Rice Embryogenic Trigger BABY BOOM1 Promotes Somatic Embryogenesis 4 by Upregulation of Auxin Biosynthesis Genes

bioRxiv preprint doi: https://doi.org/10.1101/2020.08.24.265025; this version posted August 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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-NC-ND 4.0 International license.

1 Short title: Regulation of somatic embryogenesis by OsBABY BOOM1

3 Title: Rice embryogenic trigger BABY BOOM1 promotes somatic embryogenesis 4 by upregulation of auxin biosynthesis genes

6 Imtiyaz Khanday1, 2, Christian Santos-Medellín1,3 Venkatesan Sundaresan1, 2, 4 *

7 1Department of Plant Biology, University of California, Davis, CA, USA.

8 2Innovative Genomics Institute, University of California, Berkeley, CA, USA.

9 3 Present address: Department of Plant Pathology, University of California Davis, CA, USA.

10 4Department of Plant Sciences, University of California, Davis, CA, USA.

12 *To whom correspondence should be addressed:

13 Dr. Venkatesan Sundaresan

14 Department of Plant Biology, University of California, Davis, CA, USA.

15 Tel: +1 530-754-9677

16 Email: [email protected]

18 One-sentence summary:

19 Rice BABY BOOM1 induces somatic embryogenesis from differentiated tissues by promoting 20 auxin biosynthesis through direct upregulation of YUCCA genes.

21 Author contributions: I. K. and V. S. designed experiments; I. K. performed experiments. I. K. 22 and C. S-M. analyzed the data. I. K. wrote the manuscript with inputs from V. S.

23 Funding information: 24 C.S-M. acknowledges support from the University of California Institute for Mexico 25 (UCMEXUS), Consejo Nacional de Ciencia y Tecnología (CONACYT), and Secretaría de 26 Educación Pública (México). This research was funded by grants from the National Science 27 Foundation (IOS-1547760), Innovative Genomics Institute and the U.S. Department of 28 Agriculture (USDA) Agricultural Experiment Station (CA-D-XXX-6973-H).

29 ABSTRACT

30 Somatic embryogenesis, a powerful tool for clonal propagation and for plant transformation, 31 involves cellular reprogramming of differentiated somatic cells to acquire pluripotency. Somatic 32 embryogenesis can be induced by treating explants with plant growth regulators. However, 33 several plant species including agronomically important cereal crops remain recalcitrant to 34 dedifferentiation and transformation except from embryonic tissues. Somatic embryogenesis can 35 also be induced by ectopic expression of select embryonic factors, including in cereals by BABY 36 BOOM (BBM) transcription factors. How BBM genes bypass the need for exogenous hormones 37 is not well understood. Here, we investigated downstream targets during induction of somatic 38 embryogenesis in rice by OsBBM1 ((Oryza sativa BABY BOOM1). Transient induction of 39 OsBBM1 led to the upregulation of auxin biosynthesis OsYUCCA genes. Continued induction of 40 OsBBM1 resulted in somatic embryogenesis without the need for exogenous auxins. Genetic 41 mutant analysis of OsBBM1 downstream targets, OsYUCCA6, OsYUCCA7 and OsYUCCA9, 42 show that they are required for normal rice development including root and shoot development. 43 Somatic embryogenic potential of OsYUCCA triple mutants was highly compromised despite the 44 presence of exogenous auxin. Additionally, we show that somatic embryogenesis induction by 45 exogenous auxin in rice requires functional BBM genes. Thus, OsBBM1 mediated cellular 46 reprogramming and somatic embryogenesis likely involves increased localized auxin through 47 direct upregulation of OsYUCCA genes. This study reveals mechanistic details of how somatic 48 embryogenesis is established in differentiated tissues in rice, a monocot model and 49 agronomically important cereal crop, with the potential utility to improve regeneration from 50 tissue culture for recalcitrant plants in future.

51 Keywords: Somatic embryogenesis, pluripotency, auxin biosynthesis, dedifferentiation, 52 regeneration.

54 INTRODUCTION

55 Plant cells possess the remarkable property of dedifferentiation, the ability to return to a 56 meristematic state whereby they can be redirected to various developmental fates including 57 pluripotency and totipotency. This can be achieved by endogenous or exogenous stimuli such as 58 the expression of various pluripotency factors or by treatment with plant hormones and stresses 59 (Elhiti et al., 2013; Su et al., 2020). Somatic embryogenesis is a method of clonal propagation

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60 and a powerful tool for dedifferentiation and subsequently plant regeneration. Although different 61 ways of inducing somatic embryogenesis exist in dicots (Mendez-Hernandez et al., 2019), many 62 plant species including monocots and especially agronomically important cereal crops remain 63 resistant to in vitro tissues culture, except for embryonic tissues (Eudes et al., 2003). A number 64 of plant genes such as BABY BOOM (BBM) (Boutilier et al., 2002), LEAFY COTYLEDON1 65 (LEC1), LEC2 (Lotan et al., 1998; Stone et al., 2001; Wojcikowska et al., 2013), WUSCHEL 66 (WUS) (Zuo et al., 2002), FUSCA3 (FUS3) (Luerssen et al., 1998), ABA INSENSITIVE3 (ABI3) 67 (Parcy et al., 1994; Shiota et al., 1998), EMBRYOMAKER (Tsuwamoto et al., 2010), AtMYB115, 68 AtMYB118 (Wang et al., 2009), SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE 69 (SERK) (Schmidt et al., 1997) and AGAMOUS-LIKE 15 (AGL15) (Harding et al., 2003) have 70 been shown to either induce or promote somatic embryogenesis in dicotyledonous plants. 71 Among plant hormones, treatment with auxin is most widely used to induce totipotency and 72 embryogenesis from differentiated plant cells (Wojcik et al., 2020). Stress conditions like 73 osmotic stress, temperature stress, heavy metal stress, ultraviolet irradiation, wounding and 74 chemical treatments have been used to promote somatic embryogenesis (Feher, 2015; Su et al., 75 2020).

76 BABY BOOM genes are members of the AINTEGUMENTA (ANT) clade of 77 the superfamily of APETALA 2/ETHYLENE RESPONSE FACTOR (AP2/ERF) transcription 78 factors with two AP2 domains (Dipp-Alvarez and Cruz-Ramirez, 2019). The first member of this 79 family was identified in Brassica napus microspore cultures and its ectopic expression induced 80 somatic embryogenesis on seedlings (Boutilier et al., 2002). The property of BBM genes to 81 induce somatic embryogenesis is well documented in several other dicot species (Jha and Kumar, 82 2018). While only one BBM gene has been identified in Arabidopsis, four BBM-like genes exist 83 in rice (Dipp-Alvarez and Cruz-Ramirez, 2019; Khanday et al., 2019). In Arabidopsis, BBM has 84 been shown to express in zygotic embryos (Boutilier et al., 2002; Galinha et al., 2007); however, 85 the role of AtBBM in zygotic embryogenesis has not yet been characterized, and loss-of- 86 function mutants show no phenotypic abnormalities (Galinha et al., 2007)). We previously 87 showed that BBM genes play an important role in zygotic embryogenesis in rice; specifically, 88 that paternal expression of BBM factors is essential for embryo initiation and three BBM genes, 89 OsBBM1, OsBBM2 and OsBBM3 redundantly regulate embryo development (Khanday et al., 90 2019).

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91 YUCCA (YUC) genes encode flavin-containing monooxygenase enzymes that 92 catalyze the rate-limiting step of oxidative decarboxylation of indole-3-pyruvate acid during 93 auxin biosynthesis to form indole-3-acetic acid (IAA), the predominant auxin found in plants 94 (Cao et al., 2019). The Arabidopsis genome encodes 11 YUCCA genes, whereas 14 members of 95 this family have been identified in rice (Gallavotti et al., 2008). YUC gene mediated auxin 96 biosynthesis has been shown to play diverse roles during various phases of plant development 97 and response to environmental changes (Cao et al., 2019). YUC genes have been shown to 98 regulate root and shoot development (Chen et al., 2014; Zhang et al., 2018), leaf morphogenesis 99 (Cheng et al., 2006, 2007), inflorescence development (Woodward et al., 2005; Gallavotti et al., 100 2008), floral organ development (Cheng et al., 2006), establishment of the embryonic axis 101 (Robert et al., 2013) and embryo organ initiation (Cheng et al., 2007).

102 We previously reported that ectopic expression of OsBBM1 can induce somatic 103 embryogenesis in differentiated tissues in rice (Khanday et al., 2019), thus making it a potential 104 model for inducing and understanding somatic embryogenesis in monocots. Many downstream 105 target genes regulated by AtBBM during somatic embryogenesis have been identified in 106 Arabidopsis (Passarinho et al., 2008; Horstman et al., 2017). However, a specific mechanism by 107 which BBM factors induce somatic embryogenesis and support a hormone-independent growth 108 of explants remains to be fully characterized. More generally, the mechanisms underlying 109 induction of somatic embryogenesis in monocots are not known. In this study, we characterized 110 the downstream target genes and pathways regulated by rice BBM1. We show that OsBBM1 111 directly activates the expression of auxin biosynthesis YUCCA genes to promote somatic 112 embryogenesis. We further show the OsYUCCA genes targeted by OsBBM1 are essential for 113 plant development and somatic embryogenesis.

114

115 RESULTS

116 Global Profile of Genes Deregulated on Induction of OsBBM1

117 In order to understand the gene regulatory network underlying initiation of embryogenesis in 118 somatic tissues by BBM genes, we translationally fused OsBBM1 gene product to a 119 dexamethasone (DEX) inducible rat glucocorticoid receptor (GR) (Supplemental Fig. S1A; 120 Khanday et al., 2019). Of the 10 transgenic lines raised with this construct, line #5 was found to

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121 have the best expression for the transgene GR (Supplemental Fig. S1C), thus all the subsequent 122 experiments were performed in the progenies from line #5. For controlled induction of 123 OsBBM1 expression, we took two-week old seedlings, wild-type or transgenic for the OsBBM1- 124 GR fusion and treated them for various time points with DEX or a protein synthesis inhibitor, 125 cycloheximide (CYC) or a combination of both (Supplemental Fig. S1D; see Materials and 126 Methods). We choose 6 h time point for treatments based on the induction of OsYUC7, a 127 potential target gene of OsBBM1 (Supplemental Fig. S1B and S1D). OsYUC7 expression was 128 induced in rice zygotes soon after fertilization and this expression pattern coincides with 129 expression of OsBBM1 (Anderson et al., 2017). OsYUC7 expression was also found to be highly 130 induced in OsBBM1 overexpression lines in rice (Supplemental Fig. S1B; Khanday et al., 2019). 131 Among the various time points tested, OsYUC7 attained peak induction in 6 h time point, thus 132 this time point was chosen for gene expression analysis regulated by OsBBM1 (Supplemental 133 Fig. S 1D).

134 From our RNA-seq analysis of the samples treated with DEX for 6 hours, we detected a

135 total of 298 differentially expressed (DE) genes in OsBBM1-GR seedlings at a log2 fold change 136 of >1 and FDR < 0.05, after normalization with mock and wild-type controls (see Materials and 137 Methods) ( Supplemental Data Set S1). Of these 298 DE transcripts, 162 were upregulated and 138 136 were downregulated on OsBBM1 induction (Fig. 1, A and B). In the upregulated category, 139 the expression of 138 genes was maintained in the presence of CYC, suggesting that they might 140 be direct targets of OsBBM1 (Fig. 1A; Supplemental Data Set S2). From the downregulated 141 category, the expression of 120 transcripts was also suppressed in the presence of CYC, which 142 indicates OsBBM1 can directly represses the expression of these genes (Fig. 1B; Supplemental 143 Data Set S2). A closer inspection of the genes directly regulated by OsBBM1 shows they belong 144 to different functional categories including transcription factors, cell signaling, cell cycle 145 regulation, hormone signaling, metabolism etc. (Supplemental Data Set S1 and S2). We 146 confirmed the perturbations in expression of some the representative members of these different 147 functional categories of genes in both upregulated and downregulated genes by RT-qPCR (Fig. 148 1, C and D).

149 OsBBM1 Upregulates Expression of Auxin Biosynthesis Genes

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150 Plant hormone auxin is known to regulate various developmental and physiological processes 151 (Cao et al., 2019). Among the genes whose expression is regulated by OsBBM1, we find 152 members of the YUCCA family of auxin biosynthesis genes and some auxin response factors 153 (Supplemental Data Set S1). We confirmed the upregulation of expression for three OsYUCCA 154 genes, OsYUC6 (Os07g25540), OsYUC7 (Os04g03980) and OsYUC9 (Os01g16714) by RT- 155 qPCR (Fig. 2A). The elevation in the expression of all the three genes by DEX was also 156 maintained in the presence of CYC, suggesting their direct regulation by OsBBM1 (Fig. 2A). To 157 further confirm that changes in the expression of these auxin biosynthesis genes are directly 158 modulated by OsBBM1, we probed the occupancy of OsBBM1 at cis-regulatory regions of these 159 OsYUC genes. Chromatin immune-precipitation (ChIP) was carried out with an anti-GR 160 antibody (Sorefan et al., 2009), with tissues from OsBBM1-GR DEX treated plants of similar age 161 as used for the RNA-seq experiment (see Materials and Methods). ChIP PCR reactions were 162 carried out for 2 kb upstream sequences using various primers combination (Supplementary 163 Table S2). OsBBM1 occupancy was observed in a region about 1.4 kb upstream of the 164 transcription start site (TSS) of OsYUC6 (Fig. 2B), in the immediate upstream sequences of 165 OsYUC7 (Fig. 2C) and about 1.1 kb upstream of TSS for OsYUC9 (Fig. 2D). Significant 166 enrichment at these sites was further confirmed by ChIP-qPCR over wild-type and input controls 167 (Fig. 2E). The other regions within these 2 kb upstream sequences showed no enrichment by 168 ChIP-PCR (Supplemental Fig. S2, A - C). A closer inspection of the sequences of OsBBM1 169 binding sites in the cis-regulatory regions of these OsYUCCA genes shows they contain putative

170 sequence (CACA (N)5CNA or reverse complement GTGT(N)5GNT) of AtBBM binding site 171 (Horstman et al., 2017). Two of these sequence elements are present in OsBBM1 bound regions 172 of OsYUC7 and OsYUC9, whereas only one site is present in OsYUC9.Such sequences are also 173 present in the other upstream regions of these OsYUCCA genes; however, we did not observe 174 any OsBBM1 occupancy on those sites (Supplemental Fig. S2, A - C). Thus, OsBBM1 175 enrichment is site specific in the cis-regulatory regions of these OsYUCCA genes. Together with 176 OsBBM1-GR DEX-induction data, these results confirm that OsBBM1 directly regulates the 177 expression of OsYUC6, OsYUC7 and OsYUC9 auxin biosynthesis genes.

178 OsBBM1 Expression Phenocopies Auxin Induced Somatic Embryogenesis

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179 Auxin treatment induces somatic embryogenesis during plant regeneration (Wojcik et al., 2020). 180 It is required for cellular reprogramming and induction of embryogenic callus from wild-type 181 seeds in rice (Wani et al., 2011). Ectopic expression of OsBBM1 resulted in somatic embryo 182 formation on rice leaves in the absence of auxin (Khanday et al., 2019). This result, combined 183 with our observation that OsBBM1 can directly upregulate the expression of OsYUC genes, led 184 us to examine if OsBBM1 promotes somatic embryogenesis by controlling auxin biosynthesis. 185 We germinated OsBBM1-GR seeds on mock and DEX containing media. While mock treated 186 seeds grew normally (Fig. 3A), we observed callus induction from the scutellum tissues in seeds 187 grown on DEX containing media (Fig. 3, B and C). The seedlings from zygotic embryos in DEX 188 treated seeds germinated normally; however, they were shorter and twisted compared to the 189 mock treated seeds (Fig. 3, A and B; Supplemental Fig. S3B). Interestingly, wild-type seeds 190 germinated on auxin-rich media, in addition to callus formation from scutellum, also exhibited 191 the twisted seedling phenotype (Supplemental Fig. S3A). Callus formation was also observed 192 from the first two juvenile leaves in the DEX treated OsBBM1-GR seedlings or from any leaf 193 that was in contact with the DEX containing media (Fig. 3D, Supplemental Fig. S3, C and D). 194 However, no such phenotype was observed in wild-type seeds germinated on auxin containing 195 media (Supplemental Fig. S3A). When the OsBBM1-GR induced calli were allowed to grow on 196 the DEX containing media for 3 to 4 weeks, they went on to start organogenesis ultimately 197 regenerated roots and form green foci, indicative of start of shoot regeneration (Fig. 3, E and F). 198 However, the greening calli ultimately reverted back to proliferation phase with loss of 199 chlorophyll and never completed shoot regeneration. Also, regenerated roots or roots from the 200 zygotic embryos after a prolonged time (3-4 weeks) on the DEX containing media started to 201 dedifferentiate into callus-like tissues (Supplemental Fig. S3E). Thus, OsBBM1 expression is 202 sufficient to compensate for the auxin requirement for inducing somatic embryogenesis. In 203 addition, when one-week old OsBBM1-GR seedlings grown on mock media were shifted to a 204 DEX containing medium, they developed roots from aerial tissues (Supplemental Fig. S3, F and 205 G). Moreover, OsBBM1-GR plants watered with DEX around the flowering transition developed 206 awns from lemmas, which is not observed in wild-type rice plants (Supplemental Fig. S3, H and 207 I). These vegetative and floral phenotypes have been shown to be associated with mutations 208 related to auxin signaling and response (Toriba and Hirano, 2014; Mignolli et al., 2017).

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209 We next investigated if externally applied auxin requires OsBBM1 for initiating somatic 210 embryogenesis, by subjecting Osbbm mutant seeds to auxin treatment. Three rice BBM genes, 211 OsBBM1, OsBBM2 and OsBBM3 have been shown to redundantly regulate zygotic embryo 212 development (Khanday et al., 2019). We germinated seeds on callus induction medium from a 213 mother plant segregating for OsBBM1 (OsBBM1/Osbbm1) but homozygous mutant for OsBBM2 214 (Osbbm2/Osbbm2) and OsBBM3 (Osbbm3/Osbbm3) (see Materials and Methods). While all the 215 control wild-type seeds (n=25) formed callus on auxin rich media (Supplemental Fig. S4A), 216 some of the segregating mutant seeds failed to undergo any somatic embryogenesis 217 (Supplemental Fig. S4B, red arrows). After genotyping (Supplemental Fig. S4F), seeds forming 218 callus were found to be either wild-type (OsBBM1/OsBBM1 Osbbm2/Osbbm2 Osbbm3/Osbbm3; 219 n=6/26) or heterozygous (OsBBM1/Osbbm1 Osbbm2/Osbbm2 Osbbm3/Osbbm3; n=13/26) for 220 OsBBM1 (Supplemental Fig. S4, B, C, E and F). None of the homozygous mutant seeds 221 (Osbbm1/Osbbm1 Osbbm2/Osbbm2 Osbbm3/Osbbm3; n=7/26) underwent somatic 222 embryogenesis (Supplemental Fig. S4, B, D, E and F). Thus, auxin mediated induction of 223 somatic embryogenesis displays a requirement for functional BBM genes.

224 Functional Characterization of OsYUUCA6, OsYUCCA7 and OsYUCCA9 Genes

225 Our results that OsBBM1 can directly regulate the expression of three OsYUCCA genes (Fig. 2, 226 A-E) combined with auxin perturbation phenotypes observed on induction of OsBBM1 (Fig. 3, 227 Supplemental Fig. S3), led us to investigate the functions of these YUCCA genes in rice and 228 decipher their role in promoting embryogenesis downstream of OsBBM1. We created mutants in 229 these three OsYUCCA genes using genome editing (see Materials and Methods). We created an 230 Osyuc7 Osyuc9 double mutant and an Osyuc6 Osyuc7 Osyuc9 triple mutant (Supplemental Fig. 231 S5 and S6). Ten independent transgenic lines with CRISPR-Cas9 construct were raised for 232 Osyuc7 Osyuc9 double mutant and 12 for Osyuc6 Osyuc7 Osyuc9 triple mutant. Two randomly 233 selected lines were used for detailed mutation analysis for each mutant combination 234 (Supplemental Fig. S5 and S6). T2 progenies in which CRISPR-Cas9 transgene had already 235 segregated out (Supplemental Fig. S5C) and were null mutations for all alleles of all the genes 236 (Supplemental Fig. S5, A, B and S6, C and D) were taken further for phenotypic analysis.

237 Mutations in OsYUC genes affected various vegetative characteristics of rice plants. 238 Plants double mutant for OsYUC7 and OsYUC9 were shorter compared to the wild-type plants

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239 (Fig. 4, A and E). The phenotype of reduction in height was further enhanced in Osyuc6 Osyuc7 240 Osyuc9 triple mutant plants (Fig. 4, B and E). Another prominent phenotype in these mutant 241 plants was elongation in primary root length (Fig. 4, C and D). While average root length in 12 242 day old wild-type control seedlings was 4.4 cm, it increased to 15.3 cm in Osyuc7 Osyuc9 double 243 mutant (Fig. 4, C and F). This phenotype was further elevated in Osyuc6 Osyuc7 Osyuc9 triple 244 mutant (Fig. 4D). The average root length in Osyuc6 Osyuc7 Osyuc9 triple mutant plants was 245 about 23.5 cm (Fig. 4F). Lateral root development was also impaired in rice YUC mutants. 246 Compared to the wild-type, fewer lateral roots developed in Osyuc7 Osyuc9 double mutant 247 (Supplemental Fig. S5, D and E). This lateral root development defect was more prominent in 248 Osyuc6 Osyuc7 Osyuc9 triple mutant (Supplemental Fig. S5F).

249 Next, we analyzed the effect of these OsYUCCA mutations on embryogenic callus 250 formation. Seeds from wild-type controls, Osyuc7 Osyuc9 double and Osyuc6 Osyuc7 Osyuc9 251 triple mutant plants were germinated in dark on media containing 2 mg/L of 2,4- 252 Dichlorophenoxyacetic acid (2,4-D) for callus induction. While we observed robust induction 253 and proliferation of calli from the embryonic tissues in wild-type seeds (n=37/60) (Fig. 4, G and 254 H), embryogenic calli (nodular, compact and pale) formation was severely reduced in Osyuc7 255 Osyuc9 double mutant seeds (n=8/60) (Fig. 4, I and J). In Osyuc6 Osyuc7 Osyuc9 triple mutant 256 seeds, embryogenic calli induction was drastically reduced and exhibited almost no proliferation 257 (n=3/60) (Fig. 4, K and L). Thus, despite supplementing the media with auxin to complement the 258 auxin biosynthesis deficiency caused by the OsYUCCA mutations, callus induction in these 259 mutants was severely compromised.

260 Cytokinin Supplementation Completes Shoot Regeneration in OsBBM1 Induced Somatic 261 Embryos

262 Cytokinin plant growth regulators are required for the shoot regeneration in tissue culture (Hill 263 and Schaller, 2013). The DEX induction of OsBBM1-GR resulted in somatic embryos which 264 started organogenesis, but shoot regeneration was not completed (Fig 3, E and F). Therefore, we 265 tested the possibility of promoting the shoot regeneration process to completion, by 266 supplementation of the media with cytokinin. Seeds germinated either in light or dark on media 267 containing both DEX and cytokinin 6-Benzylaminopurine (BAP), showed regeneration of 268 shootlets from somatic embryos induced by OsBBM1 expression (Fig. 3G, Supplemental Fig.

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269 S3J). When these regenerating plantlets were taken off both the treatments and grown on normal 270 media, they completed the full developmental program and formed normal seedlings (Fig. 3H). 271 From our RNA-seq analysis of DEX treated 6h leaf samples, we also found that OsBBM1 272 negatively regulates the expression of some cytokinin signaling genes, such as cytokinin 273 response regulators like OsRR4 (Os01g72330) and OsRR9 (Os11g04720)/OsRR10 274 (Os12g04500) (Fig. 2F and Supplemental Data Set S1 and S2). This putative repressive function 275 is consistent with previous results from Arabidopsis BBM, which has been shown to interact 276 with TOPLESS-related (TPR) corepressors (Causier et al., 2012), suggesting that BBM factors 277 can also act as transcriptional repressors. Thus, these results demonstrate that supplementation of 278 cytokinin can override the incomplete shoot formation in OsBBM1-induced embryogenesis, 279 which might be related to OsBBM1-mediated suppression of cytokinin response factors.

280

281 DISCUSSION

282 Although somatic embryogenesis has been a problem of long-standing interest in plant biology, 283 it is not known whether a common mechanism underlies somatic embryogenesis between 284 monocot and dicot species, due in part to limited studies on successful induction of somatic 285 embryogenesis in monocot species. Previous studies in Arabidopsis show AtBBM regulates 286 somatic embryogenesis by regulating the expression of so called LAFL genes -LEC1, LEC2, 287 FUS3 and ABI3 (Horstman et al., 2017). Further, AtLEC1 can regulate AtYUC10 expression 288 (Junker et al., 2012) and AtLEC2 expression has been shown to be associated with expression of 289 AtYUC1, AtYUC4 and AtYUC10 genes (Wojcikowska et al., 2013). Although AtBBM can bind 290 to cis-regulatory sequences of AtYUC3 and AtYUC8 genes (Horstman et al., 2017), no changes in 291 their expression or those of other YUC genes have been reported upon induction of somatic 292 embryogenesis by AtBBM (Passarinho et al., 2008). Our data show that in rice, OsBBM1 293 directly upregulates the expression of YUCCA genes- OsYUC6, OsYUC7 and OsYUC9 upon 294 induction of somatic embryos (Fig. 2). These results may indicate differences in the mechanism 295 between dicots and monocots in the control of auxin biosynthesis for induction of somatic 296 embryos by BBM genes. Additionally, our analysis shows no detectable expression of either 297 OsLEC1 (OsLEC1A, Os02g49370; OsLEC1B, Os06g17480) or OsLEC2 (Os04g58000, 298 Os04g58010 and Os01g68370) related genes in our leaf samples of 6 h DEX treatment

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299 (Supplemental Data Set S1). However, OsLEC1A and OsLEC1B expression was observed in 300 somatic embryos established subsequent to induction by OsBBM1 (Khanday et al., 2019). We 301 note that changes in AtLEC gene expression were not observed in 35S:AtBBM-GR DEX treated 302 seedlings prior to formation of somatic embryos (Passarinho et al., 2008), although the LAFL 303 genes were found to be targets of AtBBM in somatic embryos as discussed above (Horstman et 304 al., 2017). Therefore, it is possible that LEC genes are not required for the initiation of somatic 305 embryogenesis in differentiated cells by BBM factors but are important during the later stages of 306 somatic embryogenesis, possibly for maintenance of the embryonic state. The observation that 307 OsBBM1 modulates auxins by regulating the expression of OsYUC genes is also supported by 308 the fact that transient induction of OsBBM1-GR during other stages of development such as 309 seedlings and flowers also leads to auxin perturbation phenotypes (Supplemental Fig. S3, F - I). 310 These observations together with the phenotypes observed on BBM1 overexpression (Khanday et 311 al., 2019), support the evidence that YUCCA genes are immediate downstream targets of 312 OsBBM1. Of the three OsYUCCA genes upregulated by OsBBM1 (Fig. 2), OsYUC7 expression 313 is de novo induced in rice zygotes after fertilization and peaks at 9 hours after pollination which 314 correlates with OsBBM1 expression (Anderson et al., 2017). Auxin biosynthesis genes have also 315 been shown to be upregulated in maize zygotes after fertilization (Chen et al., 2017). These data 316 suggest that the initiation of zygotic embryogenesis by BBM transcription factors might also 317 involve the biosynthesis of auxin by direct activation of YUCCA genes. The differences in the 318 spectrum of YUCCA genes activated during zygotic embryogenesis compared to the direct 319 targets we observed in this study might reflect different chromatin configurations and relative 320 accessibility of these genes in the two very different cell types, i.e. egg cells and leaf cells. The 321 extent to which the pathways for the initiation of somatic and zygotic embryogenesis by BBM 322 factors is conserved remains to be elucidated.

323 Tissue culture for plant regeneration is a two-step process in which explants are 324 initially cultured on an auxin-rich medium to produce callus. These calli are then cultivated on a 325 cytokinin-rich medium to regenerate shoots (Hill and Schaller, 2013). Although OsBBM1 was 326 able to induce somatic embryogenesis, and embryos proceeded to greening and roots 327 development, the entire process of complete plantlet regeneration was never achieved without 328 the supplementation of media with cytokinin (Fig. 3). The down regulation of some cytokinin

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329 response regulator genes by OsBBM1 (Fig. 2F) might partially account for the requirement of 330 cytokinin to complete the shoot formation in OsBBM1 induced somatic embryos.

331 In Arabidopsis, it was found that efficiency of somatic embryogenesis induced by 332 external auxin is reduced by mutants in two YUC genes (Wojcikowska et al., 2013). We show 333 here that in rice, induction of somatic embryogenesis is highly compromised in Osyuc7 Osyuc9 334 double and Osyuc6 Osyuc7 Osyuc9 triple mutant combinations, despite the presence of 335 exogenous auxins in the medium (Fig. 4, I to L). The observation that endogenous IAA 336 biosynthesis is required for somatic embryogenesis in rice and cannot be substituted by 337 application of exogenous auxins is also consistent with the different developmental roles of 338 endogenous and external auxin, deduced from previous studies in Arabidopsis. Root 339 developmental defects caused by yuc mutations were not rescued by external application of 340 auxins in Arabidopsis (Cheng et al., 2006; Chen et al., 2014). Thus, tissue-specific local auxin 341 biosynthesis by YUCCA flavin monooxygenases is important both for plant development and 342 somatic embryogenesis.

343 Auxin has been shown to be main inducer of somatic embryogenesis in various 344 plant species (Wojcik et al., 2020). Auxin has also been shown to induce members of 345 PLETHORA/BBM transcription factors in Arabidopsis roots (Mahonen et al., 2014), and also 346 expression of BBM homologs in seedlings of the monocot cereal Brachypodium distachyon 347 (Kakei et al., 2015). Differentiated leaves from rice do not express OsBBM1 (Khanday et al., 348 2019) and are recalcitrant to callus induction even in the presence of auxin (Supplemental Fig. 349 S3A; (Hu et al., 2017). However, rice leaves with ectopic expression of OsBBM1, or with 350 OsBBM1-GR DEX-induction, undergo somatic embryogenesis (Fig. 3D; Khanday et al., 2019). 351 These observations combined with our results that Osbbm1 Osbbm2 Osbbm3 mutant seeds are 352 unable to undergo somatic embryogenesis (Supplemental Fig. S4), suggest that externally 353 applied auxin induces somatic embryogenesis by upregulating BBM gene expression in scutellar 354 cells, but is unable to upregulate BBM gene expression in differentiated leaf cells (Fig. 5). 355 Consistent with these observations, regeneration from tissue culture of a triple knock-out mutant 356 of OsBBM1, OsBBM2 and OsBBM3 was never achieved, even though OsBBM1-3 genes are 357 dispensable for vegetative growth and development (Khanday et al., 2019).

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358 In conclusion, OsBBM1 expression in differentiated tissues induces somatic 359 embryogenesis in the absence of exogenous auxins by promoting local auxin biosynthesis 360 through control of OsYUCCA gene expression. This function of OsBBM1 suggests a pathway 361 for induction of somatic embryogenesis that may be applicable to cereals generally, as 362 summarized in Figure 5. In scutellum or other tissues that are competent for somatic 363 embryogenesis induction by exogenous auxin, auxin induces expression of OsBBM1 (and 364 presumptively, the closely related paralogs OsBBM2 and OsBBM3). Loss of these BBM genes 365 results in failure of external auxin to induce somatic embryos. In differentiated leaf, a tissue that 366 is recalcitrant to somatic embryo induction by external auxin, the ectopic expression of OsBBM1 367 is sufficient to activate the downstream somatic embryogenesis pathway. In both cases, 368 OsBBM1 expression, promotes endogenous auxin biosynthesis through YUCCA genes, as well as 369 auto-activation of its own promoter to sustain its expression during somatic embryo formation 370 (Khanday et al., 2019). External auxin cannot substitute for the crucial role of the OsBBM1- 371 targeted YUCCA genes, as shown by mutant analysis. Elevated intracellular levels of IAA from 372 the YUCCA genes leads to dedifferentiation, and somatic embryos that produce roots and 373 greening callus with incipient shoot formation. However, complete formation of shoots requires 374 exogenous cytokinin, following which viable plants can be regenerated. This initial 375 characterization of the molecular pathway for induction of somatic embryogenesis in a cereal 376 crop can help pave the way for future experiments to develop methods for efficient tissue culture 377 and transformation in cereals and other recalcitrant plants.

378

379 MATERIALS AND METHODS

380 Plant Materials and Growth Conditions 381 All the experiments in this study were performed in rice cultivar Kitaake (Oryza sativa L. 382 subsp. japonica). All the seeds, wild-type, transgenic and mutant were initially germinated on 383 half strength Murashige and Skoog’s medium (Murashige and Skoog, 1962) containing 1% 384 sucrose and 0.3% phytagel. After 12-15 days of initial growth on media, seedlings were 385 transferred to greenhouse and grown there until seeds matured. 386 387 Chemical Treatments.

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388 Dexamethasone (DEX) and cycloheximide (CYC) treatments were carried in two-week-old wild- 389 type and OsBBM1:GR seedlings as described in Khanday et al., 2019. Leaf tissue samples for 390 RNA isolation for each biological replicate were collected from 4 plants and pooled together. 391 Tissues samples were harvested at 3h, 6h, 12h and 24 h after the start of treatment. For DEX 392 induced phenotypes (Fig. 3, A-F and Supplemental Fig. S3, B-E) OsBBM1-GR seeds were 393 grown under light on a media containing either mock or 10 µm DEX. In Fig. 3G and 394 Supplemental Fig. S3J, in addition to DEX, media also contained cytokinin 6- 395 Benzylaminopurine at 3 mg/L concentration and seeds were grown either in light (Fig. 3G) or in 396 dark (Supplemental Fig. S3J). For phenotypes in Supplemental Fig. S3, F and G, one-week-old 397 OsBBM1-GR seedlings were transferred to half strength MS media either mock treated or 398 containing 10 µM DEX and grown for one more week. For phenotypes observed in 399 Supplemental Fig. 3, H and I, plants started receiving either mock or 10 µM DEX water around 400 flowering transition (45 days after germination) until the panicles completely emerged. For 401 phenotypes shown in Fig. 4, G to L, Supplemental Fig. S3A and Supplemental Fig. 4, wild-type, 402 Osbbm or Osyuc mutant seeds were germinated on media containing 2 mg/L of 2,4- 403 Dichlorophenoxyacetic acid (2,4-D). The seeds were allowed to grow for two weeks under light 404 (Supplemental Fig. S3A), two weeks in the dark (Supplemental Fig. 4) and for six weeks in the 405 dark (Fig. 4, G to L). 406 407 Microscopy and Image Capture 408 409 Microscopic images were captured using a Zeiss Stemi SV11 stereo microscope fitted AxioCam 410 HRC camera (Zeiss). AxioVision version 4.8 software (Zeiss) was used for image capture and 411 processing. Images for plant phenotypes were taken with Cannon PowerShot SX60 HS digital 412 camera. All the images and other data panels were assembled into article figures using Adobe 413 Photoshop (Adobe Inc.). 414 415 RNA-Seq Library Preparation and Sequencing 416 RNA isolation, quality assessment, quantification and library preparations were done as 417 described (Anderson et al., 2017). Libraries were prepared from two biological replicates for 418 each sample with 80 ng of input RNA, using NuGEN Ovation RNA-seq Systems 1-16 following

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419 manufacturer instructions. Samples were multiplexed and 8 libraries per lane were run on 420 Illumina HiSeq platforms at UC Davis, Genome Center. 421 422 RNA-Seq Analysis

423 Cutadapt (Martin, 2011) was used to remove 3’ adapters and quality-trim reads at a Phred quality 424 threshold of 13. High-quality reads were then mapped to the Oryza sativa Nipponbare reference 425 genome (MSU v7.0)(Kawahara et al., 2013) using Tophat2 (Kim et al., 2013) with the minimum 426 and maximum intron sizes set at 20 and 15000 bases and the microexon search switched on. 427 Mapped reads were assigned to the MSU v7.0 gene models (Phytozome release 323) using 428 HTSeq. Differential expression analysis was performed using the edgeR package (Robinson et 429 al., 2010). Effective library sizes were calculated using the trimmed mean of M-values (TMM) 430 method and a quasi-likelihood (QL) negative binomial generalized linear model was fitted to the 431 data (Chen et al., 2016). The contrasts of DEX, DEX + CYC and CYC with their respective 432 mock samples were created for both wild-type and OsBBM1-GR genotypes. For each contrast, a 433 QL F-test was performed to detect differentially expressed genes (FDR < 0.05). Genes induced 434 by DEX or CYC treatments in wild-type were subtracted from the respective treatments in 435 OsBBM1-GR samples.

436 437 RT-PCR and RT-qPCR 438 All the cDNA synthesis, RT-PCRs, RT-qPCRs and analysis of RT-qPCRs were performed as 439 described (Khanday et al., 2019). All the RT-qPCR amplifications were done in two biological 440 replicates and each biological replicate was repeated as three technical replicates. All the primers 441 used for RT-PCRs and RT-qPCRs are listed in Supplemental Table S1. 442

443 Chromatin Immunoprecipitation (ChIP)

444 Chromatin Immunoprecipitation (ChIP) was performed as described (Khanday et al., 2013) with 445 following modifications. Wild-type and OsBBM1-GR two-week-old rice seedlings were grown 446 in 0.5X Mureshige and Skoog’s medium and treated with 10 µM DEX for 6 h. Leaf tissues were 447 harvested, and cross linked with 1% formaldehyde. For each biological replicate, chromatin was 448 prepared from 300 mg of tissues. Chromatin was sheared to ~500 bp size using a Covaris E220

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449 instrument in 1 ml Covaris milliTUBE. The instrument settings for chromatin shearing were 140 450 peak incident power (PIP), 5% duty factor, 200 cycles per burst (CBP). Chromatic complexes 451 were immunoprecipitated with 2 µg of anti-GR antibody (AB3580, Abcam) (Sorefan et al., 452 2009). Chromatin-antibody complexes were recovered using Protein A conjugated Dynabeads 453 (Invitrogen, 10001D). Primers used for ChIP-PCRs and ChIP-qPCRs are listed in Supplemental 454 Table S2. 455 456 Vector Construction, Osyucca and Osbbm Mutant Generation, and Genotyping 457 458 The construction of pZmUBI1::OsBBM1-GR (Supplemental Fig. S1A) is described in Khanday 459 et al., 2019. For generating Osyuc7 Osyuc9 double and Osyuc6 Osyuc7 Osyuc9 triple mutants, 460 single guide RNAs (sgRNAs) for gene editing for OsYUC6 (Os07g25540), OsYUC7 461 (Os04g03980) and OsYUC9 (Os01g16714) were designed using the webtool 462 https://www.genome.arizona.edu/crispr/ as described (Xie et al., 2014). The sgRNA sequences 463 are listed in Supplemental Table S3. Assembly of plasmid constructs for CRISPR-Cas9 editing 464 with these sgRNAs and rice transformations were done as described (Khanday et al., 2019). Ten 465 and twelve independent T0 transformants were generated for pCRISPR-OsYUC7 + OsYUC9 and 466 pCRISPR-OsYUC6 + OsYUC7+ OsYUC9 constructs, respectively. These T0 plants were 467 screened for mutations by genotyping PCRs, followed by sequencing of the amplicons. The 468 primers for genotyping PCRs are listed in Supplemental Table S3. T2 progenies form two lines 469 for each of the two CRSIPR-Cas9 constructs were studied in detail. Only those progenies in 470 which CRISPR-Cas9 construct was no longer present (Supplemental Fig. S5C) and had all the 471 requisite null alleles for mutations were considered for analysis (Supplemental Fig. S5 and S6). 472 Osbbm mutant generation and genotyping are described in detail in Khanday et al., 2019. Briefly, 473 a single bp deletion mutation in OsBBM1 CDS disrupts a SphI site. The DNA fragment at the 474 mutation site is amplified with the genotyping primers listed in Supplemental Table S3 and 475 restriction digested with SphI (Supplemental Fig. S4F). 476 Accession Numbers: 477 478 Accession numbers for the genes characterized in this work are listed in the main text. RNA 479 sequencing data from this article will be uploaded to GenBank data library and accession number 480 XXX updated after review. 481

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482 Supplemental Data 483 484 Supplemental Figure S1. DEX inducible gene expression system for OsBBM1. 485 Supplemental Figure S2. Occupancy of OsBBM1 on YUCCA gene loci. 486 Supplemental Figure S3. DEX induced phenotypes in OsBBM1-GR plants. 487 Supplemental Figure S4. Somatic embryogenesis in Osbbm mutants. 488 Supplemental Figure S5. Mutations in Osyucca7 Osyucca9 double mutant plants. 489 Supplemental Figure S6. Mutations in Osyucca6 Osyucca7 Osyucca9 triple mutants. 490 Supplemental Data Set S1. Genes deregulated by OsBBM1-GR in Mock vs. DEX treatment. 491 Supplemental Data Set S2. Genes directly regulated by OsBBM1-GR in Mock vs. DEX+CYC 492 treatment. 493 Supplemental Table S1. Primers used for RT-PCRs and RT-qPCRs. 494 Supplemental Table S2. Primers used for ChIP-PCRs and ChIP-qPCRS. 495 Supplemental Table S3. Sequences of sgRNAs and genotyping primers. 496 ACKNOWLEDGMENTS 497 We thank Alina Yalda for technical assistance with genotyping and green house transplantations. 498

499 Figure Legends

500 Figure 1. Changes in global gene expression in rice plants on OsBBM1-GR induction. A, RNA- 501 sequencing analysis showing number of genes up regulated on induction of OsBBM1-GR with 502 DEX alone or in the presence of CYC. B, A Venn diagram showing the down regulated 503 transcripts by OsBBM1-GR in rice leaves. The numbers are genes deregulated by OsBBM1-GR 504 under each treatment (Log2 fold >1 and FDR < 0.05). C and D, RT-qPCR validation of some of 505 the genes regulated by OsBBM1. C, Genes from up regulated category are Os04g45810, a 506 homeobox leucine zipper transcription factor, Os05g32350, a zinc finger containing protein, 507 Os04g55620, a receptor kinase, Os08g37390, a cyclin and Os02g53430, a DNA-3- 508 methyladenine glycosylase. D, From the down regulated category, the genes are Os05g10690 is a 509 MYB domain transcription factor, Os02g07820 a DUF581 domain containing protein and 510 Os05g03610 is phospholipase C protein. The log2 fold change is calculated after normalization 511 with mock and respective wild-type treatments. Error bars represent SEM, calculated from two 512 independent biological replicates, each done in triplicates. DEX and CYC treatments were done 513 for 6 h (see Materials and Methods). DEX, dexamethasone and CYC, cycloheximide.

514 Figure 2. Regulation of plant hormone pathway genes by OsBBM1. A to E, Direct up regulation 515 of auxin biosynthesis genes by OsBBM1. A, RT-qPCR analysis showing quantification of 516 transcript levels for auxin biosynthesis genes OsYUC6, OsYUC7 and OsYUC9 in OsBBM1-GR

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517 plants. Fold change in normalized expression is shown for samples treated for 6 h with DEX, 518 CYC or a combination of both the chemicals. The expression levels are normalized with mock 519 treated samples and also with the similar treatments in wild-type tissues. Error bars were 520 calculated as SEM, from two independent biological replicates. B to D, Chromatin- 521 immunoprecipitation (ChIP) assay for enrichment of OsBBM1 at three OsYUCCA loci. ChIP- 522 PCRs are shown for (B) OsYUC6, (C) OsYUC7 and (D) OsYUC9. Schematics show the 523 genomic organization of each locus. Exons are represented by blue box, introns by thin black 524 line, UTRs with unfilled boxes and upstream sequences with thick black line. The short bar 525 above the schematic shows the position of OsBBM1 occupancy on upstream sequences. E, ChIP- 526 qPCR for quantitation of OsBBM1 enrichment on the three OsYUCCA loci. The enrichment is 527 presented as percentage of input DNA. Error bars are represented as SEM, from two independent 528 biological replicates. F, Quantitation of downregulation in expression of cytokinin response 529 genes by RT-qPCR. Changes in expression are calculated as described for (A).

530 Figure 3. Phenotypic consequences of up regulation of auxin biosynthesis by OsBBM1 in rice 531 seeds. A, OsBBM1-GR seeds germinated on mock treated medium. Seedlings grow normally 532 (red arrowhead). B and C, OsBBM1-GR seeds grown on dexamethasone (DEX) containing 533 media showing callus formation from scutellum tissue of seeds (white arrow). Red arrowhead 534 points to the normally growing zygotic seedling. D, Somatic embryo formation from leaves of an 535 OsBBM1-GR seedling induced with DEX (white arrow). Red arrowhead shows the growing 536 zygotic seedling. E and F, Regenerating OsBBM1-GR calli induced with DEX. Black arrow 537 shows the chlorophyll/shoot development and red arrowhead marks the regenerating roots. G, 538 Complete plantlet formation from OsBBM1-GR DEX induced somatic embryos with addition of 539 cytokinin. White arrows show the regenerating plantlets from somatic embryos and red 540 arrowhead shows the seedling growing from the zygotic embryo. BAP is 6-Benzylaminopurine. 541 H, Complete development of DEX+BAP regenerated plantlets in (G), when taken off both the 542 treatments. Red arrowhead points to zygotic seedling and white arrows show regenerated 543 plantlets from somatic embryos. Scale bars are 1 cm in (A), (B), (C) and (H), and 2 mm in (D to

544 G).

545 Figure 4. Functional characterization of OsYUCCA6, OsYUCCA7 and OsYUCCA9 genes in rice. 546 A and B, Reduction in plant height in Osyucca mutants. A, Osyucca7 Osyucca9 double mutant 547 compared with wild-type. B, Osyucca6 Osyucca7 Osyucca9 triple mutant compared to wild-type. 548 Scale bars 10 cm. C and D, Perturbations in primary root length in Osyucca mutants. C, 549 Osyucca7 Osyucca9 double mutant. D, Osyucca6 Osyucca7 Osyucca9 triple mutant. Scale bars 5 550 cm. E, Bar graph showing the quantification of reduction in plant height in Osyucca mutants 551 (n=20 for each genotype). F, Quantitative representation of increase in root length in Osyucca 552 mutants in rice (n=20 for each genotype). G to L, Reduction in embryogenic calli induction in 553 Osyucca mutants in the presence of supplemented auxin. G and H, Wild-type showing induction 554 and proliferation of compact, pale and nodular embryogenic calli (red arrow heads) which are 555 phenotypically distinct from brown sectors of non-embryogenic calli (white arrows). I and J,

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556 Osyucca7 Osyucca9 double mutant showing some proliferation of embryogenic calli (red arrow 557 heads). K and L, Osyucca6 Osyucca7 Osyucca9 triple mutant, showing extensive brown sectors 558 of non-embryogenic calli (white arrows) without proliferation of pale embryogenic calli (red 559 arrowheads). 2,4-D is 2,4-Dichlorophenoxyacetic acid. Scale bars G to L are 1 cm.

560 Figure 5. Model explaining the mechanism of OsBBM1 action during somatic embryogenesis in 561 rice. OsBBM1 expression is required for dedifferentiation of somatic cells. In scutellum cells, 562 OsBBM1 is downstream of exogenous auxin (dotted arrow), but in differentiated leaf cells that 563 are recalcitrant to external auxin, OsBBM1 has to be expressed using a transgene as in this study. 564 OsBBM1 positively regulates the expression of auxin biosynthesis OsYUCCA (OsYUC6, 565 OsYUC7 and OsYUC9) genes. This leads to increase in the endogenous auxin levels, followed by 566 dedifferentiation, callus formation and subsequently somatic embryogenesis. OsBBM1 also 567 auto-activates its promoter to maintain expression and pluripotency during somatic 568 embryogenesis. The somatic embryos are able form roots and initiate shoot regeneration, but 569 shoot formation is not completed, possibly due to negative regulation of some cytokinin response 570 genes by OsBBM1. This block can be overcome by the addition of cytokinin in the culture media 571 and complete plant regeneration is achieved.

572

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641 Lotan T, Ohto M, Yee KM, West MA, Lo R, Kwong RW, Yamagishi K, Fischer RL, Goldberg RB, Harada JJ 642 (1998) Arabidopsis LEAFY COTYLEDON1 is sufficient to induce embryo development in 643 vegetative cells. Cell 93: 1195-1205 644 Luerssen H, Kirik V, Herrmann P, Misera S (1998) FUSCA3 encodes a protein with a conserved 645 VP1/AB13-like B3 domain which is of functional importance for the regulation of seed 646 maturation in Arabidopsis thaliana. Plant J 15: 755-764 647 Mahonen AP, Ten Tusscher K, Siligato R, Smetana O, Diaz-Trivino S, Salojarvi J, Wachsman G, Prasad K, 648 Heidstra R, Scheres B (2014) PLETHORA gradient formation mechanism separates auxin 649 responses. Nature 515: 125-129 650 Martin M (2011) Cutadapt removes adapter sequences from high-throughput sequencing reads. 651 EMBnet.journal; Vol 17, No 1: Next Generation Sequencing Data Analysis 652 Mendez-Hernandez HA, Ledezma-Rodriguez M, Avilez-Montalvo RN, Juarez-Gomez YL, Skeete A, 653 Avilez-Montalvo J, De-la-Pena C, Loyola-Vargas VM (2019) Signaling Overview of Plant Somatic 654 Embryogenesis. Front Plant Sci 10: 77 655 Mignolli F, Mariotti L, Picciarelli P, Vidoz ML (2017) Differential auxin transport and accumulation in the 656 stem base lead to profuse adventitious root primordia formation in the aerial roots (aer) mutant 657 of tomato (Solanum lycopersicum L.). J Plant Physiol 213: 55-65 658 Murashige T, Skoog F (1962) A Revised Medium for Rapid Growth and Bio Assays with Tobacco Tissue 659 Cultures. Physiologia Plantarum 15: 473-497 660 Parcy F, Valon C, Raynal M, Gaubier-Comella P, Delseny M, Giraudat J (1994) Regulation of gene 661 expression programs during Arabidopsis seed development: roles of the ABI3 locus and of 662 endogenous abscisic acid. Plant Cell 6: 1567-1582 663 Passarinho P, Ketelaar T, Xing M, van Arkel J, Maliepaard C, Hendriks MW, Joosen R, Lammers M, 664 Herdies L, den Boer B, van der Geest L, Boutilier K (2008) BABY BOOM target genes provide 665 diverse entry points into cell proliferation and cell growth pathways. Plant Mol Biol 68: 225-237 666 Robert HS, Grones P, Stepanova AN, Robles LM, Lokerse AS, Alonso JM, Weijers D, Friml J (2013) Local 667 auxin sources orient the apical-basal axis in Arabidopsis embryos. Curr Biol 23: 2506-2512 668 Robinson MD, McCarthy DJ, Smyth GK (2010) edgeR: a Bioconductor package for differential expression 669 analysis of digital gene expression data. Bioinformatics 26: 139-140 670 Schmidt ED, Guzzo F, Toonen MA, de Vries SC (1997) A leucine-rich repeat containing receptor-like 671 kinase marks somatic plant cells competent to form embryos. Development 124: 2049-2062 672 Shiota H, Satoh R, Watabe K, Harada H, Kamada H (1998) C-ABI3, the carrot homologue of the 673 Arabidopsis ABI3, is expressed during both zygotic and somatic embryogenesis and functions in 674 the regulation of embryo-specific ABA-inducible genes. Plant Cell Physiol 39: 1184-1193 675 Sorefan K, Girin T, Liljegren SJ, Ljung K, Robles P, Galvan-Ampudia CS, Offringa R, Friml J, Yanofsky MF, 676 Ostergaard L (2009) A regulated auxin minimum is required for seed dispersal in Arabidopsis. 677 Nature 459: 583-586 678 Stone SL, Kwong LW, Yee KM, Pelletier J, Lepiniec L, Fischer RL, Goldberg RB, Harada JJ (2001) LEAFY 679 COTYLEDON2 encodes a B3 domain transcription factor that induces embryo development. Proc 680 Natl Acad Sci U S A 98: 11806-11811 681 Su YH, Tang LP, Zhao XY, Zhang XS (2020) Plant cell totipotency: Insights into cellular reprogramming. J 682 Integr Plant Biol 683 Toriba T, Hirano HY (2014) The DROOPING LEAF and OsETTIN2 genes promote awn development in rice. 684 Plant J 77: 616-626 685 Tsuwamoto R, Yokoi S, Takahata Y (2010) Arabidopsis EMBRYOMAKER encoding an AP2 domain 686 transcription factor plays a key role in developmental change from vegetative to embryonic 687 phase. Plant Mol Biol 73: 481-492

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688 Wang X, Niu QW, Teng C, Li C, Mu J, Chua NH, Zuo J (2009) Overexpression of PGA37/MYB118 and 689 MYB115 promotes vegetative-to-embryonic transition in Arabidopsis. Cell Res 19: 224-235 690 Wani SH, Sanghera GS, Gosal SS (2011) An efficient and reproducible method for regeneration of whole 691 plants from mature seeds of a high yielding Indica rice (Oryza sativa L.) variety PAU 201. N 692 Biotechnol 28: 418-422 693 Wojcik AM, Wojcikowska B, Gaj MD (2020) Current Perspectives on the Auxin-Mediated Genetic 694 Network that Controls the Induction of Somatic Embryogenesis in Plants. Int J Mol Sci 21 695 Wojcikowska B, Jaskola K, Gasiorek P, Meus M, Nowak K, Gaj MD (2013) LEAFY COTYLEDON2 (LEC2) 696 promotes embryogenic induction in somatic tissues of Arabidopsis, via YUCCA-mediated auxin 697 biosynthesis. Planta 238: 425-440 698 Woodward C, Bemis SM, Hill EJ, Sawa S, Koshiba T, Torii KU (2005) Interaction of auxin and ERECTA in 699 elaborating Arabidopsis inflorescence architecture revealed by the activation tagging of a new 700 member of the YUCCA family putative flavin monooxygenases. Plant Physiol 139: 192-203 701 Xie K, Zhang J, Yang Y (2014) Genome-wide prediction of highly specific guide RNA spacers for CRISPR- 702 Cas9-mediated genome editing in model plants and major crops. Mol Plant 7: 923-926 703 Zhang T, Li R, Xing J, Yan L, Wang R, Zhao Y (2018) The YUCCA-Auxin-WOX11 Module Controls Crown 704 Root Development in Rice. Front Plant Sci 9: 523 705 Zuo J, Niu QW, Frugis G, Chua NH (2002) The WUSCHEL gene promotes vegetative-to-embryonic 706 transition in Arabidopsis. Plant J 30: 349-359 707

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Figure 1. Changes in global gene expression in rice plants on OsBBM1-GR induction. A, RNA- sequencing analysis showing number of genes up regulated on induction of OsBBM1-GR with DEX alone or in the presence of CYC. B, A Venn diagram showing the down regulated transcripts by OsBBM1-GR in rice leaves. The numbers are genes deregulated by OsBBM1-GR under each treatment (Log2 fold >1 and FDR < 0.05). C and D, RT-qPCR validation of some of the genes regulated by OsBBM1. C, Genes from up regulated category are Os04g45810, a homeobox leucine zipper transcription factor, Os05g32350, a zinc finger containing protein, Os04g55620, a receptor kinase, Os08g37390, a cyclin and Os02g53430, a DNA-3- methyladenine glycosylase. D, From the down regulated category, the genes are Os05g10690 is a MYB domain transcription factor, Os02g07820 a DUF581 domain containing protein and Os05g03610 is phospholipase C protein. The log2 fold change is calculated after normalization with mock and respective wild-type treatments. Error bars represent SEM, calculated from two independent biological replicates, each done in triplicates. DEX and CYC treatments were done for 6 h (see Materials and Methods). DEX, dexamethasone and CYC, cycloheximide.

Figure 2. Regulation of plant hormone pathway genes by OsBBM1. A to E, Direct up regulation of auxin biosynthesis genes by OsBBM1. A, RT-qPCR analysis showing quantification of transcript levels for auxin biosynthesis genes OsYUC6, OsYUC7 and OsYUC9 in OsBBM1-GR plants. Fold change in normalized expression is shown for samples treated for 6 h with DEX, CYC or a combination of both the chemicals. The expression levels are normalized with mock treated samples and also with the similar treatments in wild-type tissues. Error bars were calculated as SEM, from two independent biological replicates. B to D, Chromatin-immunoprecipitation (ChIP) assay for enrichment of OsBBM1 at three OsYUCCA loci. ChIP-PCRs are shown for (B) OsYUC6, (C) OsYUC7 and (D) OsYUC9. Schematics show the genomic organization of each locus. Exons are represented by blue box, introns by thin black line, UTRs with unfilled boxes and upstream sequences with thick black line. The short bar above the schematic shows the position of OsBBM1 occupancy on upstream sequences. E, ChIP-qPCR for quantitation of OsBBM1 enrichment on the three OsYUCCA loci. The enrichment is presented as percentage of input DNA. Error bars are represented as SEM, from two independent biological replicates. F, Quantitation of downregulation in expression of cytokinin response genes by RT-qPCR. Changes in expression are calculated as described for (A).

Figure 3. Phenotypic consequences of up regulation of auxin biosynthesis by OsBBM1 in rice seeds. A, OsBBM1-GR seeds germinated on mock treated medium. Seedlings grow normally (red arrowhead). B and C, OsBBM1-GR seeds grown on dexamethasone (DEX) containing media showing callus formation from scutellum tissue of seeds (white arrow). Red arrowhead points to the normally growing zygotic seedling. D, Somatic embryo formation from leaves of an OsBBM1-GR seedling induced with DEX (white arrow). Red arrowhead shows the growing zygotic seedling. E and F, Regenerating OsBBM1-GR calli induced with DEX. Black arrow shows the chlorophyll/shoot development and red arrowhead marks the regenerating roots. G, Complete plantlet formation from OsBBM1-GR DEX induced somatic embryos with addition of cytokinin. White arrows show the regenerating plantlets from somatic embryos and red arrowhead shows the seedling growing from the zygotic embryo. BAP is 6-Benzylaminopurine. H, Complete development of DEX+BAP regenerated plantlets in (G), when taken off both the treatments. Red arrowhead points to zygotic seedling and white arrows show regenerated plantlets from somatic embryos. Scale bars are 1 cm in (A), (B), (C) and (H), and 2 mm in (D to

G).

Figure 4. Functional characterization of OsYUCCA6, OsYUCCA7 and OsYUCCA9 genes in rice. A and B, Reduction in plant height in Osyucca mutants. A, Osyucca7 Osyucca9 double mutant compared with wild-type. B, Osyucca6 Osyucca7 Osyucca9 triple mutant compared to wild-type. Scale bars 10 cm. C and D, Perturbations in primary root length in Osyucca mutants. C, Osyucca7 Osyucca9 double mutant. D, Osyucca6 Osyucca7 Osyucca9 triple mutant. Scale bars 5 cm. E, Bar graph showing the quantification of reduction in plant height in Osyucca mutants (n=20 for each genotype). F, Quantitative representation of increase in root length in Osyucca mutants in rice (n=20 for each genotype). G to L, Reduction in embryogenic calli induction in Osyucca mutants in the presence of supplemented auxin. G and H, Wild-type showing induction and proliferation of compact, pale and nodular embryogenic calli (red arrow heads) which are phenotypically distinct from brown sectors of non-embryogenic calli (white arrows). I and J, Osyucca7 Osyucca9 double mutant showing some proliferation of embryogenic calli (red arrow heads). K and L, Osyucca6 Osyucca7 Osyucca9 triple mutant, showing extensive brown sectors of non-embryogenic calli (white arrows) without proliferation of pale embryogenic calli (red arrowheads). 2,4-D is 2,4-Dichlorophenoxyacetic acid. Scale bars G to L are 1 cm.

Figure 5. Model explaining the mechanism of OsBBM1 action during somatic embryogenesis in rice. OsBBM1 expression is required for dedifferentiation of somatic cells. In scutellum cells, OsBBM1 is downstream of exogenous auxin (dotted arrow), but in differentiated leaf cells that are recalcitrant to external auxin, OsBBM1 has to be expressed using a transgene as in this study. OsBBM1 positively regulates the expression of auxin biosynthesis OsYUCCA (OsYUC6, OsYUC7 and OsYUC9) genes. This leads to increase in the endogenous auxin levels, followed by dedifferentiation, callus formation and subsequently somatic embryogenesis. OsBBM1 also auto-activates its promoter to maintain expression and pluripotency during somatic embryogenesis. The somatic embryos are able form roots and initiate shoot regeneration, but shoot formation is not completed, possibly due to negative regulation of some cytokinin response genes by OsBBM1. This block can be overcome by the addition of cytokinin in the culture media and complete plant regeneration is achieved.

1 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.24.265025; this version posted August 25, 2020. 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-NC-ND 4.0 International license. Parsed Citations Anderson SN, Johnson CS, Chesnut J, Jones DS, Khanday I, Woodhouse M, Li C, Conrad LJ, Russell SD, Sundaresan V (2017) The Zygotic Transition Is Initiated in Unicellular Plant Zygotes with Asymmetric Activation of Parental Genomes. Dev Cell 43: 349-358 e344 Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Boutilier K, Offringa R, Sharma VK, Kieft H, Ouellet T, Zhang L, Hattori J, Liu CM, van Lammeren AA, Miki BL, Custers JB, van Lookeren Campagne MM (2002) Ectopic expression of BABY BOOM triggers a conversion from vegetative to embryonic growth. 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Plant Cell, Tissue and Organ Culture 73: 147-157 Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Feher A (2015) Somatic embryogenesis - Stress-induced remodeling of plant cell fate. Biochim Biophys Acta 1849: 385-402 Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Galinha C, Hofhuis H, Luijten M, Willemsen V, Blilou I, Heidstra R, Scheres B (2007) PLETHORA proteins as dose-dependent master regulators of Arabidopsis root development. Nature 449: 1053-1057 Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Gallavotti A, Barazesh S, Malcomber S, Hall D, Jackson D, Schmidt RJ, McSteen P (2008) sparse inflorescence1 encodes a monocot- specific YUCCA-like gene required for vegetative and reproductive development in maize. Proc Natl Acad Sci U S A 105: 15196-15201 Pubmed: Author and Title bioRxiv preprint doi: https://doi.org/10.1101/2020.08.24.265025; this version posted August 25, 2020. 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-NC-ND 4.0 International license. Google Scholar: Author Only Title Only Author and Title Harding EW, Tang W, Nichols KW, Fernandez DE, Perry SE (2003) Expression and maintenance of embryogenic potential is enhanced through constitutive expression of AGAMOUS-Like 15. Plant Physiol 133: 653-663 Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Hill K, Schaller GE (2013) Enhancing plant regeneration in tissue culture: a molecular approach through manipulation of cytokinin sensitivity. Plant Signal Behav 8: doi: 10 4161/psb 25709 Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Horstman A, Li M, Heidmann I, Weemen M, Chen B, Muino JM, Angenent GC, Boutilier K (2017) The BABY BOOM Transcription Factor Activates the LEC1-ABI3-FUS3-LEC2 Network to Induce Somatic Embryogenesis. Plant Physiol 175: 848-857 Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Hu B, Zhang G, Liu W, Shi J, Wang H, Qi M, Li J, Qin P, Ruan Y, Huang H, Zhang Y, Xu L (2017) Divergent regeneration-competent cells adopt a common mechanism for callus initiation in angiosperms. Regeneration (Oxf) 4: 132-139 Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Jha P, Kumar V (2018) BABY BOOM (BBM): a candidate transcription factor gene in plant biotechnology. Biotechnol Lett 40: 1467-1475 Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Junker A, Monke G, Rutten T, Keilwagen J, Seifert M, Thi TM, Renou JP, Balzergue S, Viehover P, Hahnel U, Ludwig-Muller J, Altschmied L, Conrad U, Weisshaar B, Baumlein H (2012) Elongation-related functions of LEAFY COTYLEDON1 during the development of Arabidopsis thaliana. Plant J 71: 427-442 Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Kakei Y, Mochida K, Sakurai T, Yoshida T, Shinozaki K, Shimada Y (2015) Transcriptome analysis of hormone-induced gene expression in Brachypodium distachyon. Sci Rep 5: 14476 Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Kawahara Y, de la Bastide M, Hamilton JP, Kanamori H, McCombie WR, Ouyang S, Schwartz DC, Tanaka T, Wu J, Zhou S, Childs KL, Davidson RM, Lin H, Quesada-Ocampo L, Vaillancourt B, Sakai H, Lee SS, Kim J, Numa H, Itoh T, Buell CR, Matsumoto T (2013) Improvement of the Oryza sativa Nipponbare reference genome using next generation sequence and optical map data. Rice (N Y) 6: 4 Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Khanday I, Skinner D, Yang B, Mercier R, Sundaresan V (2019) A male-expressed rice embryogenic trigger redirected for asexual propagation through seeds. Nature 565: 91-95 Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Khanday I, Yadav SR, Vijayraghavan U (2013) Rice LHS1/OsMADS1 controls floret meristem specification by coordinated regulation of transcription factors and hormone signaling pathways. Plant Physiol 161: 1970-1983 Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Kim D, Pertea G, Trapnell C, Pimentel H, Kelley R, Salzberg SL (2013) TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol 14: R36 Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Lotan T, Ohto M, Yee KM, West MA, Lo R, Kwong RW, Yamagishi K, Fischer RL, Goldberg RB, Harada JJ (1998) Arabidopsis LEAFY COTYLEDON1 is sufficient to induce embryo development in vegetative cells. Cell 93: 1195-1205 Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Luerssen H, Kirik V, Herrmann P, Misera S (1998) FUSCA3 encodes a protein with a conserved VP1/AB13-like B3 domain which is of functional importance for the regulation of seed maturation in Arabidopsis thaliana. Plant J 15: 755-764 Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Mahonen AP, Ten Tusscher K, Siligato R, Smetana O, Diaz-Trivino S, Salojarvi J, Wachsman G, Prasad K, Heidstra R, Scheres B (2014) PLETHORA gradient formation mechanism separates auxin responses. Nature 515: 125-129 Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Martin M (2011) Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet.journal; Vol 17, No 1: Next Generation Sequencing Data Analysis bioRxiv preprint doi: https://doi.org/10.1101/2020.08.24.265025; this version posted August 25, 2020. 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-NC-ND 4.0 International license. Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Mendez-Hernandez HA, Ledezma-Rodriguez M, Avilez-Montalvo RN, Juarez-Gomez YL, Skeete A, Avilez-Montalvo J, De-la-Pena C, Loyola-Vargas VM (2019) Signaling Overview of Plant Somatic Embryogenesis. Front Plant Sci 10: 77 Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Mignolli F, Mariotti L, Picciarelli P, Vidoz ML (2017) Differential auxin transport and accumulation in the stem base lead to profuse adventitious root primordia formation in the aerial roots (aer) mutant of tomato (Solanum lycopersicum L.). J Plant Physiol 213: 55-65 Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Murashige T, Skoog F (1962) A Revised Medium for Rapid Growth and Bio Assays with Tobacco Tissue Cultures. 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