOX1 is located in the distal end of the high recombining region of chromosome 221 2H, while HvHOX2 is closer to the centromeric region on 2H. Concertedly, all the above 222 results indicate that HvHOX2 is highly conserved compared to its paralog HvHOX1. HvHOX1, Chr.2H: 581356498-581377358) in 200 223 224 HvHOX1 and HvHOX2 are functional HD-ZIP class I transcription factors 225 In general, members of the HD-ZIP family (class I to IV) of transcription factors possess 226 a homeodomain (HD) followed by a leucine zipper motif (LZ). The LZ motif enables the 227 dimerization of HD-ZIP proteins, which bind to their specific DNA target (cis-element) 6 bioRxiv preprint doi: https://doi.org/10.1101/2021.11.08.467769; this version posted November 8, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 228 via the HD motif (Ariel et al., 2007). The HD-ZIP class I proteins - HvHOX1 and 229 HvHOX2 show a very high sequence identity between their HD (89.3 %) and LZ (90 %) 230 motifs. However, they have several amino acid changes across their protein with yet 231 unknown consequences (Supplementary Fig. 2). In particular, HvHOX1 lacks a 232 putative AHA-like motif in its C-terminus, which was predicted to be an interaction motif 233 with the basal transcriptional machinery (Arce et al., 2011; Capella et al., 2014) 234 (Supplementary Fig. 2). All these similarities and discrepancies paved the way to 235 compare the functionality of these two proteins. 236 Figure 1: HvHOX1 and HvHOX2 are functional HD-ZIP class I transcription factors. Bimolecular fluorescence complementation assay (BiFC) for HvHOX1 and HvHOX2 proteins is shown (A). The bright field panel displays the protoplast in which the results were captured; YFP (Yellow Fluorescent Protein) panel reveals the dimer formation with the yellow color fluorescence, and CFP (Cyan Fluorescent Protein) panel discloses the location of the nucleus (blue, dark spot), and the autofluorescence of chlorophyll (red signal) is seen in the chlorophyll panel. The last overlay panel exhibits the merged signals from the above three panels. nYFPYFP fused to N-terminal; cYFP- YFP fused to C-terminal end. Scale bar 10 µm. B) The DNA binding specificity of HvHOX1 and HvHOX2 proteins on HD-Zip I cis-element assessed by Electro Mobility Shift Assay (EMSA) is shown. Three different concentrations (0.5 µL, 1 µL, and 2 µL) of protein were used along with the DNA fragment containing the HD-Zip I cis-element (Binding sequence, BS). The shift of protein-DNA complex (*) denotes the specific DNA binding of these proteins. Also, a combination of HvHOX1 and HvHOX2 proteins (1 µL from each) also shows the protein-DNA complex. Dihydrofolate reductase (DHFR) was used as a negative control. BS- binding sequence (HD-Zip I cis element); Free BS- unbound BS; different numbers show the in vitro translated protein volume in µL. C) The transactivation property of HvHOX1 and HvHOX2 proteins is shown. Bar plot indicates the detected GUS activity relative to luciferase (LUC). Data shown are mean ± SE (n=3); different letters (a, b, c, and d) indicate that the mean values are significantly different at the 1% probability level, by One-way ANOVA with Newman-Keuls Multiple Comparison Test; EV- empty vector, pGAL4-4xUAS::GUS; HvHOX1- construct of GAL4DNA binding domain fused to N-terminus of HvHOX1; HvHOX2- GAL4-DNA binding domain fused to N-terminus of HvHOX2; LUC- luciferase used for normalization; GUS- β-glucuronidase 237 238 We assessed the dimerization properties of HvHOX1 and HvHOX2 with the 239 bimolecular fluorescence complementation assay. HvHOX1 and HvHOX2 were cloned 240 into split-Yellow Fluorescence Protein (YFP) vectors creating N-terminal c-myc-nYFP 241 and HA-cYFP fusions. The resulting plasmids were co-transformed with a Cyan 242 Fluorescent Protein (CFP) construct into Arabidopsis mesophyll protoplasts. The CFP 243 served as a transformation control, accumulating in the nucleus and cytoplasm. The 244 detection of yellow fluorescence in all four combinations indicated that the HvHOX1 245 and HvHOX2 proteins are able to form homo- or heterodimers (Fig. 1A). The 246 superimposed YFP channel (dimerization) on the CFP channel (strong nuclear signal) 7 bioRxiv preprint doi: https://doi.org/10.1101/2021.11.08.467769; this version posted November 8, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 247 indicated that homo- or heterodimers of both proteins are localized in the nucleus (Fig. 248 1A), which is in agreement with the nuclear localization signals predicted for both 249 proteins (Sakuma et al., 2013). This localization also implied that these dimers might 250 bind to their cis-elements to transactivate their downstream genes (Fig. 1A). A western 251 blot analysis using antibodies directed against HA and c-myc epitopes confirmed that 252 the proteins were expressed in full-length and at similar levels (Supplementary Fig. 3). 253 Following, we verified the DNA binding properties of HvHOX1, and HvHOX2 with an 254 electromobility shift assay (EMSA) using the in vitro translated proteins and 255 experimentally verified HD-Zip I cis-element from Sessa et al. (1993)(Sessa et al., 256 1993). A clear shift of protein-DNA bands (marked with *) was detected for both 257 proteins, especially in higher concentrations of proteins, which indicated binding to the 258 HD-Zip I cis-element (Fig. 1B). The result further suggested that HvHOX1 might have 259 a more potent DNA binding property than HvHOX2 (Fig. 1B). We then conducted a cis- 260 element competition assay to evaluate the binding specificity of the proteins to the HD- 261 Zip I cis-element. Intriguingly, we observed binding of HvHOX1 to HvHOX2 promoter 262 and mild interactions of HvHOX2 with the HvHOX1 and HvHOX2 promoters 263 (Supplementary Fig. 4). This suggests that in vivo, HvHOX1 potentially influences 264 HvHOX2 expression, similarly, HvHOX2 modulates HvHOX1 expression. 265 After the dimerization and DNA binding studies, we investigated the transactivation 266 property of these proteins in vivo using an Arabidopsis mesophyll protoplast system. 267 We found that both proteins have transactivating properties, which were quantified and 268 compared with the empty vector. Interestingly, the transactivation property of HvHOX2 269 was significantly higher compared to that of HvHOX1 (Fig. 1C). Collectively, all of the 270 above results exemplified that both HvHOX1 and HvHOX2 possess DNA binding 271 activity, can form homo- and heterodimers, and have transactivation potential, which 272 corroborated that both proteins are functional HD-ZIP class I transcription factors. 273 274 Two-rowed spikes have delayed lateral spikelet initiation and reduced growth 275 The size and fertility of lateral spikelets determine the row-type and intermedium-spike 276 types in barley (Komatsuda et al., 2007; Ramsay et al., 2011; Youssef et al., 2017; 277 Zwirek et al., 2019). To comprehend the differences of lateral and central spikelets in 278 two-rowed barley, we tracked these spikelets from their early initiation until pollination 279 in the two-rowed cv. Bowman. Barley spike development starts from the double ridge 280 (DR) stage, in which spikelet ridges are subtended by leaf ridges (Fig. 2A). In the next 8 bioRxiv preprint doi: https://doi.org/10.1101/2021.11.08.467769; this version posted November 8, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 281 stage, known as ‘triple mound’ (TM), the spikelet ridge differentiates into one central A B C S LSM LS CSM LS D LSM F C S G G HvHOX1 64 G L 64 ns ns ** ns 0.25 0.0625 ** 16 4 1 TM GP LP SP AP 0.0625 ns 4 *** * * *** 1 0.25 0.25 DR Lateral Central 16 Expression value (tpm) (log2 transformed) 1 * Expression value (tpm) (log2 transformed) Expression value (tpm) (log2 transformed) ns 4 I Lateral Central * *** A G H ns ** G G HvHOX2 16 S S L G LSM E LS G G L TM GP LP SP AP 0.0625 TM GP LP SP AP Figure 2: Two-rowed spikes have delayed lateral spikelet initiation and reduced growth. Early spike developmental stages of a two-rowed cultivar Bowman are shown from A to F. Double ridge (DR) is shown in A, in which the spikelet ridge (SR) is subtended by a leaf ridge (LR). The SR differentiates into one central and two lateral spikelet meristems (CSM & LSM) at triple mound stage (TM), which is displayed in B. Panel C discloses the appearance of two glume primordia (GP) from the CSM, while the two LSMs do not show any sign of differentiation. Subsequently, the CSM further differentiates and forms a lemma primordium (LP), which is shown in D. Two GP and a sign of LP initiation from the LSM can be seen in the panel E; the CSM initiated three stamen primordia along with a sign of carpel primordia development at this stage. At awn primordium stage (AP), F, the CSM completed the formation of all floral organ primordia (including the carpel), and the AP initiates from the medial point of the LP. However, the laterals are found only with two GP and a LP. Panels G, H, and I depicts the expression pattern of HvHOX1 and HvHOX2 genes in the whole spikes of DR to AP stages, HvHOX1 and HvHOX2 in the central and lateral spikelets of TM to AP stages, respectively. HvHOX1 expresses higher than HvHOX2 in the whole spikes of GP, LP, and SP stages (G). Both the genes are expressed in the dissected central and lateral spikelets from TM to AP stages. Mean values of G-I are compared with the multiple Student’s t-test; *, **, ***, mean values are significantly different at 5, 1, and 0.1% probability levels; ns-not significantly different. Scale bar in panel A whole spike 500 µm, magnified three nodes 100 µm; B & C-500 µm & 200 µm; D- 500 µm & 100 µm; E & F-200 µm & 100 µm. W-Waddington scale. 282 and two lateral spikelet meristems (CSM & LSM), in which the CSM develops as a 283 bigger structure compared to the two LSMs (Fig. 2B). This marks the first difference 284 between the central and lateral spikelets. Following the TM stage, the CSM continues 285 to differentiate into various spikelet/floret organ primordia (glume, lemma, palea, 286 stamen, pistil, and awn) (Fig. 2C-F). From the glume primordium stage, however, the 9 bioRxiv preprint doi: https://doi.org/10.1101/2021.11.08.467769; this version posted November 8, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 287 LSM exhibits a delayed differentiation indicating the suppression of LSM (Fig. 2C). At 288 the awn primordium stage (AP), the central spikelets have completed the differentiation 289 of all spikelet/floret organs, while the laterals only achieved the differentiation of glume 290 and lemma (Fig. 2F). We also compared the development of lateral spikelets between 291 the two-rowed cv. Bowman and its near-isogenic six-rowed line BW-NIL(vrs1.a) (Druka 292 et al., 2011). Close to the white anther stage (Kirby and Appleyard, 1984), the 293 difference between the laterals of two- and six-rowed spikes became apparent (Fig. 294 3A-D). The six-rowed laterals possessed primordia for all spikelet/floret organs, 295 whereas in two-rowed, laterals had retarded awn and pistil primordia (Fig. 3C & D). We 296 also verified the divergence of lateral spikelet development in another pair of two- (cv. 297 Bonus) and six-rowed (hex-v.3, vrs1 deletion mutant) barleys (Supplementary Fig. 5). 298 To fathom the sterility of lateral spikelets, we compared the histology of pistil and anther 299 growth in Bowman and its vrs1.a mutant [BW-NIL(vrs1.a)] from Waddington stage 4.5 300 (W4.5, awn primordium stage) to W10.0 (pollination) (Supplementary Fig. 6&7). The 301 delayed differentiation of lateral spikelets observed during the spikelet initiation stages 302 (TM to AP) continued in the growth stages of the reproductive organs (Fig. 3E-L). 303 Anthers of two-rowed lateral spikelets showed an impeded differentiation compared to 304 the anthers of other spikelets (Supplementary Fig. 6, A3-J3). However, the central 305 spikelet anthers of two- (Supplementary Fig. 6, A1-J1) and six-rowed (Supplementary 306 Fig. 6, A2-J2) exhibited an advanced progression rate across the stages. Notably, the 307 six-rowed lateral anther (Supplementary Fig. 6, A4-J4) followed a differentiation rate 308 between the two- and six-rowed centrals as well as the two-rowed laterals, indicating 309 that there are additional suppressors of lateral spikelet development besides HvHOX1. 310 Moreover, anthers of the two-rowed lateral spikelets stopped differentiation at W7.5 311 (Supplementary Fig. 6, E3), followed by tissue disintegration in the subsequent stages 312 (Supplementary Fig. 6, E3 to J3). In contrast, all other anthers continued their growth 313 towards pollination (Supplementary Fig. 6). A similar delay of differentiation and 314 disintegration of tissues was also observed in the pistil of two-rowed laterals at W7.5 315 (Supplementary Fig. 7, C5). Concertedly, these results substantiate that two-rowed 316 spikes have delayed lateral spikelet initiation and suppressed growth compared to their 317 central and all the spikelets of six-rowed spikes. Eventually, the reproductive organs of 318 the lateral spikelets in two-rowed cv. Bowman abort during the later growth phase. 319 10 bioRxiv preprint doi: https://doi.org/10.1101/2021.11.08.467769; this version posted November 8, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 320 HvHOX1 and HvHOX2 have a contrasting dosage of expression during spikelet 321 initiation and growth 322 We have taken the log2 transformed expression values of HvHOX1 and HvHOX2 from 323 the Bowman RNA-seq spike atlas data (Thiel et al., 2021) and reanalyzed them to find 324 their expression pattern across the spikelet initiation stages (Fig. 2G-I). In the tissue- 325 unspecific (central and lateral combined or whole spike) transcript analysis, both genes 326 showed a linear increase in expression along with the spikelet initiation stages (Fig. 327 2G). With the exception of the DR stage, HvHOX1 generally displayed higher transcript 328 levels than HvHOX2 (TM to AP). This was particularly evident in glume primordium 329 (GP), lemma primordium (LP), and stamen primordium (SP) stages (Fig. 2G). We then 330 compared the tissue-specific expression patterns of these genes in central and lateral 331 spikelets. Notably, both genes had higher levels of mRNA in the laterals than centrals 332 at TM, LP, and stamen primordium (SP) stages (Fig. 2H & I). Then, we compared the 333 expression level of these genes within the same tissues (central and lateral spikelets), 334 in which HvHOX1 showed significantly higher expression than HvHOX2 in several 335 stages (TM, LP, & SP) of lateral and at the SP stage of central spikelets 336 (Supplementary fig. 8A & B). The high expression of HvHOX1 in the laterals correlates 337 with the delayed differentiation and suppression of the lateral spikelets (compared to 338 the centrals) from the TM to AP stages in Bowman (Supplementary Fig. 8B). This 339 reinforced the role of HvHOX1 as a negative regulator of lateral spikelet development 340 in barley (Komatsuda et al., 2007; Sakuma et al., 2010; Sakuma et al., 2013). The 341 presence of HvHOX1 transcripts in central spikelets of two-rowed barleys, which are 342 fertile and do not show any developmental disorder, poses a question that has yet to 343 be solved (Komatsuda et al., 2007; Sakuma et al., 2010; Sakuma et al., 2013) (Fig. 344 2C-F, Supplementary Fig. 7&8). 345 Following the comparison on spikelet initiation stages, we explored expression levels 346 of these genes also in the spikelet growth stages of Bowman and BW-NIL(vrs1.a) (non- 347 functional HvHOX1) by doing a quantitative real-time (qRT) PCR with tissue-unspecific 348 (W5.0, W5.5, & W6.0) and tissue-specific (W7.5, W8.5, W10.0) samples (Fig. 3M-R). 349 Also, in these later stages of development, HvHOX1 exhibited significantly higher 350 expression than HvHOX2 in the whole spike at W5.0, W5.5, and W6.0, both in Bowman 351 and BW-NIL(vrs1.a) (Fig. 3M & N). Intriguingly, HvHOX1 displayed a reduced 352 expression trend both in the central and lateral spikelets of Bowman from W7.5 to 11 bioRxiv preprint doi: https://doi.org/10.1101/2021.11.08.467769; this version posted November 8, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 353 W10.0 (Fig. 3O & Q). Contrastingly, HvHOX2 had an increasing trend of expression in 354 these two tissues of Bowman (Fig. 3O & Q). More importantly, HvHOX2 showed 355 356 357 358 359 360 361 362 363 364 Figure 3: Two-rowed spikes have delayed and reduced lateral spikelet development compared to its central and six-rowed lateral spikelets. Images of panel A & C are the W5.5 stage inflorescence meristem of six-rowed mutant BW-NIL(vrs1.a), and B & D are from the two-rowed progenitor Bowman. Development of different organ primordia (AP, SP, CP, & GP) in central spikelets (yellow color) of two-rowed (D) and six-rowed (C) are visibly similar. Awn primordium (AP) and carpel primordium (CP) are formed only in lateral spikelets (blue color) of six-rowed (C) and not in two-rowed (D, marked with red arrow heads). Cross sections of anthers and carpels of BW-NIL(vrs1.a) and Bowman are shown in E-L. The W7.5 stage central spikelet anthers (E & G) and carpels (I & K) of BW-NIL(vrs1.a) and Bowman display normal development, while the lateral spikelet anther (H) and carpel (L) of Bowman show suppressed and aborted development. However, the lateral spikelet anther (F) and carpel (J) of BW-NIL(vrs1.a) seems developing normally but comparatively slower than its central spikelet organs. Expression of HvHOX1 and HvHOX2 genes in the whole spike (M & N), central spikelet (O & P), and lateral spikelet (Q & R) of Bowman and BW-NIL(vrs1.a) is shown, respectively. In whole spike of W5.0 to W6.0 stages, HvHOX1 expressed greater than HvHOX2 both in Bowman (M) and BW-NIL(vrs1.a) (N). Contrastingly, the HvHOX2 showed stronger expression than HvHOX1 in the Bowman central spikelets of W7.5 to W10.0 (O); however, in BW-NIL(vrs1.a), HvHOX2’s expression was higher only in W8.5. In the Bowman lateral spikelets of W7.5 to W10.0, HvHOX1 and HvHOX2 exhibited an anticyclic expression pattern, i.e., when HvHOX1’s expression dropped down from W7.5 to W10.0, HvHOX2’s expression started increasing. Mean values of M-R are compared with the multiple Student’s t-test; *, **, ***, mean values are significantly different at 5, 1, and 0.1% probability levels; ns-not significantly different. orange- stamen primordium; blue- carpel primordium; green: glume primordium; pink: lemma primordium; purple: palea 12 primordium; W-Waddington scale. Scale bar, A&B, 800 µm; C&D, 200 µm; E-L is 100 µm. bioRxiv preprint doi: https://doi.org/10.1101/2021.11.08.467769; this version posted November 8, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 365 greater expression than HvHOX1 in the centrals (Fig. 3O), whereas in the laterals, 366 HvHOX1 had a superior level of expression in the first two stages (W7.5 and W8.5), 367 followed by the increase of HvHOX2 at W10.0 (Fig. 3Q). Crucially, the transcript levels 368 of HvHOX1 were gradually decreased from W7.5, while HvHOX2 levels increased. 369 Similar to the Bowman centrals, HvHOX2 showed a higher trend of expression in BW- 370 NIL(vrs1.a) central spikelets. However, the expression patterns of these two genes 371 were different in BW-NIL(vrs1.a) lateral spikelets compared to Bowman (Fig. 3P & R). 372 The antagonistic expression patterns of HvHOX1 and HvHOX2, i.e., when HvHOX2 373 expression goes up, HvHOX1 expression turns down, suggests that these two genes 374 might act anti-cyclic during the later growth stages. Based on this observation and the 375 higher expression of HvHOX2 (W7.5 to W10.0) in the Bowman central spikelets (Fig. 376 3O) that show no developmental and growth aberration, we hypothesized that 377 overexpression of HvHOX2 might promote spikelet development by acting as a 378 positive regulator of spikelet development. 379 380 Promoters of HvHOX1 and HvHOX2 share similar spatiotemporal expression 381 patterns during spike growth stages 382 The expression studies of HvHOX1 and HvHOX2 (Fig. 2G-I & 3M-R) exemplified that 383 these genes have similar temporal expression during the spikelet initiation and growth 384 stages though at different amplitudes. Additionally, their central- and lateral-specific 385 transcript levels indicated that they might also share spatial boundaries across the 386 initiation and growth stages. To verify their spatial co-localization and similar temporal 387 expression patterns, promoters (HvHOX2-1929 bp; HvHOX1-991 bp) of these genes 388 were fused with a synthetic GFP (GFP) coding sequence and transformed into the two- 389 rowed cv. Golden Promise. Five and eight independent transgenic events showed GFP 390 accumulation in the T0 generation for HvHOX1, and HvHOX2 GFP constructs, 391 respectively. Three independent events from both the constructs were selected, and 392 their GFP accumulation was confirmed until T2 generation. As expected, we found that 393 promoter activity of these genes in identical tissues like the base of the central 394 spikelet’s carpel (Fig. 4A & D), the tapetal layer of the central spikelet’s anther (Fig. 4B 395 E), and rudimentary lateral anthers (Fig. 4C & F) at W8.5 stage. Collectively, the tissue- 396 specific expression analysis and the promoter activity in the transgenic plants 397 suggested that HvHOX1 and HvHOX2 might have similar spatiotemporal expression 398 patterns during spikelet growth stages. 13 bioRxiv preprint doi: https://doi.org/10.1101/2021.11.08.467769; this version posted November 8, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. Figure 4. HvHOX1 and HvHOX2 have similar spatiotemporal expression pattern during spike growth and development. HvHOX2 promoter activity (GFP expression) in central spikelet’s stamen and carpel (A), tapetum of central spikelet’s stamen (B) and lateral spikelets’ stamen (C) at W8.5 is shown. Similarly, HvHOX1 promoter activity in central spikelets’ carpel (D), tapetum of central spikelet’s stamen (E) and lateral spikelet’s stamen (F) is shown. Green color - GFP fluorescence; red color- chlorophyll autofluorescence. Scale bar 100 µm. WWaddington scale. 399 HvHOX1 has a unique co-expression module apart from a shared module with 400 HvHOX2 during spike development 401 In an effort to predict the role of HvHOX1 and HvHOX2 genes, we constructed their 402 co-expression signatures from the transcript profiles across six spikelet initiation and 403 growth stages (W2.5, W3.0, W4.5, W6.5, W7.5, and W8.5) in Bowman. We found 404 twenty co-expression modules from a set of 7,520 genes that showed a dynamic 405 expression profile (Fig. 5A & B). HvHOX1 and HvHOX2 genes clustered together in 406 one module (Figure 5A; red) along with 4,213 genes. A weighted gene co-expression 407 network analysis (WGCNA) revealed that HvHOX1 shares one part of its co- 408 expression module (Fig. 5C, shown in blue, 16 genes) with HvHOX2, while HvHOX1 409 has unique co-expressed signatures (Fig. 5C, shown in orange, 39 genes). Most 410 importantly, HvHOX2 is one of the co-expressed genes within the HvHOX1 module 411 (Fig. 5C). In other words, both genes share a similar expression signature across spike 412 development. This supports our previous transcript and GFP analyses and suggests 413 that these genes have similar spatiotemporal expression patterns. Furthermore, 414 hierarchical clustering (HCL), divided the genes in the shared module (Supplementary 14 bioRxiv preprint doi: https://doi.org/10.1101/2021.11.08.467769; this version posted November 8, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. Figure 5. Dendrogram from gene co-expression network analysis of two-rowed cv. Bowman spike tissues. Modules of the co-expressed genes were assigned colors, shown by the horizontal bars below the dendrogram. Merged modules are shown under the dynamic module profile (A). Expression heat map of the red module is shown in (B) and the coexpressed gene clusters of HvHOX1 and HvHOX2 are shown in (C). 415 fig. 9A; blue) into two sub-clusters based on their expression in central and lateral 416 spikelets, but not the unique HvHOX1 co-expressed module (Supplementary Fig. 10B). 417 This indicates that HvHOX1 may play a specific role in the lateral spikelets, while 418 HvHOX2 probably has a different function from HvHOX1. Interestingly, the shared 419 module was enriched with genes [e.g. AGAMOUS (AG), SUPPRESSOR OF 420 OVEREXPRESSION OF CONSTANS 1 (SOC1), ENOLASE 1 (ENO1), and AUXIN F- 421 BOX PROTEIN 5 (AFB5)] associated with flower development, promotion of flowering, 422 carpel and stamen identity, auxin signaling, transcription and nitrate assimilation 423 (Covington and Harmer, 2007; Dreni and Kater, 2014; Hyun et al., 2016; Gaufichon et 424 al., 2017). The HvHOX1 unique co-expressed module, on the other hand, was 425 enriched in genes [such as BREVIPEDICELLUS 1 (BP1), WRKY 12, NOVEL PLANT 426 SNARE 11 (NPSN11), FORMIN HOMOLOGY 14 (AFH14), LONELY GUY 3 (LOG3), 427 and G PROTEIN ALPHA SUBUNIT 1 (GPA1)] that are predicted to be involved in 15 bioRxiv preprint doi: https://doi.org/10.1101/2021.11.08.467769; this version posted November 8, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 428 inflorescence architecture, flower development, ABA response, cell division, cell 429 communication, senescence, and cell death (Li et al., 2010; Tokunaga et al., 2012; 430 Zhao et al., 2015; Li et al., 2016; Chakraborty et al., 2019; Wu et al., 2020) 431 (Supplementary Table 4). 432 433 HvHOX2 might be a dispensable gene during barley spikelet development 434 To understand the function of HvHOX2, we developed Hvhox2 mutants by using RNA- 435 guided Cas9 endonucleases (RGEN). A guide RNA was designed for the conserved Figure 6: HvHOX2 gene might be a team player in barley spikelet development. Figure A graphically shows the guide sequence with the Protospacer Adjacent Motif (PAM) and a putative cutting site, used to generate the single and double mutants of HvHOX1 and HvHOX2 genes, by using Cas9 endonuclease. Figures B & F are from an azygous plant and C-D & G-I are the representative images of the BG724-E07 mutants. Figures B-E compare the lateral spikelet development of wild-type, single and double mutants of HvHOX1 and HvHOX2 genes. At W4.5, the lateral spikelet primordia of HvHOX2 mutant (C) is at similar developmental stage with the wild-type (B) by having differentiated primordia for only glume and lemma. However, the mutant of HvHOX1 (D) and double mutant of HvHOX1 and HvHOX2 (E) displayed an advanced lateral spikelet development with the well differentiated primordia for glume, lemma, stamen, and carpel. The matured lateral spikelets of HvHOX2 mutant (G) and wildtype (F) are sterile and smaller compared to the fertile lateral spikelets that form grains of HvHOX1 mutant (H) and HvHOX1 and HvHOX2 double mutant (I). Scale bar-whole spikes in B, C, D, & E, 500 µm; magnified three nodes, 200 µm; HD-Homeodomain; LZ-Leucine Zipper. 16 bioRxiv preprint doi: https://doi.org/10.1101/2021.11.08.467769; this version posted November 8, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 436 homeodomain region shared by HvHOX1 and HvHOX2 for site-directed mutagenesis 437 of both genes (Fig. 6A). We created the mutants in the two-rowed cv. Golden Promise, 438 via stable transformation, using respective constructs and identified the independent 439 events BG724_E02 and BG724_E07 bearing different insertions and/or deletions by 440 sequencing 441 (heterozygous/chimeric) mutants, wild-type (T-DNA-free, non-mutant) plants, as well 442 as single and double mutants for both genes, were selected (Fig. 6A). For HvHox1, the 443 two mutants, BG724_E02 and BG724_E07, had one and eight nucleotides deletions, 444 respectively, in the target regions (Fig. 6A), which created two frame shifted mutant 445 HvHOX1 proteins (Supplementary Fig. 10A&B). 446 BG724_E02 event had seven nucleotides addition and four nucleotides deletion (Fig. 447 6A), which resulted in a mutant HvHOX2 protein that had one amino acid addition and 448 one amino acid exchange in the first HD (Supplementary Fig. 10C). Similar to the 449 Hvhox1 BG724_E02 mutant, Hvhox2 BG724_E07 mutant possessed one nucleotide 450 deletion (Fig. 6A) and formed a frame shifted protein (Supplementary Fig. 10D). The 451 spikelet development of these plants was compared at W4.5 and after spike maturity. 452 It was found that the central and lateral spikelets of the Hvhox2 mutants (Fig. 6C, 453 Supplementary Fig. 11B) displayed a similar stage of differentiation at W4.5 as in the 454 spikes of wild-type plants (Fig. 6B, Supplementary Fig. 11A). Analogous to the pattern 455 of spikelet differentiation, the matured spikes of Hvhox2 mutants (Fig. 6G, 456 Supplementary Fig. 11F) possessed smaller (compared to the centrals) and sterile 457 lateral spikelets like in spikes of wild-type plants (Fig. 6F, Supplementary Fig. 11E), 458 implying that HvHOX2 might neither promote nor suppress spikelet primordia 459 differentiation and growth. However, Hvhox1 single (Fig. 6D, Supplementary Fig. 11C) 460 and double mutants (Hvhox1/Hvhox2) (Fig. 6E, Supplementary Fig. 11D) exhibited 461 advanced lateral spikelet differentiation compared to wild-type plants (Fig. 6B, 462 Supplementary Fig. 11A) and Hvhox2 mutants (Fig. 6C, Supplementary Fig. 11B). 463 Interestingly, the lateral spikelet differentiation of Hvhox1 (Fig. 6D, Supplementary Fig. 464 11C) and double mutants (Hvhox1/Hvhox2) (Fig. 6E, Supplementary Fig. 11D) were 465 at a similar stage at W4.5, which reiterated the fact that HvHOX1 is suppressing lateral 466 spikelet development in two-rowed spikes, irrespective of the HvHOX2 function. As 467 expected, spikes of Hvhox1 single (Fig. 6H, Supplementary Fig. 11G) and double 468 mutant (Hvhox1/Hvhox2) (Fig. 6I, Supplementary Fig. 11H) had bigger and fertile 469 spikelets (grains) like six-rowed barley. We explored Hvhox2 mutants by screening its their target regions. Among 17 the progenies of these primary With regards to Hvhox2, the bioRxiv preprint doi: https://doi.org/10.1101/2021.11.08.467769; this version posted November 8, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 470 coding sequence in 5500 second-generation (M2) TILLING (Targeting Induced Local 471 Lesions in Genomes) mutant lines of cv. Barke (Gottwald et al., 2009), and found only 472 four mutations. Among these, three were synonymous, and one was non-synonymous 473 (P197S, line 11869) nucleotide substitutions (Supplementary Fig. 12). Interestingly, 474 the mutant line 11869 did not show aberrations during spike development and growth 475 in the M3 generation, which supported our RGEN Hvhox2 mutants. Taken together, 476 our RGEN mutant analyses suggest that HvHOX2, at its native expression level, 477 appears dispensable for barley spikelet development. 478 479 Overexpression of HvHOX2 can promote lateral spikelet development 480 Our qRT expression study conducted during spike growth stages revealed that higher 481 transcript levels of HvHOX2 than HvHOX1 in central spikelets might be associated with 482 the proper development of those spikelets in two-rowed barley (Fig. 3D, G, K & O). To 483 validate this ‘HvHOX2-dosage’-hypothesis, we tagged the HvHOX1 promoter (991 bp 484 – also used for assessing the spatiotemporal activity of HvHOX1 promoter) with the 485 coding sequence of HvHOX2 and used these constructs to create transgenic plants of 486 cv. Golden Promise. We used the HvHOX1 promoter because HvHOX1 expresses 487 higher in the lateral spikelets (Supplementary Fig. 8B, Fig. 3Q), so this promoter might 488 increase the transcript levels of HvHOX2 in the lateral spikelets of transgenic plants. 489 As a result, the smaller and sterile lateral spikelets might be restored to fertile and 490 bigger spikelets. Eight independent transgene-positive events were selected and 491 screened for the restored lateral spikelets. Two events, E189 (at T2) and E541 (at T1), 492 showed partial promotion of lateral spikelets compared to a wild-type control plant 493 E511 (Fig. 7). The spikes of the two events displayed an advanced lateral spikelet 494 differentiation at W4.5 compared to the spike of wild-type (E511) plants (Fig. 7A-C). 495 Interestingly, the lateral spikelets of both the events had a quantitative difference in 496 development, in which E189 showed a mild promotion, while E541 possessed a bit 497 stronger improvement compared to the spikes of control plants (Fig. 7B & C). The 498 matured spikes of E189 and E541 had partially restored lateral spikelets that are bigger 499 and occasionally developed small awns in contrast to the spikes of control plants (Fig. 500 7D-F). The matured lateral spikelets of E189 were smaller than E541, which followed 501 the similar pattern of developmental differentiation observed during spikelet 502 differentiation (Fig. 7B&C and E&F). Then, we quantified the transcripts of HvHOX1, 503 HvHOX2, and HvHOX2-T (HvHOX1pro::HvHOX2) in W6.5 (tissue-unspecific) (Fig. 18 bioRxiv preprint doi: https://doi.org/10.1101/2021.11.08.467769; this version posted November 8, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. Figure 7: Overexpression of HvHOX2 partially restored the lateral spikelet development in two-rowed barley. A comparison of wild-type (A) and two transgenic plants E189 (B) E541 (C) lateral spikelet primordia differentiation is displayed. The lateral spikelet primordia of transgenic plants E189 and E541 exhibited an advanced development compared to the wild-type at W4.5. At this stage, the wild-type laterals are found only with the differentiated glume and lemma primordia, while both the transgenic plants already initiated three stamen primordia along with the glume and lemma primordia. Matured spikes and triple spikelets of wild-type (D), E189 (E), and E541 (F) are shown. The lateral spikelets of both the transgenic plants are bigger compared to the wild-type and occasionally found with short awns. Quantification of endogenous HvHOX1 and HvHOX2 and transgenic HvHOX2-T expression performed in a wild-type and two independent transgenic plants’ (E189 & E541) whole spike at W6.5 (G), central spikelet (H) and lateral spikelets (I) at W8.0 is shown. The overexpression of transgenic HvHOX2-T did not greatly change the endogenous HvHOX1 and HvHOX2 expression in the whole spike at W6.5 (G) and lateral spikelets of W8.0 (I). However, in the central spikelets of W8.0, the transgenic HvHOX2-T expression drastically lowered the HvHOX1 expression (H). In G, H & I, the mean values of HvHOX1 from the transgenic plants were compared to the wild-type. Similarly, both the endogenous and transgenic HvHOX2 of transgenic plants were compared with the wild-type HvHOX2 expression. Mean values of G-I are compared with the multiple Student’s t-test; *, ***, mean values are significantly different at 5 and 0.1% probability levels . In A, B, & C, the scale bars of whole spike images represent 500 µm, and in magnified three node images they represent 200 µm. W-Waddington scale. 504 7G) and W8.0 (tissue-specific) (Fig. 7H&I). It revealed that both the events (E189 & 505 E541) had HvHOX2-T transcripts in the two stages and tissues analyzed (Fig. 7G-I). 506 Most importantly, there was no difference in the expression levels of HvHOX1 and 507 HvHOX2 genes in the lateral spikelets of transgenic events compared to the azygous 508 plant (Fig. 7I). However, we found a significant reduction of HvHOX1 transcripts in the 19 bioRxiv preprint doi: https://doi.org/10.1101/2021.11.08.467769; this version posted November 8, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 509 central spikelets (Fig. 7H). We also observed that event E189 had a four-fold higher 510 expression of HvHOX2-T than E541 at W6.5 (Fig. 7G), which was similarly seen in the 511 lateral spikelets at W8.0, where E189 had 1.6-fold higher expression than E541 (Fig. 512 7I). We hypothesize that this difference in expression is mainly due to the 513 developmental disparity between E189 and E541 (Fig. 7B & C). Thus, our 514 overexpression study supports the idea that increasing the dosage of HvHOX2 515 transcripts promotes lateral spikelet development in two-rowed barley. 516 517 Discussion 518 HvHOX1 and HvHOX2, two functional HD-ZIP class I transcription factors, may 519 act antagonistically to each other 520 Based on the sequence similarity of HvHOX2 to its orthologs in grass species, it was 521 proposed that HvHOX2 might have a similar molecular role in the Poaceae (Sakuma 522 et al., 2010). However, HvHOX1, specific to the Triticeae tribe, showed a very high 523 sequence variation, at least in barley (Komatsuda et al., 2007; Saisho et al., 2009; 524 Casas et al., 2018). It was hypothesized that HvHOX1 and HvHOX2 are duplicated 525 genes, in which HvHOX2 might be retaining the ancestral sequence and promotion of 526 development, while HvHOX1 became neofunctionalized as a suppressor of lateral 527 spikelets (Sakuma et al., 2010; Sakuma et al., 2013). Our nucleotide diversity study 528 also supports this postulation, as we found a higher nucleotide diversity for HvHOX1 529 than HvHOX2 (Supplementary Fig. 1, Supplementary Table 3). Despite a few amino 530 acid changes between HvHOX1 and HvHOX2 proteins (Supplementary Fig. 3), both 531 of them can bind to their HD-ZIP class I-specific cis-element, make dimers, and 532 transactivate their downstream genes (Fig. 1, Supplementary Fig. 3&4), thus 533 confirming that both are functional HD-ZIP class I TFs. Also, our expression studies 534 suggested that both the genes have similar spatiotemporal expression patterns during 535 spikelet initiation (Fig. 2) and growth stages (Fig. 3 & 4) that could facilitate the 536 interaction between them. Similarly, our gene co-expression network (GCN) analysis 537 revealed that most likely, these genes are sharing similar gene networks, as they fall 538 into the same cluster of co-expressed genes and share a common network of genes 539 (Fig. 5). This finding reaffirms the hypothesis that both genes might have originated 540 from a common ancestral gene (Sakuma et al., 2010; Sakuma et al., 2013). Crucially, 541 HvHOX1 has a unique network of genes (Fig. 5C) that are highly expressed in lateral 542 spikelets (Supplementary Fig. 9B) and are enriched with genes that are involved in the 20 bioRxiv preprint doi: https://doi.org/10.1101/2021.11.08.467769; this version posted November 8, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 543 suppression of development and exert cell death (Supplementary Table 4) 544 (Thirulogachandar et al., 2017). It also corroborates that HvHOX1 might have acquired 545 a new role as a suppressor of lateral spikelets in barley. Contrastingly, the genes in 546 the shared network of HvHOX1 and HvHOX2 are expressed both in the central and 547 lateral spikelets, and they are predicted to function towards the promotion of 548 development and flowering. It also suggests that along with the suppressors, there 549 might also be some promoters that are highly expressed in the lateral spikelets. 550 Presumably, this is the first insight into the antagonistic behavior of these two genes 551 during barley spikelet development. 552 Additionally, our analyses of differentially expressed genes between Bowman and BW- 553 NIL(vrs1.a) (Supplementary Fig. 13&14) and wild-type and HvHOX2 overexpressing 554 transgenic plants (Supplementary Fig. 15) pointed out that HvHOX1 and HvHOX2 555 might work antagonistically to each other during spikelet development. There are many 556 examples in plants in which homologous/paralogous genes are antagonists. In 557 Arabidopsis, WRKY12 and WRKY13 oppositely modulate flowering time under SD 558 conditions; WRKY12 promotes flowering, whereas WRKY13 delays this process (Li et 559 al., 2016). Likewise, TERMINAL FLOWER 1 (TFL1) and FLOWERING LOCUS T (FT) 560 are homologous PEBP class proteins, which are antagonistic to each other; TFL1 561 being a repressor and FT an activator of flowering (Hanzawa et al., 2005). Another 562 example is the closely related MADS-box proteins SHORT VEGETATIVE PHASE 563 (SVP) and AGAMOUS-LIKE 24 (AGL24), which perform opposite roles during the floral 564 transition, acting as repressor and promotor of flowering, respectively (Hartmann et al., 565 2000; Yu et al., 2002; Michaels et al., 2003; Lee et al., 2007). Recently, in rice, it was 566 found that Teosinte branched 2 (Tb2) counteracts with its paralog Tb1 to influence tiller 567 number(Lyu et al., 2020). All these instances corroborate that gene duplication events 568 followed by neofunctionalization might generate homologous/paralogous genes that 569 can act antagonistically to each other and modulate specific developmental pathways. 570 To understand the evolutionary importance of these genes, a new sub-category under 571 neofunctionalization might be necessary for which we propose to group them as 572 ‘antifunctionalized’ homologs. Thus, our studies suggest that the paralogous HD-ZIP 573 class I transcription factors, HvHOX1, and HvHOX2 are antifunctionalized and may act 574 against each other during barley spikelet development. 575 576 21 bioRxiv preprint doi: https://doi.org/10.1101/2021.11.08.467769; this version posted November 8, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 577 Dosage of HvHOX1 and HvHOX2 transcripts influence spikelet development 578 HvHOX1 was previously proposed as a negative regulator of lateral spikelet, 579 specifically pistil/carpel development, in barley (Komatsuda et al., 2007; Sakuma et al., 580 2010; Sakuma et al., 2013). We found evidence supporting the claim that from the 581 initiation of TM (Fig. 2G & H) to W8.5 (Fig. 3M & Q), HvHOX1 transcripts are enriched 582 in the lateral spikelets of two-rowed barley. This correlated well with the delayed 583 meristem differentiation (Fig. 2B-F) and anther and carpel development within the 584 lateral spikelets (Fig. 3E-L, Supplementary Fig. 6&7). More importantly, the abortion of 585 lateral spikelets’ anther and pistil/carpel at W7.5 (Fig. 3E-L, Supplementary Fig. 6&7), 586 and the gradual reduction of HvHOX1 expression in lateral spikelets from W7.5 (Fig. 587 3Q), reaffirm that HvHOX1 is highly expressed in the reproductive organs of lateral 588 spikelets. We also identified HvHOX1 transcripts in central spikelets during early and 589 late spikelet development (Fig. 2H; Fig. 3O & Q). However, we observed no disorder 590 during spikelet differentiation (Fig. 2B-F, 3C & D) or growth of reproductive organs (Fig. 591 3E-L, Supplementary Fig. 6&7) in two-rowed barley. Also, previous studies did not 592 report any developmental irregularities in central spikelets of two-rowed barley 593 (Komatsuda et al., 2007; Sakuma et al., 2010; Sakuma et al., 2013; Zwirek et al., 594 2019). We, therefore, hypothesized that this could be due to a lower dosage of 595 HvHOX1 transcripts (compared to the laterals) (Fig. 2H & 3O) and some more positive 596 regulators, which act antagonistically to HvHOX1 in central spikelets. It led us to 597 examine the expression of HvHOX2 - a paralog of HvHOX1, which was proposed to 598 be promoting the development in barley (Sakuma et al., 2010; Sakuma et al., 2013). A 599 similar (i.e., non-significant) level of HvHOX2 transcripts as HvHOX1 during the early 600 spikelet differentiation stages (Supplementary Fig. 8A) (except at SP stage), and a 601 higher dosage of HvHOX2 transcripts in the central spikelets across the growth of 602 reproductive organs (Fig. 3O) supports the claim of a promoting HvHOX2 function. 603 Furthermore, we recognized an anti-cyclic expression pattern between these two 604 genes during the growth stages (Fig. 3Q) and binding of HvHOX1 protein on HvHOX2 605 promoter and vice versa (Supplementary Fig. 4) indicating that these genes influence 606 the expression pattern of each other. A similar expression pattern of these two genes 607 had already been reported in other two-rowed barleys(Sakuma et al., 2013). 608 Our RGEN mutant study suggests that HvHOX2 is rather dispensable during barley 609 spikelet development because the two Hvhox2 mutants retained a canonical spikelet 610 development in laterals and centrals of wild-type plants (Fig. 6, Supplementary Fig. 22 bioRxiv preprint doi: https://doi.org/10.1101/2021.11.08.467769; this version posted November 8, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 611 10). Interestingly, ubiquitous overexpression of orthologous HOX2 genes in 612 wheat(Wang et al., 2017) and rice(Shao et al., 2018) reduced the inflorescence length 613 and complexity. However, when HvHOX2 transcripts were increased transgenically, 614 HvHOX2 can restore and promote barley lateral spikelet development in a dosage- 615 dependent manner (Fig. 7). A significant reduction of HvHOX1 transcripts in the central 616 spikelets of HvHOX2 overexpression mutants (Fig. 7H) reinstated that these two genes 617 can influence each other's expression level. We also observed a reduction of HvHOX1 618 transcripts in the lateral spikelets of HvHOX2 overexpression plants. Specifically, 3.5 619 and 6.9 times (mean transcript values) of reduction in transcripts were identified in the 620 HvHOX2 overexpressing plants E189 and E541, respectively; however, the declines 621 were not statistically significant (Fig. 7I). We hypothesize that this could be due to the 622 solid lateral-specific expression of HvHOX1, which is under the control of VRS4 623 (HvRA2) and VRS3 – two upstream regulators of HvHOX1 (Koppolu et al., 2013; 624 Sakuma et al., 2013; Bull et al., 2017; van Esse et al., 2017). The reduction level of 625 HvHOX1 transcripts and the degree of lateral spikelet promotion in the two HvHOX2 626 overexpression events indicated that HvHOX1 regulates lateral spikelet development 627 based on the dosage of its expression, which was also shown previously (Sakuma et 628 al., 2013). Taken together, our expression and transgenic studies suggest that the 629 transcript levels of HvHOX1 and HvHOX2 influence lateral spikelet development in 630 two-rowed barley in a dosage-dependent fashion. 631 632 Methods: 633 Plant materials and their growth conditions 634 Barley cultivars, Bonus, Bowman, and Golden Promise, were used in this study as two- 635 rowed representatives and induced mutant hex-v.3 (progenitor cv. Bonus), cultivar 636 Morex and Bowman backcross-derived line BW-NIL(vrs1.a) / BW 898 (Druka et al., 637 2011) were used as six-rowed representatives. Wild species of Hordeum were 638 obtained from Dr. Roland von Bothmer, Swedish University of Agricultural Sciences, 639 Alnarp, Sweden (Supplementary table 2). Arabidopsis thaliana Col-0 plants were used 640 for protoplast isolations and grown on a 1:3 vermiculite: soil mixture in a phytochamber 641 (8 hr light/16 hr dark at 20° C and 18° C, respectively; 60 % humidity). See the 642 supplemental methods for detailed information. 643 644 23 bioRxiv preprint doi: https://doi.org/10.1101/2021.11.08.467769; this version posted November 8, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 645 Microscopic studies 646 Please refer to the supplementary experimental procedures for histology of anther, 647 carpel, and spike development, as well as different microscopic methods like light, 648 scanning electron, and fluorescence. 649 650 Nucleic acid analysis 651 In the Supplemental methods, one can find methods for genomic DNA extraction, 652 Southern hybridization, RNA extraction, and qRT-PCR. 653 654 Nucleotide diversity (π) calculation 655 The whole-genome resequencing (WGS) data and SNP matrix for 200 diverse barley 656 genotypes were downloaded from Jayakodi et al., 2020. The sequencing reads were 657 aligned to the reference cv. Morex, as described (Jayakodi et al., 2020). The effectively 658 covered areas of the barley genome were identified by the regions covered by at least 659 two reads in ≥80% of the WGS accessions. The nucleotide diversity (π) was calculated 660 on a 10 kb window with a step size of 2 kb with a custom script. Only the windows with 661 ≥2 kb effectively covered region were considered. Please refer to the Supplemental 662 methods for further nucleotide diversity analyses, including TILLING and resequencing 663 of HvHOX1 and HvHOX2 in various genotypes and species. 664 665 Microarray probe preparation and data analysis 666 The microarray probe preparation, hybridization, and data analysis were done as 667 previously reported (Thirulogachandar et al., 2017). An elaborate method of the data 668 preparation and co-expression network construction is given in the supplemental 669 methods 670 671 Data analysis 672 The qRT data were analyzed using the Prism software, version 8.4.2 (GraphPad 673 Software, LLC). Mean value comparison of different traits was made with the multiple 674 Student’s t-tests, paired Student’s t-test (parametric), and a one-way ANOVA with 675 Tukey’s multiple comparison test (alpha=5%). 676 677 678 24 bioRxiv preprint doi: https://doi.org/10.1101/2021.11.08.467769; this version posted November 8, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 679 Gene ontology enrichment analysis 680 The Gene Ontology (GO) enrichment analysis of differentially expressed genes and 681 gene modules was done using the agriGO platform (v2) (Tian et al., 2017). The 682 selected genes’ Arabidopsis IDs were queried against the Arabidopsis genome locus 683 (TAIR9) reference set with the Fisher statistical test, Hochberg (FDR) multi-test 684 adjustment method, and a significance level 0.05. The Plant GO slim “GO type” has 685 been selected with a minimum number of entries. For final interpretation, the GO 686 enrichment of biological processes was used. 687 688 Transgenic and targeted mutagenesis 689 In silico identification of genes and promoters used for generating the transgenic plants 690 used in this study are given in the Supplemental methods. Also, the methods of cloning 691 various constructs, guide RNA design and preparation Cas9-triggered mutagenesis, 692 as well as plant transformation are shown in the supplemental methods. 693 694 Analysis of proteins 695 The preparation details of constructs used for the transactivation assay, electrophoretic 696 mobility 697 complementation assay are given in the supplemental methods. shift assay (EMSA), Western blot, and bimolecular fluorescence 698 699 Author Contributions 700 V.T., N.S., T.S. G.G., and T.K. conceptualized the study. The study was supervised by 701 N.S., T.S., and M.K. Microscopic analyses were done by V.T., and T.R. Transcriptome 702 data were generated by V.T., and G.G., which was analyzed by V.T., and S.K. 703 Transgenics and targeted gene-specific mutants were generated by G.H. and J.K. and 704 analyzed by V.T. RGEN mutants were molecularly characterized by J.R. 705 Resequencing of genes, and TILLING analysis were performed by R.K., T.S., S.S., 706 T.K., and M.J. Constructs for protein characterization were prepared by G.G., V.T., 707 P.S.R., and C.S. Transactivation and BiFC experiments were conducted by L.E-L., 708 G.G., and J.L., and M.K. performed DNA binding study (EMSA). All the data were 709 compiled, interpreted and drafted by V.T. The manuscript was reviewed by all the 710 authors. 711 712 25 bioRxiv preprint doi: https://doi.org/10.1101/2021.11.08.467769; this version posted November 8, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 713 Acknowledgments 714 We express our sincere gratitude to Jana Lorenz, Mandy Püffeld, Gabi Einert, Corinna 715 Trautewig, and Angelika Püschel for their excellent technical support. We would like to 716 thank Karin Lipfert, Heike Müller, Gudrun Schütze and Andreas Bähring for 717 photography and graphical works. We are grateful to Sabine Sommerfeld und Sibylle 718 Freist for technical assistance in barley transformation. We also extend our thanks to 719 Dr. Nils Stein for providing access to TILLING screening platform and Stefan Ortleb for 720 creating the spike movie. Work in the N.S. laboratory has been supported by a grant 721 from the Ministry of Education Saxony-Anhalt (IZN), while work in the T.S. laboratory 722 has been supported by the HEISENBERG Program of the German Research 723 Foundation (DFG), grant nos. SCHN 768/8-1 and SCHN 768/15-1 and the IPK core 724 budget. 725 726 Competing interests 727 The authors declare no competing interests. 728 729 Accession number 730 HvVHOX1 (VRS1) (Version: AB259782.1, GI: 119943316) 731 HvHOX2 (Version: AB490233.1, GI: 266265607) 732 733 Supplemental data 734 Supplemental Figure S1: Comparison of HvHOX1 and HvHOX2 nucleotide diversity 735 in 200 domesticated barleys 736 Supplemental Figure S2: Pairwise alignment of HvHOX1 and HvHOX2 proteins 737 Supplemental Figure S3: Western blot for HvHOX1 and HvHOX2 proteins 738 Supplemental Figure S4: EMSA competition assay of in vitro translated HvHOX1 and 739 HvHOX2 740 Supplemental Figure S5. Two-rowed spikes have delayed lateral spikelet 741 development compared to its central spikelet and six-rowed lateral spikelets 742 Supplemental Figure S6. Transverse sections of anthers from central and lateral 743 spikelets of Bowman and BW-NIL(vrs1.a) 744 Supplemental Figure S7: Transverse sections of carpels from central and lateral 745 spikelets of Bowman and BW-NIL(vrs1.a) 746 Supplemental Figure S8: Comparison of HvHOX1 and HvHOX2 expression pattern 747 during early spike development 26 bioRxiv preprint doi: https://doi.org/10.1101/2021.11.08.467769; this version posted November 8, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 748 Supplemental Figure S9. Hierarchial clustering of HvHOX1 and HvHOX2 shared and 749 HvHOX1 unique modules 750 Supplemental Figure S10: Sequence alignment of the wild-type and mutant proteins 751 of HvHOX1 and HvHOX2 resulted from the RGEN study 752 Supplemental Figure S11: The HvHOX2 gene might be a team player in barley 753 spikelet development 754 Supplemental Figure S12: Multiple sequence alignment of the orthologous HvHOX2 755 proteins and HvHOX2 from a TILLING mutant 11869 756 Supplemental Figure S13. Gene ontology of Differentially expressed genes in W2.5, 757 W3.0 and W4.5 in Bowman and BW-NIL(vrs1.a). 758 Supplemental Figure S14. Gene ontology of differentially expressed genes in W7.5 759 and W8.5 lateral spikelets of Bowman. 760 Supplemental Figure S15. Gene ontology of differentially expressed genes in W8.0 761 lateral spikelets of Transgenic plant E189 vs control plant E511. 762 Supplemental Methods. Additional methods and analyses used in this study. 763 Supplemental Table S1. HvHOX2 SNP haplotypes identified in 83 diverse spring 764 barley collection 765 Supplemental Table S2. List of Hordeum species used in this study. 766 Supplemental Table S3. Nucleotide diversity of HvHOX1 and HvHOX2 in Hordeum 767 species. 768 Supplemental Table S4. List of genes coexpressed with HvHOX1 and HvHOX2 769 genes during spike development in cv. Bowman 770 Supplemental Table S5. Primers used in this study. 771 772 27 bioRxiv preprint doi: https://doi.org/10.1101/2021.11.08.467769; this version posted November 8, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 773 774 775 776 777 778 779 780 781 782 783 784 785 786 787 788 789 790 791 792 793 794 795 796 797 798 799 800 801 802 803 804 805 806 807 808 809 810 811 812 813 814 815 816 817 818 819 820 821 822 References Arce, A.L., Raineri, J., Capella, M., Cabello, J.V., and Chan, R.L. (2011). Uncharacterized conserved motifs outside the HD-Zip domain in HD-Zip subfamily I transcription factors; a potential source of functional diversity. BMC Plant Biol 11, 42. Ariel, F.D., Manavella, P.A., Dezar, C.A., and Chan, R.L. (2007). The true story of the HD-Zip family. Trends Plant Sci 12, 419-426. Bonnett, O.T. (1935). The development of the barley spike. Journal of Agricultural Research 51, 451-457. Bull, H., Casao, M.C., Zwirek, M., Flavell, A.J., Thomas, W.T.B., Guo, W.B., Zhang, R.X., Rapazote-Flores, P., Kyriakidis, S., Russell, J., Druka, A., McKim, S.M., and Waugh, R. (2017). Barley SIX-ROWED SPIKE3 encodes a putative Jumonji C-type H3K9me2/me3 demethylase that represses lateral spikelet fertility. Nat Commun 8. Capella, M., Re, D.A., Arce, A.L., and Chan, R.L. (2014). Plant homeodomain-leucine zipper I transcription factors exhibit different functional AHA motifs that selectively interact with TBP or/and TFIIB. Plant Cell Rep 33, 955-967. Casas, A.M., Contreras-Moreira, B., Cantalapiedra, C.P., Sakuma, S., Gracia, M.P., Moralejo, M., Molina-Cano, J.L., Komatsuda, T., and Igartua, E. (2018). Resequencing theVrs1 gene in Spanish barley landraces revealed reversion of six-rowed to two-rowed spike. Molecular Breeding 38, 51. Chakraborty, N., Kanyuka, K., Jaiswal, D.K., Kumar, A., Arora, V., Malik, A., Gupta, N., Hooley, R., and Raghuram, N. (2019). GCR1 and GPA1 coupling regulates nitrate, cell wall, immunity and light responses in Arabidopsis. Sci Rep-Uk 9, 117. Clifford, H., Soderstrom, T., Hilu, K., Campbell, C., and Barkworth, M. (1987). Grass systematics and evolution (Washington DC: Random House (Smithsonian Institution Press)). Covington, M.F., and Harmer, S.L. (2007). The circadian clock regulates auxin signaling and responses in Arabidopsis. PLoS biology 5, e222. Dreni, L., and Kater, M.M. (2014). MADS reloaded: evolution of the AGAMOUS subfamily genes. New Phytologist 201, 717-732. Druka, A., Franckowiak, J., Lundqvist, U., Bonar, N., Alexander, J., Houston, K., Radovic, S., Shahinnia, F., Vendramin, V., Morgante, M., Stein, N., and Waugh, R. (2011). Genetic dissection of barley morphology and development. Plant Physiol 155, 617-627. Forster, B.P., Franckowiak, J.D., Lundqvist, U., Lyon, J., Pitkethly, I., and Thomas, W.T. (2007). The barley phytomer. Ann Bot 100, 725-733. Gaufichon, L., Marmagne, A., Belcram, K., Yoneyama, T., Sakakibara, Y., Hase, T., Grandjean, O., Clément, G., Citerne, S., and Boutet‐Mercey, S. (2017). ASN 1‐ encoded asparagine synthetase in floral organs contributes to nitrogen filling in Arabidopsis seeds. The Plant Journal 91, 371-393. Gottwald, S., Bauer, P., Komatsuda, T., Lundqvist, U., and Stein, N. (2009). TILLING in the two-rowed barley cultivar 'Barke' reveals preferred sites of functional diversity in the gene HvHox1. BMC Res Notes 2, 258. Hanzawa, Y., Money, T., and Bradley, D. (2005). A single amino acid converts a repressor to an activator of flowering. Proc Natl Acad Sci U S A 102, 7748-7753. Hartmann, U., Höhmann, S., Nettesheim, K., Wisman, E., Saedler, H., and Huijser, P. (2000). Molecular cloning of SVP: a negative regulator of the floral transition in Arabidopsis. The Plant Journal 21, 351-360. 28 bioRxiv preprint doi: https://doi.org/10.1101/2021.11.08.467769; this version posted November 8, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 823 824 825 826 827 828 829 830 831 832 833 834 835 836 837 838 839 840 841 842 843 844 845 846 847 848 849 850 851 852 853 854 855 856 857 858 859 860 861 862 863 864 865 866 867 868 869 870 871 872 873 Hyun, Y., Richter, R., Vincent, C., Martinez-Gallegos, R., Porri, A., and Coupland, G. (2016). Multi-layered regulation of SPL15 and cooperation with SOC1 integrate endogenous flowering pathways at the Arabidopsis shoot meristem. Developmental cell 37, 254-266. Jayakodi, M., Padmarasu, S., Haberer, G., Bonthala, V.S., Gundlach, H., Monat, C., Lux, T., Kamal, N., Lang, D., Himmelbach, A., Ens, J., Zhang, X.-Q., Angessa, T.T., Zhou, G., Tan, C., Hill, C., Wang, P., Schreiber, M., Boston, L.B., Plott, C., Jenkins, J., Guo, Y., Fiebig, A., Budak, H., Xu, D., Zhang, J., Wang, C., Grimwood, J., Schmutz, J., Guo, G., Zhang, G., Mochida, K., Hirayama, T., Sato, K., Chalmers, K.J., Langridge, P., Waugh, R., Pozniak, C.J., Scholz, U., Mayer, K.F.X., Spannagl, M., Li, C., Mascher, M., and Stein, N. (2020). The barley pan-genome reveals the hidden legacy of mutation breeding. Nature 588, 284-289. Kellogg, E.A., Camara, P.E.A.S., Rudall, P.J., Ladd, P., Malcomber, S.T., Whipple, C.J., and Doust, A.N. (2013). Early inflorescence development in the grasses (Poaceae). Frontiers in Plant Science 4. Kirby, E.M., and Appleyard, M. (1984). Cereal development guide. Cereal development guide. 2nd Edition. Komatsuda, T., Pourkheirandish, M., He, C., Azhaguvel, P., Kanamori, H., Perovic, D., Stein, N., Graner, A., Wicker, T., Tagiri, A., Lundqvist, U., Fujimura, T., Matsuoka, M., Matsumoto, T., and Yano, M. (2007). Six-rowed barley originated from a mutation in a homeodomain-leucine zipper I-class homeobox gene. Proceedings of the National Academy of Sciences 104, 1424-1429. Koppolu, R., and Schnurbusch, T. (2019). Developmental pathways for shaping spike inflorescence architecture in barley and wheat. Journal of Integrative Plant Biology 61, 278-295. Koppolu, R., Anwar, N., Sakuma, S., Tagiri, A., Lundqvist, U., Pourkheirandish, M., Rutten, T., Seiler, C., Himmelbach, A., Ariyadasa, R., Youssef, H.M., Stein, N., Sreenivasulu, N., Komatsuda, T., and Schnurbusch, T. (2013). Six-rowed spike4 (Vrs4) controls spikelet determinacy and row-type in barley. Proceedings of the National Academy of Sciences 110, 13198-13203. Lee, J.H., Yoo, S.J., Park, S.H., Hwang, I., Lee, J.S., and Ahn, J.H. (2007). Role of SVP in the control of flowering time by ambient temperature in Arabidopsis. Genes & development 21, 397-402. Li, W., Wang, H., and Yu, D. (2016). Arabidopsis WRKY Transcription Factors WRKY12 and WRKY13 Oppositely Regulate Flowering under Short-Day Conditions. Mol Plant 9, 1492-1503. Li, Y., Shen, Y., Cai, C., Zhong, C., Zhu, L., Yuan, M., and Ren, H. (2010). The type II Arabidopsis formin14 interacts with microtubules and microfilaments to regulate cell division. The Plant Cell 22, 2710-2726. Lyu, J., Huang, L., Zhang, S., Zhang, Y., He, W., Zeng, P., Zeng, Y., Huang, G., Zhang, J., Ning, M., Bao, Y., Zhao, S., Fu, Q., Wade, L.J., Chen, H., Wang, W., and Hu, F. (2020). Neo-functionalization of a Teosinte branched 1 homologue mediates adaptations of upland rice. Nat Commun 11. Michaels, S.D., Ditta, G., Gustafson‐Brown, C., Pelaz, S., Yanofsky, M., and Amasino, R.M. (2003). AGL24 acts as a promoter of flowering in Arabidopsis and is positively regulated by vernalization. The Plant Journal 33, 867-874. Ramsay, L., Comadran, J., Druka, A., Marshall, D.F., Thomas, W.T., Macaulay, M., MacKenzie, K., Simpson, C., Fuller, J., Bonar, N., Hayes, P.M., Lundqvist, U., Franckowiak, J.D., Close, T.J., Muehlbauer, G.J., and Waugh, R. (2011). INTERMEDIUM-C, a modifier of lateral spikelet fertility in barley, is an ortholog 29 bioRxiv preprint doi: https://doi.org/10.1101/2021.11.08.467769; this version posted November 8, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 874 875 876 877 878 879 880 881 882 883 884 885 886 887 888 889 890 891 892 893 894 895 896 897 898 899 900 901 902 903 904 905 906 907 908 909 910 911 912 913 914 915 916 917 918 919 920 921 922 923 of the maize domestication gene TEOSINTE BRANCHED 1. Nat Genet 43, 169172. Saisho, D., Pourkheirandish, M., Kanamori, H., Matsumoto, T., and Komatsuda, T. (2009). Allelic variation of row type gene Vrs1 in barley and implication of the functional divergence. Breeding Sci 59, 621-628. Sakuma, S., Salomon, B., and Komatsuda, T. (2011). The domestication syndrome genes responsible for the major changes in plant form in the Triticeae crops. Plant Cell Physiol 52, 738-749. Sakuma, S., Pourkheirandish, M., Matsumoto, T., Koba, T., and Komatsuda, T. (2010). Duplication of a well-conserved homeodomain-leucine zipper transcription factor gene in barley generates a copy with more specific functions. Funct Integr Genomics 10, 123-133. Sakuma, S., Pourkheirandish, M., Hensel, G., Kumlehn, J., Stein, N., Tagiri, A., Yamaji, N., Ma, J.F., Sassa, H., Koba, T., and Komatsuda, T. (2013). Divergence of expression pattern contributed to neofunctionalization of duplicated HD-Zip I transcription factor in barley. New Phytol 197, 939-948. Sakuma, S., Lundqvist, U., Kakei, Y., Thirulogachandar, V., Suzuki, T., Hori, K., Wu, J., Tagiri, A., Rutten, T., Koppolu, R., Shimada, Y., Houston, K., Thomas, W.T.B., Waugh, R., Schnurbusch, T., and Komatsuda, T. (2017). Extreme Suppression of Lateral Floret Development by a Single Amino Acid Change in the VRS1 Transcription Factor. Plant Physiol 175, 1720-1731. Sakuma, S., Golan, G., Guo, Z., Ogawa, T., Tagiri, A., Sugimoto, K., Bernhardt, N., Brassac, J., Mascher, M., Hensel, G., Ohnishi, S., Jinno, H., Yamashita, Y., Ayalon, I., Peleg, Z., Schnurbusch, T., and Komatsuda, T. (2019). Unleashing floret fertility in wheat through the mutation of a homeobox gene. Proceedings of the National Academy of Sciences 116, 5182-5187. Sessa, G., Morelli, G., and Ruberti, I. (1993). The Athb-1 and-2 HD-Zip domains homodimerize forming complexes of different DNA binding specificities. The EMBO journal 12, 3507. Shao, J., Haider, I., Xiong, L., Zhu, X., Hussain, R.M.F., Övernäs, E., Meijer, A.H., Zhang, G., Wang, M., Bouwmeester, H.J., and Ouwerkerk, P.B.F. (2018). Functional analysis of the HD-Zip transcription factor genes Oshox12 and Oshox14 in rice. PLOS ONE 13, e0199248. Thiel, J., Koppolu, R., Trautewig, C., Hertig, C., Kale, S.M., Erbe, S., Mascher, M., Himmelbach, A., Rutten, T., Esteban, E., Pasha, A., Kumlehn, J., Provart, N.J., Vanderauwera, S., Frohberg, C., and Schnurbusch, T. (2021). Transcriptional landscapes of floral meristems in barley. Science Advances 7, eabf0832. Thirulogachandar, V., Alqudah, A.M., Koppolu, R., Rutten, T., Graner, A., Hensel, G., Kumlehn, J., Brautigam, A., Sreenivasulu, N., Schnurbusch, T., and Kuhlmann, M. (2017). Leaf primordium size specifies leaf width and vein number among row-type classes in barley. Plant J 91, 601-612. Tian, T., Liu, Y., Yan, H., You, Q., Yi, X., Du, Z., Xu, W., and Su, Z. (2017). agriGO v2. 0: a GO analysis toolkit for the agricultural community, 2017 update. Nucleic acids research 45, W122-W129. Tokunaga, H., Kojima, M., Kuroha, T., Ishida, T., Sugimoto, K., Kiba, T., and Sakakibara, H. (2012). Arabidopsis lonely guy (LOG) multiple mutants reveal a central role of the LOG‐dependent pathway in cytokinin activation. The Plant Journal 69, 355-365. Ullrich, S.E. (2011). Barley. Production, improvement, and uses. (Oxford: WileyBlackwell). 30 bioRxiv preprint doi: https://doi.org/10.1101/2021.11.08.467769; this version posted November 8, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 924 925 926 927 928 929 930 931 932 933 934 935 936 937 938 939 940 941 942 943 944 945 946 947 948 949 950 951 952 953 van Esse, G.W., Walla, A., Finke, A., Koornneef, M., Pecinka, A., and Von Korff, M. (2017). Six-rowed spike 3 (VRS3) is a histone demethylase that controls lateral spikelet development in barley. Plant physiology, pp. 00108.02017. Waddington, S.R., Cartwright, P.M., and Wall, P.C. (1983). A Quantitative Scale of Spike Initial and Pistil Development in Barley and Wheat. Ann Bot-London 51, 119-130. Wang, Y., Yu, H., Tian, C., Sajjad, M., Gao, C., Tong, Y., Wang, X., and Jiao, Y. (2017). Transcriptome Association Identifies Regulators of Wheat Spike Architecture. Plant Physiology 175, 746-757. Wu, T.-Y., Krishnamoorthi, S., Goh, H., Leong, R., Sanson, A.C., and Urano, D. (2020). Crosstalk between heterotrimeric G protein-coupled signaling pathways and WRKY transcription factors modulating plant responses to suboptimal micronutrient conditions. Journal of experimental botany 71, 3227-3239. Youssef, H.M., Eggert, K., Koppolu, R., Alqudah, A.M., Poursarebani, N., Fazeli, A., Sakuma, S., Tagiri, A., Rutten, T., and Govind, G. (2017). VRS2 regulates hormone-mediated inflorescence patterning in barley. Nature Genetics 49, 157161. Yu, H., Xu, Y., Tan, E.L., and Kumar, P.P. (2002). AGAMOUS-LIKE 24, a dosagedependent mediator of the flowering signals. Proceedings of the National Academy of Sciences 99, 16336-16341. Zhao, M., Yang, S., Chen, C.-Y., Li, C., Shan, W., Lu, W., Cui, Y., Liu, X., and Wu, K. (2015). Arabidopsis BREVIPEDICELLUS interacts with the SWI2/SNF2 chromatin remodeling ATPase BRAHMA to regulate KNAT2 and KNAT6 expression in control of inflorescence architecture. PLoS genetics 11, e1005125. Zwirek, M., Waugh, R., and McKim, S.M. (2019). Interaction between row‐type genes in barley controls meristem determinacy and reveals novel routes to improved grain. New Phytologist 221, 1950-1965. 31 Parsed Citations Arce, A.L., Raineri, J., Capella, M., Cabello, J.V., and Chan, R.L. (2011). Uncharacterized conserved motifs outside the HD-Zip domain in HD-Zip subfamily I transcription factors; a potential source of functional diversity. BMC Plant Biol 11, 42. Google Scholar: Author Only Title Only Author and Title Ariel, F.D., Manavella, P.A., Dezar, C.A., and Chan, R.L. (2007). The true story of the HD-Zip family. Trends Plant Sci 12, 419-426. Google Scholar: Author Only Title Only Author and Title Bonnett, O.T. (1935). The development of the barley spike. Journal of Agricultural Research 51, 451-457. Google Scholar: Author Only Title Only Author and Title Bull, H., Casao, M.C., Zwirek, M., Flavell, A.J., Thomas, W.T.B., Guo, W.B., Zhang, R.X., Rapazote-Flores, P., Kyriakidis, S., Russell, J., Druka, A., McKim, S.M., and Waugh, R. (2017). Barley SIX-ROWED SPIKE3 encodes a putative Jumonji C-type H3K9me2/me3 demethylase that represses lateral spikelet fertility. Nat Commun 8. Google Scholar: Author Only Title Only Author and Title Capella, M., Re, D.A., Arce, A.L., and Chan, R.L. (2014). Plant homeodomain-leucine zipper I transcription factors exhibit different functional AHA motifs that selectively interact with TBP or/and TFIIB. Plant Cell Rep 33, 955-967. Google Scholar: Author Only Title Only Author and Title Casas, A.M., Contreras-Moreira, B., Cantalapiedra, C.P., Sakuma, S., Gracia, M.P., Moralejo, M., Molina-Cano, J.L., Komatsuda, T., and Igartua, E. (2018). Resequencing theVrs1 gene in Spanish barley landraces revealed reversion of six-rowed to two-rowed spike. Molecular Breeding 38, 51. Google Scholar: Author Only Title Only Author and Title Chakraborty, N., Kanyuka, K., Jaiswal, D.K., Kumar, A., Arora, V., Malik, A., Gupta, N., Hooley, R., and Raghuram, N. (2019). GCR1 and GPA1 coupling regulates nitrate, cell wall, immunity and light responses in Arabidopsis. Sci Rep-Uk 9, 1-17. Google Scholar: Author Only Title Only Author and Title Clifford, H., Soderstrom, T., Hilu, K., Campbell, C., and Barkworth, M. (1987). Grass systematics and evolution (Washington DC: Random House (Smithsonian Institution Press)). Covington, M.F., and Harmer, S.L. (2007). The circadian clock regulates auxin signaling and responses in Arabidopsis. PLoS biology 5, e222. Google Scholar: Author Only Title Only Author and Title Dreni, L., and Kater, M.M. (2014). MADS reloaded: evolution of the AGAMOUS subfamily genes. New Phytologist 201, 717-732. Google Scholar: Author Only Title Only Author and Title Druka, A., Franckowiak, J., Lundqvist, U., Bonar, N., Alexander, J., Houston, K., Radovic, S., Shahinnia, F., Vendramin, V., Morgante, M., Stein, N., and Waugh, R. (2011). Genetic dissection of barley morphology and development. Plant Physiol 155, 617-627. Google Scholar: Author Only Title Only Author and Title Forster, B.P., Franckowiak, J.D., Lundqvist, U., Lyon, J., Pitkethly, I., and Thomas, W.T. (2007). The barley phytomer. Ann Bot 100, 725733. Google Scholar: Author Only Title Only Author and Title Gaufichon, L., Marmagne, A., Belcram, K., Yoneyama, T., Sakakibara, Y., Hase, T., Grandjean, O., Clément, G., Citerne, S., and Boutet‐ Mercey, S. (2017). ASN 1‐encoded asparagine synthetase in floral organs contributes to nitrogen filling in Arabidopsis seeds. The Plant Journal 91, 371-393. Google Scholar: Author Only Title Only Author and Title Gottwald, S., Bauer, P., Komatsuda, T., Lundqvist, U., and Stein, N. (2009). TILLING in the two-rowed barley cultivar 'Barke' reveals preferred sites of functional diversity in the gene HvHox1. BMC Res Notes 2, 258. Google Scholar: Author Only Title Only Author and Title Hanzawa, Y., Money, T., and Bradley, D. (2005). A single amino acid converts a repressor to an activator of flowering. Proc Natl Acad Sci U S A 102, 7748-7753. Google Scholar: Author Only Title Only Author and Title Hartmann, U., Höhmann, S., Nettesheim, K., Wisman, E., Saedler, H., and Huijser, P. (2000). Molecular cloning of SVP: a negative regulator of the floral transition in Arabidopsis. The Plant Journal 21, 351-360. Google Scholar: Author Only Title Only Author and Title Hyun, Y., Richter, R., Vincent, C., Martinez-Gallegos, R., Porri, A., and Coupland, G. (2016). Multi-layered regulation of SPL15 and cooperation with SOC1 integrate endogenous flowering pathways at the Arabidopsis shoot meristem. Developmental cell 37, 254-266. Google Scholar: Author Only Title Only Author and Title Jayakodi, M., Padmarasu, S., Haberer, G., Bonthala, V.S., Gundlach, H., Monat, C., Lux, T., Kamal, N., Lang, D., Himmelbach, A., Ens, J., Zhang, X.-Q., Angessa, T.T., Zhou, G., Tan, C., Hill, C., Wang, P., Schreiber, M., Boston, L.B., Plott, C., Jenkins, J., Guo, Y., Fiebig, A., Budak, H., Xu, D., Zhang, J., Wang, C., Grimwood, J., Schmutz, J., Guo, G., Zhang, G., Mochida, K., Hirayama, T., Sato, K., Chalmers, K.J., Langridge, P., Waugh, R., Pozniak, C.J., Scholz, U., Mayer, K.F.X., Spannagl, M., Li, C., Mascher, M., and Stein, N. (2020). The barley pan-genome reveals the hidden legacy of mutation breeding. Nature 588, 284-289. Google Scholar: Author Only Title Only Author and Title Kellogg, E.A., Camara, P.E.A.S., Rudall, P.J., Ladd, P., Malcomber, S.T., Whipple, C.J., and Doust, A.N. (2013). Early inflorescence development in the grasses (Poaceae). Frontiers in Plant Science 4. Google Scholar: Author Only Title Only Author and Title Kirby, E.M., and Appleyard, M. (1984). Cereal development guide. Cereal development guide. 2nd Edition. Google Scholar: Author Only Title Only Author and Title Komatsuda, T., Pourkheirandish, M., He, C., Azhaguvel, P., Kanamori, H., Perovic, D., Stein, N., Graner, A., Wicker, T., Tagiri, A., Lundqvist, U., Fujimura, T., Matsuoka, M., Matsumoto, T., and Yano, M. (2007). Six-rowed barley originated from a mutation in a homeodomain-leucine zipper I-class homeobox gene. Proceedings of the National Academy of Sciences 104, 1424-1429. Google Scholar: Author Only Title Only Author and Title Koppolu, R., and Schnurbusch, T. (2019). Developmental pathways for shaping spike inflorescence architecture in barley and wheat. Journal of Integrative Plant Biology 61, 278-295. Google Scholar: Author Only Title Only Author and Title Koppolu, R., Anwar, N., Sakuma, S., Tagiri, A., Lundqvist, U., Pourkheirandish, M., Rutten, T., Seiler, C., Himmelbach, A., Ariyadasa, R., Youssef, H.M., Stein, N., Sreenivasulu, N., Komatsuda, T., and Schnurbusch, T. (2013). Six-rowed spike4 (Vrs4) controls spikelet determinacy and row-type in barley. Proceedings of the National Academy of Sciences 110, 13198-13203. Google Scholar: Author Only Title Only Author and Title Lee, J.H., Yoo, S.J., Park, S.H., Hwang, I., Lee, J.S., and Ahn, J.H. (2007). Role of SVP in the control of flowering time by ambient temperature in Arabidopsis. Genes & development 21, 397-402. Google Scholar: Author Only Title Only Author and Title Li, W., Wang, H., and Yu, D. (2016). Arabidopsis WRKY Transcription Factors WRKY12 and WRKY13 Oppositely Regulate Flowering under Short-Day Conditions. Mol Plant 9, 1492-1503. Google Scholar: Author Only Title Only Author and Title Li, Y., Shen, Y., Cai, C., Zhong, C., Zhu, L., Yuan, M., and Ren, H. (2010). The type II Arabidopsis formin14 interacts with microtubules and microfilaments to regulate cell division. The Plant Cell 22, 2710-2726. Google Scholar: Author Only Title Only Author and Title Lyu, J., Huang, L., Zhang, S., Zhang, Y., He, W., Zeng, P., Zeng, Y., Huang, G., Zhang, J., Ning, M., Bao, Y., Zhao, S., Fu, Q., Wade, L.J., Chen, H., Wang, W., and Hu, F. (2020). Neo-functionalization of a Teosinte branched 1 homologue mediates adaptations of upland rice. Nat Commun 11. Google Scholar: Author Only Title Only Author and Title Michaels, S.D., Ditta, G., Gustafson‐Brown, C., Pelaz, S., Yanofsky, M., and Amasino, R.M. (2003). AGL24 acts as a promoter of flowering in Arabidopsis and is positively regulated by vernalization. The Plant Journal 33, 867-874. Google Scholar: Author Only Title Only Author and Title Ramsay, L., Comadran, J., Druka, A., Marshall, D.F., Thomas, W.T., Macaulay, M., MacKenzie, K., Simpson, C., Fuller, J., Bonar, N., Hayes, P.M., Lundqvist, U., Franckowiak, J.D., Close, T.J., Muehlbauer, G.J., and Waugh, R. (2011). INTERMEDIUM-C, a modifier of lateral spikelet fertility in barley, is an ortholog of the maize domestication gene TEOSINTE BRANCHED 1. Nat Genet 43, 169-172. Google Scholar: Author Only Title Only Author and Title Saisho, D., Pourkheirandish, M., Kanamori, H., Matsumoto, T., and Komatsuda, T. (2009). Allelic variation of row type gene Vrs1 in barley and implication of the functional divergence. Breeding Sci 59, 621-628. Google Scholar: Author Only Title Only Author and Title Sakuma, S., Salomon, B., and Komatsuda, T. (2011). The domestication syndrome genes responsible for the major changes in plant form in the Triticeae crops. Plant Cell Physiol 52, 738-749. Google Scholar: Author Only Title Only Author and Title Sakuma, S., Pourkheirandish, M., Matsumoto, T., Koba, T., and Komatsuda, T. (2010). Duplication of a well-conserved homeodomainleucine zipper transcription factor gene in barley generates a copy with more specific functions. Funct Integr Genomics 10, 123-133. Google Scholar: Author Only Title Only Author and Title Sakuma, S., Pourkheirandish, M., Hensel, G., Kumlehn, J., Stein, N., Tagiri, A., Yamaji, N., Ma, J.F., Sassa, H., Koba, T., and Komatsuda, T. (2013). Divergence of expression pattern contributed to neofunctionalization of duplicated HD-Zip I transcription factor in barley. New Phytol 197, 939-948. Google Scholar: Author Only Title Only Author and Title Sakuma, S., Lundqvist, U., Kakei, Y., Thirulogachandar, V., Suzuki, T., Hori, K., Wu, J., Tagiri, A., Rutten, T., Koppolu, R., Shimada, Y., Houston, K., Thomas, W.T.B., Waugh, R., Schnurbusch, T., and Komatsuda, T. (2017). Extreme Suppression of Lateral Floret Development by a Single Amino Acid Change in the VRS1 Transcription Factor. Plant Physiol 175, 1720-1731. Google Scholar: Author Only Title Only Author and Title Sakuma, S., Golan, G., Guo, Z., Ogawa, T., Tagiri, A., Sugimoto, K., Bernhardt, N., Brassac, J., Mascher, M., Hensel, G., Ohnishi, S., Jinno, H., Yamashita, Y., Ayalon, I., Peleg, Z., Schnurbusch, T., and Komatsuda, T. (2019). Unleashing floret fertility in wheat through the mutation of a homeobox gene. Proceedings of the National Academy of Sciences 116, 5182-5187. Google Scholar: Author Only Title Only Author and Title Sessa, G., Morelli, G., and Ruberti, I. (1993). The Athb-1 and-2 HD-Zip domains homodimerize forming complexes of different DNA binding specificities. The EMBO journal 12, 3507. Google Scholar: Author Only Title Only Author and Title Shao, J., Haider, I., Xiong, L., Zhu, X., Hussain, R.M.F., Övernäs, E., Meijer, A.H., Zhang, G., Wang, M., Bouwmeester, H.J., and Ouwerkerk, P.B.F. (2018). Functional analysis of the HD-Zip transcription factor genes Oshox12 and Oshox14 in rice. PLOS ONE 13, e0199248. Google Scholar: Author Only Title Only Author and Title Thiel, J., Koppolu, R., Trautewig, C., Hertig, C., Kale, S.M., Erbe, S., Mascher, M., Himmelbach, A., Rutten, T., Esteban, E., Pasha, A., Kumlehn, J., Provart, N.J., Vanderauwera, S., Frohberg, C., and Schnurbusch, T. (2021). Transcriptional landscapes of floral meristems in barley. Science Advances 7, eabf0832. Google Scholar: Author Only Title Only Author and Title Thirulogachandar, V., Alqudah, A.M., Koppolu, R., Rutten, T., Graner, A., Hensel, G., Kumlehn, J., Brautigam, A., Sreenivasulu, N., Schnurbusch, T., and Kuhlmann, M. (2017). Leaf primordium size specifies leaf width and vein number among row-type classes in barley. Plant J 91, 601-612. Google Scholar: Author Only Title Only Author and Title Tian, T., Liu, Y., Yan, H., You, Q., Yi, X., Du, Z., Xu, W., and Su, Z. (2017). agriGO v2. 0: a GO analysis toolkit for the agricultural community, 2017 update. Nucleic acids research 45, W122-W129. Google Scholar: Author Only Title Only Author and Title Tokunaga, H., Kojima, M., Kuroha, T., Ishida, T., Sugimoto, K., Kiba, T., and Sakakibara, H. (2012). Arabidopsis lonely guy (LOG) multiple mutants reveal a central role of the LOG‐dependent pathway in cytokinin activation. The Plant Journal 69, 355-365. Google Scholar: Author Only Title Only Author and Title Ullrich, S.E. (2011). Barley. Production, improvement, and uses. (Oxford: Wiley-Blackwell). Google Scholar: Author Only Title Only Author and Title van Esse, G.W., Walla, A., Finke, A., Koornneef, M., Pecinka, A., and Von Korff, M. (2017). Six-rowed spike 3 (VRS3) is a histone demethylase that controls lateral spikelet development in barley. Plant physiology, pp. 00108.02017. Google Scholar: Author Only Title Only Author and Title Waddington, S.R., Cartwright, P.M., and Wall, P.C. (1983). A Quantitative Scale of Spike Initial and Pistil Development in Barley and Wheat. Ann Bot-London 51, 119-130. Google Scholar: Author Only Title Only Author and Title Wang, Y., Yu, H., Tian, C., Sajjad, M., Gao, C., Tong, Y., Wang, X., and Jiao, Y. (2017). Transcriptome Association Identifies Regulators of Wheat Spike Architecture. Plant Physiology 175, 746-757. Google Scholar: Author Only Title Only Author and Title Wu, T.-Y., Krishnamoorthi, S., Goh, H., Leong, R., Sanson, A.C., and Urano, D. (2020). Crosstalk between heterotrimeric G proteincoupled signaling pathways and WRKY transcription factors modulating plant responses to suboptimal micronutrient conditions. Journal of experimental botany 71, 3227-3239. Google Scholar: Author Only Title Only Author and Title Youssef, H.M., Eggert, K., Koppolu, R., Alqudah, A.M., Poursarebani, N., Fazeli, A., Sakuma, S., Tagiri, A., Rutten, T., and Govind, G. (2017). VRS2 regulates hormone-mediated inflorescence patterning in barley. Nature Genetics 49, 157-161. Google Scholar: Author Only Title Only Author and Title Yu, H., Xu, Y., Tan, E.L., and Kumar, P.P. (2002). AGAMOUS-LIKE 24, a dosage-dependent mediator of the flowering signals. Proceedings of the National Academy of Sciences 99, 16336-16341. Google Scholar: Author Only Title Only Author and Title Zhao, M., Yang, S., Chen, C.-Y., Li, C., Shan, W., Lu, W., Cui, Y., Liu, X., and Wu, K. (2015). Arabidopsis BREVIPEDICELLUS interacts with the SWI2/SNF2 chromatin remodeling ATPase BRAHMA to regulate KNAT2 and KNAT6 expression in control of inflorescence architecture. PLoS genetics 11, e1005125. Google Scholar: Author Only Title Only Author and Title Zwirek, M., Waugh, R., and McKim, S.M. (2019). Interaction between row‐type genes in barley controls meristem determinacy and reveals novel routes to improved grain. New Phytologist 221, 1950-1965. Google Scholar: Author Only Title Only Author and Title