Molecular and physiological characterization of brassinosteroid receptor BRI1 mutants inSorghum bicolor

brassinosteroids, BRI1, C4 crops, embryonic root, meristem, receptor kinase, 11 Sorghum bicolor (sorghum) 12 Total word count (excluding summary, references , and legends) 4759 No. of figures 7 (Fig 1-7 in color) Summary: 149 No. of tables 0

890 No. of supporting information files: 15 (Fig S1 -S7, Table S1 -S5, Videos S1-S3)

1109

1658

864

30 13 14 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted April 26, 2024. ; https://doi.org/10.1101/2024.04.22.590590doi: bioRxiv preprint 2 SUMMARY 15  The high s equence and structural similarities between BRI1 brassinosteroid 16 receptors of Arabidopsis (AtBRI1) and sorghum (SbBRI1) prompted us to study 17 the functionally conserved roles of BRI in both organisms. 18  Introducing sorghum SbBRI1 in Arabidopsis bri1 mutants restores defective 19 growth and developmental phenotypes to WT levels. 20  Sorghum mutants for SbBRI1 receptors show defective BR sensitivity and 21 impaired growth and development throughout the entire sorghum life cycle. 22 Embryonic analysis of sorghum primary roots permit to trace back root growth 23 and development to early stages, revealing the functionally conserved roles of 24 SbBRI1 receptor in BR perception during meristem development . RNA-seq 25 analysis uncovers the downstream regulation of the SbBRI1 pathway in cell wall 26 biogenesis during cell growth. 27  Together, these results uncover that sorghum SbBRI1 receptor protein play 28 functionally conserved roles in plant growth and development , while encourage 29 the study of BR pathways in sorghum and its implications for improving resilience 30 in cereal crops. 31 32

33 Brassinosteroids (BRs) hormones are essential for plant growth and development and for 34 the adaptation of plants to environmental stress (Planas-Riverola et al., 2019), endorsing 35 their interest in crop improvement (Gupta et al. , 2020) . In Arabidopsis thaliana 36 (Arabidopsis), BRs are perceived in the plasma membrane, via leucine -rich-repeat 37 receptor-like-kinase (LRR -RLK) proteins of the BRI1 (BRASSINOSTEROID 38 INSENSITIVE 1) family. Other BRI1-like homologues, namely BRI1-LIKE 1, 2 and 3 39 (BRL1/2/3) are present in Arabidopsis, from which only BRL1 and 3 can bind BR 40 molecules with high affinity. Contrasting with the ubiquitous expression pattern in BRI1 41 (Li & Chory, 1997) , BRL1 and BRL3 are enriched in the vasculature and stem cells 42 (Caño-Delgado et al., 2004; Fàbregas et al., 2013; Salazar-Henao et al., 2016). BRs bind 43 to BRI1-like receptors at a hydrophobic pocket known as “island domain”, created by a 44 stretch of ~70 amino acids that interrupt the LRR tandems (Kinoshita et al., 2005). The 45 ligand binding creates a docking platform that favors BRI1 heterodimerization with the 46 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted April 26, 2024. ; https://doi.org/10.1101/2024.04.22.590590doi: bioRxiv preprint 3 co-receptor BAK1 (Li et al., 2002; Hothorn et al., 2011; Bojar et al., 2014). This initiate 47 a BR signaling cascade, t hat that triggers dephosphorylation and activation of 48 transcription factors BRI1 -EMS SUPRESSOR1 (BES1) and BRASSINAZOLE 49 RESISTANT1 (BZR1) (Wang et al., 2002; Yin et al., 2002) by dephosphorylation and 50 subsequent translocation of BES1 and BZR1 to the nucleus to control BR-regulated gene 51 expression. 52 The last twenty years of molecular anaysis of Brassinosteroids in the roots have uncover 53 that BR hormones operates with high spatio-temporal resolution (Planas-Riverola et al., 54 2019). Using the Arabidopsis root as a model, the important roles of BRs in the different 55 cellular processes that govern root growth and development have been shown (González-56 García et al. , 2011; Hacham et al. , 2011) . BRs promote root growth by controlling 57 meristem development and the shift to cell elongation (Vilarrasa-Blasi et al. , 2014; 58 Pavelescu et al. , 2018; Betegón‐Putze et al. , 2021; Nolan et al. , 2023) . Cell wall 59 modifications during cell elongation are modulated by brassinosteroids in a cell-specific 60 fashion (Li et al. , 2021; Kelly -Bellow et al. , 2023) , yet the precise regulati on of this 61 process is not clear (Graeff et al., 2020) . In the root meristem, BR hormones act in a 62 paracrine way (Lozano-Elena et al. , 2018) and can travel from cell to cell via 63 plasmodesmata (PD), where they modulate its mobility by controlling its permeability 64 (Wang et al., 2023). 65 Mutants defective in BR signaling or synthesi s have been generated in other monocot 66 crops, showing a characteristic stunted growth. A potent drive in the research for 67 semi-dwarf mutants is their agronomical value as lodging resistant and more productive 68 plants, as seen since the Green Revolution (Salas Fernandez et al., 2009). The semi-dwarf 69 phenotype of the recessive uzu barley gene, used by Japanese breeders since the 1930s, 70 is caused by a missense mutation in the kinase domain of HvBRI1 (Chono et al., 2003). 71 Other barley dwarf mutants are allelic mutants of HvBRI1, or BR-deficient mutants with 72 altered functions of three BR biosynthetic genes (Dockter et al., 2014). In maize, where 73 BR-insensitive mutants have never been found in germplasm collections, compromising 74 BRI1 homologs function with RNAi results in various degrees of dwarfism in the 75 transgenic lines and diminished root growth inhibition by BL (Kir et al., 2015). The two 76 classical dwarf maize na1 and brd1 carry mutations in BR biosynthetic genes (Hartwig 77 et al., 2011; Makarevitch et al., 2012). In rice, most of the homologs of Arabidopsis BR 78 biosynthetic genes have been characterized, with BR -deficient mutants also presenting 79 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted April 26, 2024. ; https://doi.org/10.1101/2024.04.22.590590doi: bioRxiv preprint 4 reduced height and internode elongation (Castorina & Consonni, 2020) . A similar 80 phenotype was observed in the d61 dwarf line, an OsBRI1 mutant that presents reduced 81 root sensitivity to brassinolide (BL) treatments (Yamamuro et al., 2000). 82 Sorghum bicolor (sorghum) is a monocotyledon C4 cereal closely related to maize and 83 sugarcane that ranks as the fifth most cultivated crop in the world (FAOSTAT statistical 84 database., 2020; Chadalavada et al., 2021), emerging also as a bioenergy-producing crop 85 in soils not fit to food-crop growth (Rocateli et al., 2012). Due to its resistance to drought 86 and elevated temperatures, it is extensively cultivated in arid areas of the planet . The 87 diploid sorghum genome presents minimal gene redundancy (Paterson et al., 2009), and 88 its small size and complete sequence coverage allows for amenable functional genomics 89 for other related crops such as maize or sugarcane (Swigoňová et al., 2004; Hughes et al., 90 2014). Still, it remains highly recalcitrant to genetic transformation reporting very low 91 rates of success (O’Kennedy et al., 2006; Raghuwanshi & Birch, 2010). Despite sorghum 92 potential for climate resilient agriculture, BR signaling components remain understudied, 93 although recently there have been some efforts in this direction (Yamaguchi et al., 2016; 94 Blasco-Escámez et al., 2017; Hirano et al., 2017). 95 Here, we report the functional characterization of br assinosteroid receptor kinase BRI1 96 in sorghum (SbBRI1). Driving the expression of SbBRI1 under the promoter 35S , the 97 developmental defects caused by a knockdown of BRI1 ( bri1-301) in Arabidopsis were 98 restored to WT levels. Genetic and physiological analysis of two sorghum alleles carrying 99 BRI1 mutations, SbBRI1-Pro407Leu (ems87) and SbBRI1-Val403Met (ems72) indicate 100 conserved roles for SbBRI1 in promoting growth and development. Embryonic analysis 101 of Sorghum root reveals the SbBRI1 receptor roles in meristem cell division and stem 102 cell maintenance at the root apex . Collectively, our study unveil functionally conserved 103 roles for SbBRI1 in promoting plant growth and development , while set ting a solid 104 foundation for the cellular analysis of roots in sorghum. 105 106

107 BRI1 receptor sequence conservation in Sorghum bicolor 108 To address the conservation of the BRI1 protein sequence among land plant species, a 109 phylogenetic tree was built based on ortholog protein sequences (Fig. 1a). SbBRI1 is 110 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted April 26, 2024. ; https://doi.org/10.1101/2024.04.22.590590doi: bioRxiv preprint 5 closely related to maize BRI1 (Zea mays) (93.6% of identity) and other monocot crops 111 such as barley or wheat (Hordeum vulgaris or Triticum aestivum) (79.6% and 79.2% 112 respectively). Similar conservation in the extracellular domain can be also observed in 113 other plant species. (Fig. S1a). AtBRI1 and SbBRI1 share a protein sequence identity of 114 52.6%. Both leucine-rich-repeats in the extracellular domain, intramembrane and kinase 115 intracellular domains are strongly conserved. The majority of differences are found at the 116 extracellular domain, where SbBRI1 has only 19 LRRs at the N-terminal in comparison 117 to the 24 LRRs found in AtBRI1 (Fig. 1b, S1b). The high degree of similarity in the kinase 118 intracellular region suggests functional conservation during evolution (Ferreira-Guerra 119 et al., 2020) . In addition , structure modelling of the extracellular domain based on 120 Arabidopsis BRI1 crystal (PDB: 3RJ0 (Hothorn et al., 2011)) reveals a high degree of 121 structural similarity between both BRI1 proteins (Fig. 1b), keeping the horseshoe -like 122 structure and the island domain highly conserved. The amino acidic substitutions of the 123 two independent alleles carrying BRI1 mutations used in this st udy are shown in purple 124 (Fig. 1b), which fall on a conserved LRR region in the extracellular domain of the protein. 125 Furthermore, t he conservation of the island domain, critical for hormone binding 126 (Kinoshita et al., 2005), suggests a conservation of the protein function as BR receptor. 127 128 Sorghum BRI1 protein restore bri1 mutant phenotypes of Arabidopsis. 129 To analyze the functional conservation of SbBRI1, a 35S:SbBRI1-GFP construct was 130 introduced in Arabidopsis bri1-301 mutants (35S:SbBRI1-GFP; bri1-301, hereinafter 131 named SbBRIox; bri1-301, see Methods). Adult homozygous SbBRI1ox; bri1-301 plants 132 exhibited a WT phenotype, in terms of overall size, rosette structure, and flowering time 133 (Fig. 2a, Fig. S2a), restoring the dwarf phenotype of Arabidopsis bri1-301 mutant (Kang 134 et al., 2010), supporting that SbBRI1 can function as a BR receptor in promoting plant 135 growth similarly to AtBRI1. 136 The primary root of SbBRI1 ox; bri1-301 is longer than bri1-301 roots, similar to WT 137 (Fig. 2b). Upon BL application, a dose-dependent reduction in root length was observed 138 in WT and AtBRI1 overexpressor plants (35S:BRI1-GFP; Col -0, herein named 139 AtBRIox), whereas bri1-301 mutants were insensitive to BRs (Fig. 2c; González-García 140 et al., 2011). The roots of SbBRI1ox; bri1-301 plants behaved similarly to AtBRI1ox and 141 WT (Col-0), of Arabidopsis (Fig. 2b), thus rescuing bri1-301 phenotype of BL 142 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted April 26, 2024. ; https://doi.org/10.1101/2024.04.22.590590doi: bioRxiv preprint 6 insensitivity. Upon BL application the root meristem length (Fig. 2d, e) and QC division 143 rate (Fig. 2d, f) of SbBRIox; bri1-301 plants were restored to WT levels. 144 To further substantiate sorghum SbBRI1 functionality in Arabidopsis, we analyzed its 145 ability to activate the downstream BR signaling pathway by assessing t he 146 phosphorylation status of BES1 in SbBRI1ox; bri1 -301 plants. Upon BL application, 147 SbBRI1 was found to dephosphorylate BES1 protein in WT Col-0 and similarly in 148 AtBRI1ox; bri1-301 lines (Fig. S 2b). Together, our results indicate that SbBRI1 can 149 function a s BR receptor activating downstream ef fectors in the pathway similar to 150 AtBRI1. 151 152 Characterization of bri1 receptor mutants in sorghum. 153 To investigate the native role of BRI1 receptor kinase in sorghum, mutant lines for 154 SbBRI1 from an EMS-mutagenized population of BTx623 variety (Jiao et al., 2016) were 155 identified. Two different SbBRI1 homozygous mutants were selected, ems72 (V403M) 156 and ems87 (P407L) both possessing a point mutation in the extracellular LRR domain of 157 the SbBRI1 receptor (Fig. 1 b). To reduce the number of background mutations , two 158 rounds of genetic backcrosses were performed using the non -mutagenized BTx623 159 variety as parental (Fig. S6, see Methods) . Subsequent experiments were done by 160 comparing the backcrossed bri1 mutants for SbBRI1 with their SbBRI1 WT direct 161 siblings, herein named bri1-ems72 and WT-ems72. 162 In greenhouse conditions, mature bri1-ems72 plants showed a reduction in overall plant 163 growth, (Fig. 3a) during all developmental stages until maturity (Fig. 3b, c, d) . Mature 164 plants of bri1-ems72 and 87 exhibited shorter panicles and a significantly reduced grain 165 yield (Fig. 3e, h, Figure S3a, b ), that might be attributed to a reduced grain number and 166 not a reduced grain size or weight (Fig. S3 c, d, e ). Overall, our results indicate that 167 sorghum SbBRI1 has a promoting role in plant growth by controling organ size. 168 Next, explanted embryos grown in vitro were used to study the embryonic root 169 development and standardize root growth analysis ( see Methods). The embryonic roots 170 of the WT sorghum BTx623 variety were found to germinate more homogeneously and 171 be shorter than when using whole seeds (Fig. S4a), proving to be an amenable method of 172 working in vitro with organs from non -model species and indicating that sorghum 173 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted April 26, 2024. ; https://doi.org/10.1101/2024.04.22.590590doi: bioRxiv preprint 7 embryonic root can be used as a model in developmental studies. In control conditions, 174 eight-day-old embryonic roots of bri1-ems72 mutants were shorter than WT-ems72 and 175 showed reduced sensitivity to 4nM BL (Fig. 3g, h, k, l). Furthermore, when grown in a 176 hydroponics setting, bri1-ems87 also showed insensitivity to low BL concentrations 177 (0.04nM and 0.4 nM of BL) (Fig. S4b, c). Together with the observed structural and 178 functional conservation of SbBRI1 protein, genetic analysis shows the role of BRI1 179 receptor in sorghum BL perception and yield. 180 181 Cellular anatomy of sorghum primary root in WT and SbBRI1 mutants. 182 To further investigate the development of the primary root apex and the overall root 183 architecture of bri1 mutants in sorghum, mPS -PI staining protoc ol was adapted to 184 embryonic roots from (Truernit et al., 2008; Kirschner et al., 2017) (Fig. 4). WT BTx623 185 roots shows a meristematic region of approximately fifteen cells, measured at the 186 epidermis cell layer (Fig. 4a). In the medial plane, the stem cell niche was clearly 187 visualized. The QC region and distally localized columella stem cells appear underneath, 188 followed by the differentiated columella cells presenting their characteristically stained 189 starch granules (Supplementary Video 1, Supplementary Video 2). Upon exogenous BL 190 application, the meristem region size varies accordingly with described in Arabidopsis 191 (González-García et al., 2011), and progressive disorganization was observed at the QC 192 and stem cell region (Fig. 4b-e). 193 To precisely define the QC region of the Sorghum root meristem, 5’-ethynyl-2’-194 deoxyuridine (EdU) cell proliferation assay was performed in embryonic roots (Fig. 4f-195 i). EdU is a thymidine analogue uptaken by the cell after growth media supplementation 196 and incorporated in the genome of dividing cells, labelling it and thus allowing detection 197 of said cells by confocal microscopy (in yellow) . Together with all root nuclei 198 counterstaining by DAPI (in purple), the technique allows the tracking of cell division in 199 a given timeframe (12 hours, see Methods). In 8-day-old Sorghum WT BTx623 roots, the 200 QC appears to be comprised of about 20 cells in its medial plane , and presents a 201 semispherical shape under 3D imaging (Fig. 4f, Supplementary Video 1). Ever-increasing 202 QC division was found under incremental BL supplementation, up to the point of root 203 exhaustion at 400nM of BL, showing a significantly reduced cell division rate in an 204 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted April 26, 2024. ; https://doi.org/10.1101/2024.04.22.590590doi: bioRxiv preprint 8 almost depleted meristem (Fig. 4i), accompanied by a severe cell disorganization of the 205 stem cell region (Fig. 4e, i). 206 Upon BL application, WT-ems72 shows rapid meristem shortening and cell 207 disorganization (Fig. 5a, c), a response similar to that of BTx623, but more sensitive, 208 probably due to background mutations . While bri1-ems72 presents a n overall similar 209 response (Fig. 5b), meristem cell number at 4nM BL concentrations is significantly 210 greater (Fig. 5c). No differences were found in meristem size response to BL (Fig. 5d). 211 At 40nM BL concentration, there are not significant differences between genotypes and 212 overall disorganization appears widely . These results also highlight the role of SbBRI1 213 in regulating root meristem cell division. 214 In control conditions, ems72 shows generally lesser cell division than BTx623 (Fig. 6). 215 However, w hen supplemented with 4nM BL, overall cell division increases in the 216 meristem (Fig. 6a, b, d, e), whereas bri1-ems72 consistently shows a reduced cell division 217 compared to its WT counterpart (Fig. 6g, h). At greater BL concentrations, the meristem 218 region is usually exhausted, showing little to no meristematic cell division and a 219 disorganized structure ( Fig. 6c, f, Fig. S5a, b). Reduced BR sensitivity in bri1-ems72 220 resulted in a reduction of the percentage of exhausted roots observed at 40nM BL (Fig. 221 6g, h). Our results uncover the roles of BRI1 receptor in Sorghum in meristem division 222 and overall root growth, while opening embryonic roots analysis in sorghum at a cellular 223 resolution to advance in our present understanding of root growth and development in 224 cereal crops. 225 226 Transcriptomic analysis shows conserved roles of SbBRI1 in cell wall loosening 227 during cell elongation in sorghum. 228 To study the role of BRI1 in sorghum in more molecular detail , RNA-seq analysis was 229 used to investigate which processes are transcriptionally regulated by SbBRI1 in bri1-230 ems72 roots against their sibling WT-ems72, (see Methods). Differential expression 231 analysis revealed a limited number of deregulated genes (50 downregulated, 254 232 upregulated, Supplementary Table 4, Supplementary Table 5 ), probably because of 233 transcriptional noise caused by the background mutations (FC>1.5, p value<0.05). GO 234 enrichment analyses, based on 226 high confidence Arabidopsis orthologs of upregulated 235 sorghum genes revealed enrichment in “plant-type cell wall loosening” (GO:0009828), 236 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted April 26, 2024. ; https://doi.org/10.1101/2024.04.22.590590doi: bioRxiv preprint 9 “response to wounding” (GO:0009611), “cell wall macromolecule metabolic process” 237 (GO:0044036), and “regulation of root development” (GO:2000280) categories (Fig. 7a), 238 pointing towards the action of BRI1 on cell wall function. No enrichment nor BR-related 239 transcripts (such as BRI1, BRL) were found deregulated among downregulated genes in 240 our analysis. Gene deployment of the most significantly enriched GO categories (Fig. 7 241 b, c, d, e) show that the most deregulated ortholog genes per each category correspond to, 242 respectively, expansin family members as AT1G20190 or AT1G65680 (EXPB2), 243 chitinase enzymes as AT5G24090 (CHIA) or AT3G54420 (CHIV), fucosyltransferases 244 as AT1G14100 (FUT8) and plasmodesmal-mediated primary root growth AT1G09560 245 (GLP5). As the sorghum genome is not as well annotated as Arabidopsis or other crop 246 species, a search of the same deregulated sorghum gene s reveals no different genes than 247 using orthologs from Arabidopsis. Mostly expansins, chitinases and other enzymes are 248 present. Together, our findings support the hypothesis that BRI1 functions of cell wall 249 biosynthesis and remodeling are conserved in Sorghum bicolor. 250 251

252 In sorghum, there are two distinct LRR-RLs like AtBRI1, are the SbBRI1 and a putative 253 SbBRL1, the later sharing highest homology to the BRI1-LIKE (BRL) family in 254 Arabidopsis. Here, the genetic analysis of SbBRI1 mutants showing a reduced BR 255 sensitivity conveys with similar previously described data for HvBRI1 and OsBRI1 256 mutants (Chono et al. , 2003; Tong & Chu, 2012) . The existing relationship between 257 modified BRI1 receptor levels and proper seed and panicle development (Sun et al., 2021) 258 is supportive of a direct relationship between BR biosynthesis and yield (Zhang et al., 259 2014; Singh et al. , 2016) . In Sorghum, SbBRI1 shares high amino acid sequence 260 identity(52.6%) to AtBRI1 and similar three-dimensional structure including the island 261 domain critical for BL binding (Kinoshita et al., 2005) and activation of the BR-mediated 262 phosphorylation cascade (Z. Wang et al., 2002; Yin et al., 2002) . Our data showing 263 sorghum BRI overexpression in the Arabidopsis bri1 receptor mutants resulting in 264 phenotypes similar to WT to AtBRI1 overexpression (Wang et al., 2001) conveys on the 265 functional conservation of the BR receptors in plants. 266 The meristem development phenotypes of SbBRI1 receptor mutants allied with the ones 267 reported for bri1 mutants of Arabidopsis (González-García et al., 2011; Lozano-Elena et 268 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted April 26, 2024. ; https://doi.org/10.1101/2024.04.22.590590doi: bioRxiv preprint 10 al., 2018). The roles of Arabidopsis BRI1 in cell wall elongation (González-García et al., 269 2011; Li et al., 2021) and biosynthesis (Vert et al., 2005; Sun et al., 2010; Wolf et al., 270 2012) are similar to the one s identified in this study for SbBRI1, further suggesting the 271 receptor function conservation during evolution (Ferreira-Guerra et al., 2020). Whether 272 other key BR-signaling pathway components, such as BAK1 or BES1/BZR1, are at least 273 partially conserved in sorghum is yet to be determined. 274 The BTx623 sorghum variety used in this study, that has been widely used in agriculture, 275 presents a spontaneous mutation in the gene DW1, historically traced back to 1905 (R.E. 276 Karper & Quinby, 1946). DW1 encodes a protein that positively regulates BR signaling 277 by inhibiting BIN2 activit y (Hirano et al. , 2017) and causes partial dwarfism, and 278 therefore increased lodgi ng resistance and improved crop productivity (Hilley et al. , 279 2016). When working with a gene theorized to be upstream of this described mutation, 280 such as SbBRI1, it can be expected that the observed phenotypes be mitigated by other 281 mutations such as the one present in BTx623. Nonetheless , our study shows that a 282 compromised BRI1 function causes defective growth in all plant stages in sorghum, 283 comparable with the ones reported in other species, clearing a path for future crop studies 284 regarding brassinosteroids and developmental or stress -related processes (Vriet et al., 285 2012). 286 The analysis of root s in crop plants has been challenging due to size and/or cell wall 287 composition, which increase s the difficulty of a deep analysis like it is routinely 288 performed in Arabidopsis roots. Obtaining good resolution imaging at a cellular level in 289 cereals is challenging. Here, the use of freshly explanted sorghum embryos as a source 290

allowed to synchronize the germination, standardize in vitro growth and focus 291 the study on the primary root, which arises from the scutellar node of the embryo 292 (Hochholdinger et al., 2004). Embryonic root in maize support s plant growth by itself 293 (Hetz et al. , 1996) , as we proved to be sufficient in sorghum. Our work provides 294 adaptations for mPS-PI and EdU staining protocols for sorghum roots , allowing highly 295 precise visualization at the cell-specific level, such as presented in (Kirschner et al., 2017) 296 for barley. Routinely implementing these cell biology tools can be a turning point in our 297 approach to cell -level studies of tissues in non-model plant organisms. In most crops, 298 studies with root systems have earnt less attention as compared to the ones focused on 299 aerial plant organs. Given the importance of the root for overall plant growth and 300 adaptation to the changing environment (including BR pathway and receptors), the advent 301 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted April 26, 2024. ; https://doi.org/10.1101/2024.04.22.590590doi: bioRxiv preprint 11 of studies highlighting the importance of plant hormones for root development and 302 adaptation to abiotic stress resistance (Gupta et al., 2020) might help to generate greater-303 yielding cereals, by delv ing into the knowledge of tissue -specific plant responses to 304 drought (Fàbregas et al., 2018; Gupta et al., 2023). 305 Together, these results highlight the importance of BR hormone signaling pathways in 306 plant growth and development across phyla, and its implications in crop genetic 307 improvement (Martignago et al., 2020). While the complexities of the molecular plant 308 responses to drought are still being deciphered in Arabidopsis, transferring the current 309 knowledge to crops of key importance for global food security will be central in years to 310 come. 311 312

313 Informatic tools and modeling. 314 BRI1 alignment of different species was generated using Geneious Prime software 315 v.2020.0.4. (https://geneious.com). SbBRI1 ectodomain structure was modelled based on 316 the AtBRI1 ectodomain crystal structure bound to BL (PDB:3RJ0) using Modeller v9.23 317 (Sali, s. f.). Representation of the model and mapping of the mutated residues were 318 generated with UCSF Chimera (Pettersen et al., 2004) 319 Plant material 320 Arabidopsis seedlings, ecotype Columbia, were sterilized with 35% bleach in deionized, 321 sterile water (dH2O), plated in MS0.5- media, and grown vertically at 22ºC in long day 322 conditions for 6 days. 323 For sorghum whole seedlings, seeds were sterilized with 35% bleach in deionized sterile 324 water for 10 minutes, washed five times in dH2O, and embedded overnight at 4ºC in dark. 325 Then, seeds were placed in MS0.5- and grown at 28ºC for 8 days before imaging. 326 BTx623 variety s orghum panicles between 13 -17 days post anthesis were cut from the 327 plant and sterilized with 50% bleach in deionized sterile water with 0.5% Triton X-100 328 for 10 minutes with agitation, and then washed 5 times with dH2O before embryo 329 extraction. Embryos were then plated in MS0.5 - media, and grown vertically for 8 days 330 at 28ºC, in 12/12 light conditions. For RNA-seq, sorghum BC2F2 embryos of ems72 line 331 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted April 26, 2024. ; https://doi.org/10.1101/2024.04.22.590590doi: bioRxiv preprint 12 were grown as explained and individually collected at 8 days after germination. Aerial 332 organs were also individually collected for PCR genotyping of bri1 (Fig. S6). For each 333 biological replicate, a minimum of six bri1-ems72 and WT-ems72 roots were pooled to 334 homogenize as much as possible the genetic background of the samples. For sorghum 335 phenotyping, the same strategy was followed, however, BC2F2 plants were genotyped in 336 the same way and their homozygous BC2F3 offspring was used to perform the 337 experiments. A graphical methodology can be found in Fig. S6, genotyping primers can 338 be found in Supplementary Table 1. 339 Complementation of bri1 mutants in Arabidopsis 340 Sorghum BRI1 coding sequence was cloned via Gateway Recombination Cloning 341 Technology (Invitrogen) to obtain pEN-L1L2-SbBRI1 entry clone. By LR recombination 342 reaction with pGWB406 (Nakagawa et al. , 2007) together with p4P1r -35S, the 343 destination vector p3 5S:SbBRI1-GFP was obtained. Arabidopsis bri1-301 plants were 344 transformed by floral dip to introduce the p35S:SbBRI1 -GFP vector, and stable lines 345 carrying single copy of the transgene were selected for this study. Supplementary Table 346 3 includes used primer sequences and Fig. S7 shows the plasmid maps. 347 Western Blotting 348 Total protein was extracted from 6 days old Arabidopsis seedlings and resolved by SDS-349 PAGE. Western Blot analysis was performed as described in (Fàbregas et al., 2013) using 350 primary antibodies against BES1 (kindly provided by Prof. Yanhai Yin) H3, (Yin et al., 351 2002) and GFP (Sigma-Aldrich) and anti-rabbit secondary antibody IgG (GE Healthcare 352 UK). 353 Microscopy 354 6-day-old Arabidopsis seedlings and 8-day-old root tips cut from sorghum embryos were 355 submit to mPS-PI protocol, following Truernit et al., 2008 . Standard protocol , as 356 described, was used for Arabidopsis seedlings. For sorghum roots, protocol for floral 357 stalks was followed with the following modifications: 50% methanol, 10% acetic acid 358 fixation time was extended to 48 hours minimum, 1% periodic acid fixation was carried 359 out under vacuum conditions for a minimum of 1 hour, and Schiff reagent with propidium 360 iodide incubation was carried out under agitation for at least 3 hours to ensure staining of 361 the more internal cell layers of the root tip . After chloral hydrate clearing, stained roots 362 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted April 26, 2024. ; https://doi.org/10.1101/2024.04.22.590590doi: bioRxiv preprint 13 were mounted in excavated slides using Hoyer’s media, left to dry and observed using an 363 Olympus FV1000 confocal microscope ( 20X and 40X objective, oil immersion, laser 364 excitation set to 536-617nM). Images were taken in the medial plane of the root and 365 different data was measured with ImageJ software. 366 For EdU staining, 7 and a half-day-old sorghum seedlings were transferred from 0.5MS- 367 with vitamins media to plates of the same media , also containing 10µM EdU in DMSO 368 (plus 400nM BL for the treated samples) or the corresponding amount of DMSO as mock 369 and grown in that media for 12 hours. For the staining protocol we followed (Kirschner 370 et al., 2017) staining of barley roots with a few modifications: root tips were cut and 371 fixated for at least one hour under vacuum, permeabilized for another hour under vacuum, 372 and submitted to Click-it reaction under vacuum as per Click-It Edu Alexa Fluor 488 373 Imaging Kit (Thermofisher) including nuclei counterstaining . Samples were then 374 submitted to mild fixation with paraformaldehyde and cleared for around 15 -20 days 375 following (Warner et al. , 2014) . Microscopy was performed using Olympus FV1000 376 confocal microscope (40X objective, oil immersion, laser excitation set to 495/519nM) . 377 Images were taken in the medial plane of the root and different data measured with ImageJ 378 software. For Fig. 6a, laser intensity was modified for better visualization as this image 379 is a collage of many confocal pictures of the same root. 380 Measuring and statistics 381 For in vitro plate images, root length was measured using ImageJ software 382 (https://imagej.nih.gov/ij/). For cell division measures in Figure 6 , ITCN plugin for 383 ImageJ was used in the image channel corresponding to EdU staining using whole image 384 keeping constant internal parameters. All graphics were generated using RStudio default 385 plotting tools. Statistics were calculated in RStudio using Agricolae package for Tukey’s 386 HSD test, and online with a 2x3 Fisher’s test (Freeman & Halton, 1946; Soper, 2022)A p 387 value of at least p<0.05 is used as a threshold in all statistics unless otherwise stated. All 388 experiments were carried out using at least three independent biological replicates. 389 Transcriptomic profiling analysis 390 For sorghum RNA-seq, RNA was extracted from sorghum roots using Plant Easy Mini 391 Kit (Qiagen). Stranded libraries were prepared using TrueSeq Stranded mRNA KIT 392 (Ilumina). Paired -end sequencing (2x100bp) was performed usi ng NovaSeq6000 393 (Ilumina) at minimum depth of 45M. Raw reads were quality-checked using FastQC tool 394 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted April 26, 2024. ; https://doi.org/10.1101/2024.04.22.590590doi: bioRxiv preprint 14 (v0.11.2), trimmed 13bp at the 3’ end quality-trimmed and filtered at a minimum quality 395 score of 28 (Phred+33, minimum read length of 75bp) using trimmomati c software 396 (v0.38). Filtered reads were mapped to Sorghum bicolor v3.0.1 genome release using 397 HISAT2 (2.1.0) aligner and quantified at a gene level using FeatureCount software 398 (v1.6.2) on Sorghum bicolor v3.1.1 annotation file (Retrieved form Phytozome). F or 399 differential expression analysis, low-abundant raw counts were filtered out and remaining 400 counts normalized by library size and TMM. Pairwise differential expression analysis 401 was performed using edgeR package (v3.34.1) in R (v4.1.1). Gene Ontology enrichment 402 analysis and representations were performed in R using clusterProfiler package (v4.0.5) 403 based on best Arabidopsis orthologs hits for differentially regulated sorghum genes and 404 the org.At.tair.db annotation package (v3.13.0, TAIR annotation release fr om April 405 2021). Both, raw reads files and processed counts have been deposited at Gene Omnibus 406 Expression (GEO) database with identifier GSEXXXXX. Enriched GO categories were 407 obtained using Arabidopsis orthologs and double checked in PantherDB (Mi et al., 2019) 408 and Morokoshi Transcriptomic Database (Makita et al., 2015) 409 410

411 The authors thanks, Prof. Yanhai Yin (Iowa State Univ.) for kindly providing us with the 412 anti-BES1 antibodies, and Dr. Mar Marqués-Bueno for technical and hands-on advice on 413 Western Blot. 414 415 FUNDING 416 AIC-D is a recipient of a BIO2016 -78150-P grant funded by the Spanish Ministry of 417 Economy and Competitiveness and Agencia Estatal de Investigación (MINECO/AEI) 418 and Fondo Europeo de Desarrollo Regional (FEDER), and a European Research Council, 419 ERC Consolidator Grant (ERC -2015-CoG – 683163). JBF-M is supported by the grant 420 2017SGR718 from Secretaria d’Universitats i Recerca del Departament d’Empresa i 421 Coneixement de la Generalitat de Catalunya and by the ERC - 2015-CoG – 683163 422 granted to the AIC -D laboratory. N.L has received funding from the European Union’s 423 Horizon 2020 research and innovation programme under the Marie Skłodowska -Curie 424 grant agreement No 945043 and was additionally supported by grant CEX2019-000902-425 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted April 26, 2024. ; https://doi.org/10.1101/2024.04.22.590590doi: bioRxiv preprint 15 S funded by MCIN/AEI/10.13039/501100011033. AR-M received a predoctoral 426 fellowship from Fundación Tatiana Pérez de Guzmán el Bueno. DB-E and DM are funded 427 by the ERC -2015-CoG – 683163 granted to the AIC -D laboratory. This project has 428 received funding from the European Research Council (ERC) under the European 429 Union’s Horizon 2020 research and innovation programme (Grant Agreement No 430 683163). This work was supported by the CERCA Programme from the Generalitat de 431 Catalunya. We acknowledge financial support from the Spanish Ministry of Economy 432 and Competitiveness (MINECO), through the “Severo Ochoa Programme for Centres of 433 Excellence in R&D” 2016-2019 (SEV-2015-0533). 434 435 AUTHOR CONTRIBUTION 436 AR-M, DM and AIC-D outlined the manuscript, AR-M and AIC-D wrote the manuscript. 437 AR-M performed the microscopy analysis and most of the data analysis. AR-M and DM 438 performed WB experiments and the complementation analysis. DM cloned SbBRI1 CDS. 439 DB-E, NL and JBF-M performed the sorghum EMS mutant research in adult plants. FL-440 E analyzed the raw transcriptomic data. AIC-D designed the research. All authors 441 reviewed and edited the manuscript. 442 Present address: 2 Center for Plant Systems Biology, VIB, 9025, Ghent, Belgium. 3 443 Rhine-Waal University of Applied Science, Life Science Faculty, Marie -Curie Straße, 444 47533 Kleve, Germany. 4 Institute of Chemistry and Bioanalytics, School of Life 445 Sciences, University of Applied Sciences and Arts Northwestern Switzerland (FHNW), 446 4132 Muttenz, Switzerland. 5 Department of Biosciences, University of Milan, Milan, 447 Italy. 448 449

450 Betegón‐Putze I, Mercadal J, Bosch N, Planas‐Riverola A, Marquès‐Bueno M, Vilarrasa‐451 Blasi J, Frigola D, Burkart RC, Martínez C, Conesa A, et al. 2021. Precise transcriptional 452 control of cellular quiescence by BRAVO/WOX5 complex in Arabidopsis roots. 453 Molecular Systems Biology 17. 454 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted April 26, 2024. ; https://doi.org/10.1101/2024.04.22.590590doi: bioRxiv preprint 16 Blasco-Escámez D, Lozano-Elena F, Fàbregas N, Caño-Delgado AI. 2017. The Primary 455 Root of Sorghum bicolor (L. Moench) as a Model System to Study Brassinosteroid 456 Signaling in Crops. In: Russinova E, Caño -Delgado AI, eds. Methods in Molecular 457 Biology. Brassinosteroids. New York, NY: Springer New York, 181–192. 458 Bojar D, Martinez J, Santiago J, Rybin V, Bayliss R, Hothorn M. 2014. Crystal structures 459 of the phosphorylated BRI 1 kinase domain and implications for brassinosteroid signal 460 initiation. The Plant Journal 78: 31–43. 461 Caño-Delgado A, Yin Y, Yu C, Vafeados D, Mora-García S, Cheng J-C, Nam KH, Li J, 462 Chory J. 2004. BRL1 and BRL3 are novel brassinoste roid receptors that function in 463 vascular differentiation in Arabidopsis. Development 131: 5341–5351. 464 Castorina G, Consonni G. 2020. The Role of Brassinosteroids in Controlling Plant Height 465 in Poaceae: A Genetic Perspective. International Journal of Molecular Sciences 21: 1191. 466 Chadalavada K, Kumari BDR, Kumar TS. 2021. Sorghum mitigates climate variability 467 and change on crop yield and quality. Planta 253: 113. 468 Chono M, Honda I, Zeniya H, Yoneyama K, Saisho D, Takeda K, Takatsuto S, Hoshino 469 T, Watanabe Y. 2003. A Semidwarf Phenotype of Barley uzu Results from a Nucleotide 470 Substitution in the Gene Encoding a Putative Brassinosteroid Receptor. Plant Physiology 471 133: 1209–1219. 472 Dockter C, Gruszka D, Braumann I, Druka A, Druka I, Franckowiak J, Gough SP, 473 Janeczko A, Kurowska M, Lundqvist J, et al. 2014. Induced Variations in Brassinosteroid 474 Genes Define Barley Height and Sturdiness, and Expand the Green Revolution Genetic 475 Toolkit. Plant Physiology 166: 1912–1927. 476 Fàbregas N, Li N, Boeren S, Nash TE, Goshe MB, Clouse SD, de Vries S, Caño-Delgado 477 AI. 2013. The BRASSINOSTEROID INSENSITIVE1 –LIKE3 Signalosome Complex 478 Regulates Arabidopsis Root Development. The Plant Cell 25: 3377–3388. 479 Fàbregas N, Lozano -Elena F, Blasco -Escámez D, Tohge T, Martínez -Andújar C, 480 Albacete A, Osorio S, Bustamante M, Riechmann JL, Nomura T, et al. 2018. 481 Overexpression of the vascular brassinosteroid receptor BRL3 confers drought resistance 482 without penalizing plant growth. Nature Communications 9: 4680. 483 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted April 26, 2024. ; https://doi.org/10.1101/2024.04.22.590590doi: bioRxiv preprint 17 FAOSTAT statistical database. 2020. Rome: Food and Agriculture Organization of the 484 United Nations. 485 Ferreira-Guerra M, Marquès-Bueno M, Mora-García S, Caño-Delgado AI. 2020. Delving 486 into the evolutionary origin of steroid sensing in plants. Current Opinion in Plant Biology 487 57: 87–95. 488 Freeman G, Halton J. 1946. Note on exact treatment of contingency, goodness of fit and 489 other problems of significance. Biometrika 38: 141–149. 490 González-García M -P, Vilarrasa -Blasi J, Zhiponova M, Divol F, Mora -García S, 491 Russinova E, Caño -Delgado AI. 2011. Brassinosteroids control meristem size by 492 promoting cell cycle progression in Arabidopsis roots. Development 138: 849–859. 493 Graeff M, Rana S, Marhava P, Moret B, Hardtke CS. 2020. Local and Systemic Effects 494 of Brassinosteroid Perception in Developing Phloem. Current Biology 30: 1626-1638.e3. 495 Gupta A, Rico-Medina A, Caño-Delgado AI. 2020. The physiology of plant responses to 496 drought. Science 368: 266–269. 497 Gupta A, Rico-Medina A, Lozano-Elena F, Marqués-Bueno M, Fontanet JB, Fàbregas N, 498 Alseekh S, Fernie AR, Caño-Delgado AI. 2023. Brassinosteroid receptor BRL3 triggers 499 systemic plant adaptation to elevated temperature from the phloem cells. Plant Biology. 500 Hacham Y, Holland N, Butterfield C, Ubeda -Tomas S, Bennett MJ, Chory J, Savaldi -501 Goldstein S. 2011. Brassinosteroid perception in the epidermis controls root meristem 502 size. Development 138: 839–848. 503 Hartwig T, Chuck GS, Fujioka S, Klempien A, Weizbauer R, Potluri DPV, Choe S, Johal 504 GS, Schulz B. 2011. Brassinosteroid control of sex determination in maize. Proceedings 505 of the National Academy of Sciences of the United States of America 108: 19814–19819. 506 Hetz W, Hochholdinger F, Schwall M, Günter F. 1996. Isolati on and characterization of 507 rtcs, a maize mutant deficient in the formation of nodal roots. The Plant Journal : 845–508 857. 509 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted April 26, 2024. ; https://doi.org/10.1101/2024.04.22.590590doi: bioRxiv preprint 18 Hilley J, Truong S, Olson S, Morishige D, Mullet J. 2016. Identification of Dw1, a 510 Regulator of Sorghum Stem Internode Length (PK Subudhi , Ed.). PLOS ONE 11: 511 e0151271. 512 Hirano K, Kawamura M, Araki -Nakamura S, Fujimoto H, Ohmae -Shinohara K, 513 Yamaguchi M, Fujii A, Sasaki H, Kasuga S, Sazuka T. 2017. Sorghum DW1 positively 514 regulates brassinosteroid signaling by inhibiting the nuclear localizatio n of 515 BRASSINOSTEROID INSENSITIVE 2. Scientific Reports 7: 126. 516 Hochholdinger F, Park WJ, Sauer M, Woll K. 2004. From weeds to crops: genetic 517 analysis of root development in cereals. Trends in Plant Science 9: 42–48. 518 Hothorn M, Belkhadir Y, Dreux M, Dabi T, Noel JosephP, Wilson IA, Chory J. 2011. 519 Structural basis of steroid hormone perception by the receptor kinase BRI1. Nature 474: 520 467–471. 521 Hughes TE, Langdale JA, Kelly S. 2014. The impact of widespread regulatory 522 neofunctionalization on homeolog gene evolu tion following whole-genome duplication 523 in maize. Genome Research 24: 1348–1355. 524 Jiao Y, Burke JJ, Chopra R, Burow G, Chen J, Wang B, Hayes C, Emendack Y, Ware D, 525 Xin Z. 2016. A Sorghum Mutant Resource as an Efficient Platform for Gene Discovery 526 in Grasses. The Plant Cell: tpc.00373.2016. 527 Kang B, Wang H, Nam KH, Li J, Li J. 2010. Activation -Tagged Suppressors of a Weak 528 Brassinosteroid Receptor Mutant. Molecular Plant 3: 260–268. 529 Kelly-Bellow R, Lee K, Kennaway R, Barclay JE, Whibley A, Bushell C, Spooner J, Yu 530 M, Brett P, Kular B, et al. 2023. Brassinosteroid coordinates cell layer interactions in 531 plants via cell wall and tissue mechanics. Science 380: 1275–1281. 532 Kinoshita T, Caño-Delgado A, Seto H, Hiranuma S, Fujioka S, Yoshida S, Chory J. 2005. 533 Binding of brassinosteroids to the extracellular domain of plant receptor kinase BRI1. 534 Nature 433: 167–171. 535 Kir G, Ye H, Nelissen H, Neelakandan AK, Kusnandar AS, Luo A, Inzé D, Sylvester 536 AW, Yin Y, Becraft PW. 2015. RNA Interference Knockdown of BRASSINOSTEROID 537 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted April 26, 2024. ; https://doi.org/10.1101/2024.04.22.590590doi: bioRxiv preprint 19 INSENSITIVE1 in Maize Reveals Novel Functions for Brassinosteroid Signaling in 538 Controlling Plant Architecture. Plant Physiology 169: 826–839. 539 Kirschner GK, Stahl Y, Von Korff M, Simon R. 2017. Unique and Conserved Features 540 of the Barley Root Meristem. Frontiers in Plant Science 8: 1240. 541 Li J, Chory J. 1997. A Putative Leucine -Rich Repeat Receptor Kinase Involved in 542 Brassinosteroid Signal Transduction. Cell 90: 929–938. 543 Li Z, Sela A, Fridman Y, Höfte H, Savaldi -Goldstein S, Wolf S. 2021. Optimal BR 544 signalling is required for adequate cell wall orientation in the Arabidopsis root meristem. 545 Development. 546 Li J, Wen J, Lease KA, Doke JT, Tax FE, Walker JC. 2002. BAK1, an Arabidopsis LRR 547 Receptor-like Protein Kinase, Interacts with BRI1 and Modulates Brassinosteroid 548 Signaling. Cell 110: 213–222. 549 Lozano-Elena F, Planas -Riverola A, Vilarrasa -Blasi J, Schwab R, Caño -Delgado AI. 550 2018. Paracrine brassinosteroid signaling at the stem cell niche controls cellular 551 regeneration. Journal of Cell Science: jcs.204065. 552 Makarevitch I, Thompson A, Muehlbauer GJ, Springer NM. 2012. Brd1 Gene in Maize 553 Encodes a Brassinosteroid C-6 Oxidase (S-B Wu, Ed.). PLoS ONE 7: e30798. 554 Makita Y, Shimada S, Kawashima M, Kondou-Kuriyama T, Toyoda T, Matsui M. 2015. 555 MOROKOSHI: Transcriptome Database in Sorghum bicolor. Plant and Cell Physiology 556 56: e6–e6. 557 Martignago D, Rico -Medina A, Blasco -Escámez D, Fontanet -Manzaneque JB, Caño -558 Delgado AI. 2020. Drought Resistance by Engineering Plant Tissue-Specific Responses. 559 Frontiers in Plant Science 10: 1676. 560 Nakagawa T, Suzuki T, Murata S, Nakamura S, Hino T, Maeo K, Tabata R, Kawai T, 561 Tanaka K, Niwa Y, et al. 2007. Improved Gateway Binary Vectors: High -Performance 562 Vectors for Creation of Fusion Constructs in Transgenic Analysis of Plants. Bioscience, 563 Biotechnology, and Biochemistry 71: 2095–2100. 564 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted April 26, 2024. ; https://doi.org/10.1101/2024.04.22.590590doi: bioRxiv preprint 20 Nolan TM, Vukašinović N, Hsu C -W, Zhang J, Vanhoutte I, Shahan R, Taylor IW, 565 Greenstreet L, Heitz M, Afanassiev A, et al. 2023. Brassinosteroid gene regulatory 566 networks at cellular resolution in the Arabidopsis root. Science 379: eadf4721. 567 O’Kennedy MM, Grootboom A, Shewry PR. 2006. Harnessing sorghum and millet 568 biotechnology for food and health. Journal of Cereal Science 44: 224–235. 569 Paterson AH, Bowers JE, Bruggmann R, Dubchak I, Grimwood J, Gundlach H, Haberer 570 G, Hellsten U, Mitros T, Poliakov A, et al. 2009. The Sorghum bicolor genome and the 571 diversification of grasses. Nature 457: 551–556. 572 Pavelescu I, Vilarrasa‐Blasi J, Planas‐Riverola A, González‐García M, Caño‐Delgado AI, 573 Ibañes M. 2018. A Sizer model for cell differentiation in Arabidopsis thaliana root 574 growth. Molecular Systems Biology 14. 575 Pettersen E, Goddard T, Huang C, Couch G, Greenblatt D, Meng C, Ferrin T. 2004. USCF 576 Chimera - a visualization system for exploratory research and analysis. 577 Planas-Riverola A, Gupta A, Betegón -Putze I, Bosch N, Ibañes M, Caño -Delgado AI. 578 2019. Brassinosteroid signaling in plant development and adaptation to stress. 579 Development 146: dev151894. 580 Raghuwanshi A, Birch RG. 2010. Genetic transformation of sweet sorghum. Plant Cell 581 Reports 29: 997–1005. 582 R.E. Karper, Quinby JR. 1946. The history and evolution of Milo in the United States. 583 Journal of the american society of agronomy. 584 Rocateli AC, Raper RL, Balkcom KS, Arriaga FJ, Bransby DI. 2012. Biomass sorghum 585 production and components under different irrigation/tillage systems for the southeastern 586 U.S. Industrial Crops and Products 36: 589–598. 587 Salas Fernandez MG, Becraft PW, Yin Y, Lübberstedt T. 2009. From dwarves to giants? 588 Plant height manipulation for biomass yield. Trends in Plant Science 14: 454–461. 589 Salazar-Henao JE, Lehner R, Betegón-Putze I, Vilarrasa-Blasi J, Caño-Delgado AI. 2016. 590 BES1 regulates the localization of the brassin osteroid receptor BRL3 within the 591 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted April 26, 2024. ; https://doi.org/10.1101/2024.04.22.590590doi: bioRxiv preprint 21 provascular tissue of the Arabidopsis primary root. Journal of Experimental Botany 67: 592 4951–4961. 593 Singh A, Breja P, Khurana JP, Khurana P. 2016. Wheat Brassinosteroid -Insensitive1 594 (TaBRI1) Interacts with Members of TaSERK Gene Family and Cause Early Flowering 595 and Seed Yield Enhancement in Arabidopsis (PK Trivedi, Ed.). PLOS ONE 11: 596 e0153273. 597 Soper D. 2022. Fisher’s exact test calculator for a 2x3 contingency table. 598 Sun Y, Fan X-Y, Cao D-M, Tang W, He K, Zhu J-Y, He J-X, Bai M-Y, Zhu S, Oh E, et 599 al. 2010. Integration of Brassinosteroid Signal Transduction with the Transcription 600 Network for Plant Growth Regulation in Arabidopsis. Developmental Cell 19: 765–777. 601 Sun H, Xu H, Li B, Shang Y, Wei M, Zhang S, Zhao C, Qin R, Cui F, Wu Y. 2021. The 602 brassinosteroid biosynthesis gene, ZmD11, increases seed size and quality in rice and 603 maize. Plant Physiology and Biochemistry 160: 281–293. 604 Swigoňová Z, Lai J, Ma J, Ramakrishna W, Llaca V, Bennetzen JL, Messing J. 2004. 605 Close Split of Sor ghum and Maize Genome Progenitors. Genome Research 14: 1916–606 1923. 607 Tong H, Chu C. 2012. Brassinosteroid Signaling and Application in Rice. Journal of 608 Genetics and Genomics 39: 3–9. 609 Truernit E, Bauby H, Dubreucq B, Grandjean O, Runions J, Barthélémy J, Palauqui J-C. 610 2008. High -Resolution Whole -Mount Imaging of Three -Dimensional Tissue 611 Organization and Gene Expression Enables the Study of Phloem Development and 612 Structure in Arabidopsis. The Plant Cell 20: 1494–1503. 613 Vert G, Nemhauser JL, Geldner N, Hong F, Cho ry J. 2005. MOLECULAR 614 MECHANISMS OF STEROID HORMONE SIGNALING IN PLANTS. Annual Review 615 of Cell and Developmental Biology 21: 177–201. 616 Vilarrasa-Blasi J, González -García M-P, Frigola D, Fàbregas N, Alexiou KG, López -617 Bigas N, Rivas S, Jauneau A, Lohmann JU, Benfey PN, et al. 2014. Regulation of Plant 618 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted April 26, 2024. ; https://doi.org/10.1101/2024.04.22.590590doi: bioRxiv preprint 22 Stem Cell Quiescence by a Brassinosteroid Signaling Module. Developmental Cell 30: 619 36–47. 620 Vriet C, Russinova E, Reuzeau C. 2012. Boosting Crop Yields with Plant Steroids. The 621 Plant Cell 24: 842–857. 622 Wang Z -Y, Nakano T, Gendron J, He J, Chen M, Vafeados D, Yang Y, Fujioka S, 623 Yoshida S, Asami T, et al. 2002. Nuclear-Localized BZR1 Mediates Brassinosteroid -624 Induced Growth and Feedback Suppression of Brassinosteroid Biosynthesis. 625 Developmental Cell 2: 505–513. 626 Wang Y, Perez-Sancho J, Platre MP, Callebaut B, Smokvarska M, Ferrer K, Luo Y, Nolan 627 TM, Sato T, Busch W, et al. 2023. Plasmodesmata mediate cell -to-cell transport of 628 brassinosteroid hormones. Nature Chemical Biology 19: 1331–1341. 629 Wang Z-Y, Seto H, Fujioka S, Yoshida S, Chory J. 2001. BRI1 is a critical component of 630 a plasma-membrane receptor for plant steroids. Nature 410: 380–383. 631 Warner CA, Biedrzycki ML, Jacobs SS, Wisser RJ, Caplan JL, Sherrier DJ. 2014. An 632 Optical Clearing Technique for Plant Ti ssues Allowing Deep Imaging and Compatible 633 with Fluorescence Microscopy. Plant Physiology 166: 1684–1687. 634 Wolf S, Mravec J, Greiner S, Mouille G, Höfte H. 2012. Plant Cell Wall Homeostasis Is 635 Mediated by Brassinosteroid Feedback Signaling. Current Biology 22: 1732–1737. 636 Yamaguchi M, Fujimoto H, Hirano K, Araki-Nakamura S, Ohmae-Shinohara K, Fujii A, 637 Tsunashima M, Song XJ, Ito Y, Nagae R, et al. 2016. Sorghum Dw1, an agronomically 638 important gene for lodging resistance, encodes a novel protein involved in cell 639 proliferation. Scientific Reports 6: 28366. 640 Yamamuro C, Ihara Y, Wu X, Noguchi T, Fujioka S, Takatsuto S, Ashikari M, Kitano H, 641 Matsuoka M. 2000. Loss of Function of a Rice brassinosteroid insensitive1 Homolog 642 Prevents Internode Elongation and Bending of the Lamina Joint. The Plant Cell 12: 1591–643 1605. 644 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted April 26, 2024. ; https://doi.org/10.1101/2024.04.22.590590doi: bioRxiv preprint 23 Yin Y, Wang Z -Y, Mora -Garcia S, Li J, Yoshida S, Asami T, Chory J. 2002. BES1 645 Accumulates in the Nucleus in Response to Brassinosteroids to Regulate Gene 646 Expression and Promote Stem Elongation. Cell 109: 181–191. 647 Zhang C, Bai M, Chong K. 2014. Brassinosteroid-mediated regulation of agronomic traits 648 in rice. Plant Cell Reports 33: 683–696. 649 650 FIGURE LEGENDS 651 Figure 1. BRI1 structure is conserved in Sorghum bicolor. 652 A) Sorghum BRI1 is more closely related to BRI1 from Zea mays and other crops than to 653 Arabidopsis thalian a (both underlined in red) . B) In silico 3D modelling of the LRR 654 region of both A rabidopsis and SbBRI1 reveals a great conservation in structure, 655 including the island domain , shown together with BL . Both studied EMS alleles are 656 shown. 657 658 Figure 2. SbBRI1 can complement its AtBRI1 mutant counterpa rt phenotypes at 659 macro and microscopical levels. 660 A) 35S:SbBRI1-GFP; bri1-301 (SbBRI1ox) can complement adult Arabidopsis bri1-301 661 phenotype. White bars represent 4cm. B, C) SbBRI1ox complements root length 662 phenotype of A rabidopsis bri1-301 in control conditions . (B) and after BL 663 supplementation (C). White bar represents 1cm. D, E, F) SbBRI1ox complements 664 meristem size (E) and QC division phenotype (F) of Arabidopsis bri1-301 in control 665 conditions and after BL supplementation. Black bars in (D) represents 100µm in root tip 666 panels and 20µm in small QC panels. 667 668 Figure 3. bri1-ems sorghum shows reduced plant height, yield, and reduced root 669 length and a defective BL response. 670 A) 4-month-old bri1-ems72 plants display reduced plant height. B, C, D) bri1-ems72 671 plants show reduced plant height during all developmental stages when compared against 672 their WT counterparts and against WT control. E) bri1-ems72 panicles present reduced 673 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted April 26, 2024. ; https://doi.org/10.1101/2024.04.22.590590doi: bioRxiv preprint 24 yield when compared with their WT counterparts. Total yield weight is sh own in H). F, 674 G, I, J) Embryonic roots of bri1-ems72 present reduced length and reduced sensitivity to 675 BL supplementation when compared with their WT counterparts. 676 Figure 4. Root meristem architecture and BR response of BTx623 WT sorghum is 677 shown by adapting common Arabidopsis confocal microscopy techniques. 678 A) Root architecture of BTx623 after mPS-PI staining. From upper to lower, arrowpoints 679 mark: xylem start, meristem region border, and QC region. B, C, D, E ) Root meristem 680 architecture of BTx623 grown at different BL concentrations. At increased BL amounts, 681 Root thinning, meristem shrinkening and cell disorganization is observed. F) EdU 682 staining of root meristem region of BTx623 sorghum reveals (in yellow) the cells dividing 683 in a given timeframe (12h) and, by DAPI counterstaining, (in purple) an undivided set of 684 cells that conforms the QC region. When grown at increased BL concentrations, G H, I) 685 EdU staining shows an increased cell division in the QC region (G, H) up to the point of 686 total meristem exhaustion (I). Black and white bars in (B) to (I) represent 80µm, while 687 white bars in small QC panels represent 40µm. 688 689 Figure 5. SbBRI1 is involved in root sensitivity of BRs. 690 A, B) Root architecture of WT-ems72 and bri1-ems72 shows a reduced response of bri1-691 ems72 to BL supplementation. C, D) bri1-ems72 shows smaller cell number reduction 692 than WT-ems72 when supplemented with BL (C) while the meristem length size 693 reduction shows no si gnificant differences (D), fundamentally suggesting an impaired 694 sensitivity to BL in bri1-ems72. Black bars in (A) and (B) root tip panels represent 80µm, 695 while black bars in small QC panels represent 40µm. 696 697 Figure 6. Confocal analysis of root meristem region revealed SbBRI1 role in 698 meristem development. 699 A, B, C) WT-ems72 QC region presents increased cell division grown under increasing 700 BL supplementation. D, E, F) bri1-ems72 QC region presents increased cell division 701 grown under increasing BL supplementation. G, H) A comparison of dividing cell 702 number in WT-ems72 and bri1-ems72 reveals a reduced division rate in bri1-ems72 at 703 lower BL concentrations, together with a reduced bri1-ems72 exhaustion rate at higher 704 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted April 26, 2024. ; https://doi.org/10.1101/2024.04.22.590590doi: bioRxiv preprint 25 BL concentrations, fundamentally suggesting an insensitivity to BL due to the lack of a 705 fully functional BRI1. White bars in root tip panels represent 80µm, while white bars in 706 QC panels represent 40µm. 707 Figure 7. RNA-seq of bri1vsWT sorghum roots reveal BRI1 role in root growth 708 A) GOEA map of deregulated GO categories in sorghum bri1-ems72 vs WT-ems72 709 RNA-seq shows a role of SbBRI1 in cell wall metabolism regulation. B), C), D) E) 710 Deployment of genes within the most deregulated GO terms from our upregulated set of 711 genes. Sorghum gene name and closest Arabidopsis ortholog names are shown. 712 713 714 SUPPLEMENTARY INFORMATION 715 Supplementary Figure 1: A) Sorghum and Arabidopsis BRI1 share a common sequence 716 of kinase, transmembrane and island domain, suggesting functional conservation. 717 Extracelular domain is mostly conserved with the exception of five LRR domains. B) 718 Alignment of multiple BRI1 sequences fro m different species showing a general 719 conservation of the intracellular and extracellular domains. 720 Supplementary Figure 2: A) SbBRI1 complements rosette size and flowering time in 721 bri1-301 Arabidopsis. B) BES1 dephosphorilation after BL treatment in SbBRI1 722 complemented lines of Arabidopsis bri1-301 shows a functional conservation of BRI1 in 723 sorghum. 724 Supplementary Figure 3: A) bri1-ems87 panicles present reduced yield when compared 725 with their WT counterpart. C) Seed weight of 50 seeds of WT-ems and bri1-ems mutants. 726 D, E) size comparison of seeds of WT-ems and bri1-ems mutants. 727 Supplementary Figure 4: A) Comparison of in vitro grown BTx623 from whole 728 seedling vs from explanted embryo. B) Hydroponic grown sorghum seedlings show a 729 insensitivity to BL of bri1-ems87. C) bri1-ems87 sorghum root shows insensitivity to low 730 concentrations of BL in terms of root length. 731 Supplementary Figure 5: A) Example at 40nm BL of root meristem exhaustion in WT-732 ems72 and bri1-ems72 (in yellow, dividing cells), B) and with bright field bottom 733 channel. C) Example of exhausted WT-ems72 and bri1-ems72 roots using mPS-PI show 734 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted April 26, 2024. ; https://doi.org/10.1101/2024.04.22.590590doi: bioRxiv preprint 26 a great cell architecture disorganization in the root tip. D) At elevated BL concentrations, 735 no differences were found between WT-ems72 and bri1-ems72. 736 Supplementary Figure 6: Diagram of backcrossing process to select our working 737 seed lines. EMS-mutated M4 seeds of both bri1 alleles from Jiao et al, 2016, were 738 backcrossed against their unmutagenized parental BTx623 and PCR -genotyped for bri1 739 mutations. Homozygous bri1 line was selected for a second round of backcrossing and 740 their progeny PCR-genotyped. F2 of this second backcross was PCR-genotyped using the 741 aerial organs and used at seedling stage to perform RNA -seq of the root organ of both 742 bri1 and WT alleles of the BRI1 gene. Sibling progeny of both were grown and used to 743 obtain F3 seeds for a comparative phenotypical analysis of BRI1. 744 Supplementary Figure 7: A) After BP reaction, SbBRI1 coding sequence was cloned 745 into pEN -L1L2 plasmid, giv ing pEN -L1L2-SbBRI1. B) pEN-L1L2-SbBRI1 and the 746 p4P1r-35S vector were LR -recombined with destination vector pGWB406 to obtain 747 p35S:SbBRI1-GFP 748 749 SUPPLEMENTARY FILES 750 Supplementary Table 1. Primer sequences used for sequencing of bri1 alleles in 751 Arabidopsis and sorghum. 752 Supplementary Table 2. Protein sequences used for alignments. 753 Supplementary Table 3: Primer sequences used for cloning. 754 Supplementary Table 4: List of upregulated genes in bri1 vs WT RNA-seq. 755 Supplementary Table 5: List of downregulated genes in bri1 vs WT RNA-seq. 756 Supplementary Video 1: mPS-PI of sorghum root meristem reveals the root architecture 757 of the stem region. At 60X, the QC region can be extrapolated by comparing with EdU 758 staining. 759 Supplementary Video 2: mPS-PI of sorghum root meristem reveals the root architecture 760 of the stem region at 40X. 761 Supplementary Video 3: EdU staining of sorghum root meristem shows an undividing 762 region (QC) surrounded by stem cells. Z-stack obtained from 40X confocal microscopy. 763 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted April 26, 2024. ; https://doi.org/10.1101/2024.04.22.590590doi: bioRxiv preprint 27 764 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted April 26, 2024. ; https://doi.org/10.1101/2024.04.22.590590doi: bioRxiv preprint At BRI1 crystal SbBRI1 (3RJ0) homology model P407L V403M (a) (b) Figure 1. BRI1 structure is conserved in Sorghum bicolor. (a) Sorghum BRI1 is more closely related to BRI1 from Zea Mays and other crops than to Arabidopsis thaliana (both underlined in red). (b) In silico 3D modelling of the LRR region of both Arabidopsis and SbBRI1 reveals a great conservation in structure, including the island domain, shown together with BL. Both studied EMS alleles are shown. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted April 26, 2024. ; https://doi.org/10.1101/2024.04.22.590590doi: bioRxiv preprint 4nM BL Control bri1-301Col-025 DAG AtBRI1OX SbBRI1OX#3 SbBRI1OX#8 #3 #8 Col-0 bri1-301 AtBRI1OX SbBRI1OX Col-0 bri1-301 AtBRI1OX SbBRI1OX #3 #8 - + - + - + - + - + *** * *** *** *** (a) (b) (c) (e) (f) (d) Figure 2. SbBRI1 can complement its AtBRI1 mutant counterpart phenotypes at macro and microscopical levels. (a) 35S:SbBRI1-GFP; bri1-301 (SbBRI1ox) can complement adult Arabidopsis bri1-301 phenotype. White bars represent 4cm. (b,c) SbBRI1ox complements root length phenotype of Arabidopsis bri1-301 in control conditions (b) and after BL supplementation (c). White bar represents 1cm. (d,e,f) SbBRI1ox complements meristem size (e) and QC division phenotype (f) of Arabidopsis bri1-301 in control conditions and after BL supplementation. Black bars in (d) represents 100µm in root tip panels and 20µm in small QC panels. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted April 26, 2024. ; https://doi.org/10.1101/2024.04.22.590590doi: bioRxiv preprint WT-ems72 bri1-ems72 Ctrl 4nM 40nM Ctrl 4nM 40nM WT-ems72 bri1- ems72 BTx623 WT-ems72 bri1-ems72 bri1-ems72WT-ems72 bri1-ems72WT-ems72 Ctrl 4nM BL40nM BL Ctrl 4nM BL40nM BL BTx623 bri1-ems72WT-ems72 BTx623 bri1-ems72WT-ems72 BTx623 (a) (b) (c) (d) (e) (f) (g) (h) (i) (j) Figure 3. bri1-ems sorghum shows reduced plant height, yield, and reduced root length and a defective BL response. (a) 4-month-old bri1-ems72 plants display reduced plant height. (b, c, d) bri1-ems72 plants show reduced plant height during all developmental stages when compared against their WT counterparts and against WT control. (e) bri1-ems72 panicles present reduced yield when compared with their WT counterparts. Total yield weight is shown in (h). (f, g, i, j) Embryonic roots of bri1-ems72 present reduced length and reduced sensitivity to BL supplementation when compared with their WT counterparts. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted April 26, 2024. ; https://doi.org/10.1101/2024.04.22.590590doi: bioRxiv preprint BTx623 Ctrl 4nM BL 40nM BL 400nM BL (a) (b) (c) (d) (e) (f) (g) (h) (i) Figure 4. Root meristem architecture and BR response of BTx623 WT sorghum is shown by adapting common Arabidopsis confocal microscopy techniques. (a) Root architecture of BTx623 after mPS-PI staining. From upper to lower, arrowpoints mark: xylem start, meristem region border, and QC region. (b, c, d, e) Root meristem architecture of BTx623 grown at different BL concentrations. At increased BL amounts, Root thinning, meristem shrinkening and cell disorganization is observed. (f) EdU staining of root meristem region of BTx623 sorghum reveals (in yellow) the cells dividing in a given timeframe (12h) and, by DAPI counterstaining, (in purple) an undivided set of cells that conforms the QC region. When grown at increased BL concentrations, (g, h, i) EdU staining shows an increased cell division in the QC region (g, h) up to the point of total meristem exhaustion (i). Black and white bars in (b) to (i) represent 80µm, while white bars in small QC panels represent 40µm. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted April 26, 2024. ; https://doi.org/10.1101/2024.04.22.590590doi: bioRxiv preprint WT-ems72 bri1-ems72 Ctrl 4nM BL Ctrl 4nM BL (a) (b) (d)(c) WT-ems72 Ctrl WT-ems72 4nM BL bri1-ems72 Ctrl bri1-ems72 4nM BL WT-ems72 Ctrl WT-ems72 4nM BL bri1-ems72 Ctrl bri1-ems72 4nM BL Figure 5. SbBRI1 is involved in root sensitivity of BRs. (a, b) Root architecture of WT-ems72 and bri1-ems72 shows a reduced response of bri1- ems72 to BL supplementation. (c, d) bri1-ems72 shows smaller cell number reduction than WT-ems72 when supplemented with BL (c) while the meristem length size reduction shows no significant differences (d), fundamentally suggesting an impaired sensitivity to BL in bri1-ems72. Black bars in (a) and (b) root tip panels represent 80µm, while black bars in small QC panels represent 40µm. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted April 26, 2024. ; https://doi.org/10.1101/2024.04.22.590590doi: bioRxiv preprint WT-ems72 bri1-ems72 Ctrl 4nM BL 40nM BLWT-ems72bri1-ems72 (a) (d) (e) (f) (b) (c) (g) (h) Figure 6. Confocal analysis of root meristem region revealed SbBRI1 role in meristem development. (a, b, c) WT-ems72 QC region presents increased cell division grown under increasing BL supplementation. (d, e ,f) bri1-ems72 QC region presents increased cell division grown under increasing BL supplementation. (g, h) A comparison of dividing cell number in WT-ems72 and bri1-ems72 reveals a reduced division rate in bri1-ems72 at lower BL concentrations, together with a reduced bri1-ems72 exhaustion rate at higher BL concentrations, fundamentally suggesting an insensitivity to BL due to the lack of a fully functional BRI1. White bars in root tip panels represent 80µm, while white bars in QC panels represent 40µm. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted April 26, 2024. ; https://doi.org/10.1101/2024.04.22.590590doi: bioRxiv preprint (a) (b) (c) (d) GO:0009828 "plant-type cell wall loosening" GO:0009611 "response to wounding" GO:0044036 "cell wall macromolecule metabolic process" (e) GO:2000280 "regulation of root development " p.value p.value p.value p.value Figure 7. RNA-seq of bri1vsWT sorghum roots reveal BRI1 role in root growth. (a) GOEA map of deregulated GO categories in sorghum bri1-ems72 vs WT-ems72 RNA-seq shows a role of SbBRI1 in cell wall metabolism regulation. (b, c, d, e)Deployment of genes within the most deregulated GO terms from our upregulated set of genes. Sorghum gene name and closest Arabidopsis ortholog names are shown. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted April 26, 2024. ; https://doi.org/10.1101/2024.04.22.590590doi: bioRxiv preprint