This is the accepted version of the article: Osnato, Michela; Matías Hernández, Luis; Aguilar Jaramillo, Andrea Elizabeth; [et al.]. «Genes of the RAV family control heading date and carpel development in rice». Plant Physiology, Vol. 183, issue 4 (Aug. 2020), p. 1663–1680. DOI 10.1104/pp.20.00562 This version is avaible at https://ddd.uab.cat/record/231010 under the terms of the license Plant Physiology Preview. Published on June 18, 2020, as DOI:10.1104/pp.20.00562 1 Short title: RAVs regulate heading date and carpel development 2 3 Corresponding author: 4 Soraya Pelaz: Centre for Research in Agricultural Genomics, CSIC-IRTA-UAB-UB, 5 Campus UAB, Bellaterra (Cerdanyola del Vallès) 08193 Barcelona, and 6 ICREA, Pg. Lluís Companys 23, 08010 Barcelona, Spain. 7 Phone: (34) 93 563 6600 e-mail: soraya.pelaz@cragenomica.es 8 Michela Osnato: Centre for Research in Agricultural Genomics, CSIC-IRTA-UAB-UB, 9 Campus UAB, Bellaterra (Cerdanyola del Vallès) 08193 Barcelona, Spain. Phone: (34) 10 93 563 6600 e-mail: michela.osnato@cragenomica.es 11 12 Title: Genes of the RAV family control heading date and carpel development 13 in rice 14 15 Author names and affiliations: 1,2 16 Michela Osnato , Luis Matias-Hernandez1, Andrea Elizabeth Aguilar-Jaramillo1, Mar- 17 tin M. Kater2 and Soraya Pelaz1,3 1 18 Centre for Research in Agricultural Genomics (CRAG), CSIC-IRTA-UAB-UB, Campus 19 UAB, Barcelona (SPAIN). 2Department BioSciences, University of Milan; Via Celoria 20 26, 20133, Milan (ITALY). 3ICREA, Pg. Lluís Companys 23, 08010 Barcelona, (SPAIN). 21 Corresponding authors: Michela Osnato and Soraya Pelaz 22 23 One-sentence summary 24 RELATED TO ABI3 AND VP1 (RAV) gene function is conserved in the regulation of 25 flowering time in monocots and dicots, although via different mechanisms, and reveal 26 roles in rice gynoecium development. 27 28 Footnotes in the following order: 29 Author contributions 30 MO, MK and SP designed the experiments. MO performed and analyzed most of the 31 experiments. LM and AA performed analyses of Arabidopsis transgenic plants express- 32 ing OsRAV genes. MO, MK and SP discussed the experiments and wrote the paper. 33 34 Responsibilities of the Author for Contact 35 The authors will ensure communication and share data upon request. 36 37 Funding Information 1 Downloaded from on June 22, 2020 - Published by www.plantphysiol.org Copyright © 2020 American Society of Plant Biologists. All rights reserved. Copyright 2020 by the American Society of Plant Biologists 38 This work was supported by the Spanish Ministry of Economy and Competitive- 39 ness/European Regional Development Fund (grants BFU2015-64409-P and and 40 PGC2018-095804-B-I00), the Catalonian Government (CERCA Programme, Consoli- 41 dated Research Group no. 2014 SGR 1406 and Investigator Training Program PhD 42 fellowship to A.E.A.-J.), FLOWER POWER to MK and SP, EvoRepRice (FIRST) to MK 43 and EMBO short-term fellowship to MO. We acknowledge financial support from the 44 Spanish Ministry of Economy and Competitiveness, through the “Severo Ochoa Pro- 45 gramme for Centres of Excellence in R&D” 2016-2019 (SEV‐2015‐0533). A.E.A.-J. 46 performed this work within the frame of a Ph.D. Program of the Universitat Autònoma 47 de Barcelona. 48 49 Email address of author for contact 50 michela.osnato@cragenomica.es; soraya.pelaz@cragenomica.es 51 52 Abstract 53 In plants, correct formation of reproductive organs is critical for successful seed-set 54 and perpetuation of the species. Plants have evolved different molecular mechanisms 55 to coordinate flower and seed development at the proper time of the year. Among the 56 plant-specific RELATED TO ABI3 AND VP1 (RAV) family of transcription factors, only 57 TEMPRANILLO1 (TEM1) and TEMPRANILLO2 (TEM2) have been shown to affect 58 reproductive development in Arabidopsis (Arabidopsis thaliana). They negatively regu- 59 late floral transition through direct repression of FLOWERING LOCUS T and GIBBER- 60 ELLIN 3-OXIDASE1/2, encoding major components of the florigen. Here we identify 61 RAV genes from rice (Oryza sativa), and unravel their regulatory roles in key steps of 62 reproductive development. Our data strongly suggest that, like TEMs, 63 OsRAV9/OsTEM1 has a conserved function as a repressor of photoperiodic flowering 64 upstream of the floral activators OsMADS14 and Hd3a, through a mechanism reminis- 65 cent of that one underlying floral transition in temperate cereals. Furthermore, 66 OsRAV11 and OsRAV12 may have acquired a novel function in the differentiation of 67 the carpel and the control of seed size, acting downstream of floral homeotic factors. 68 Alternatively, this function may have been lost in Arabidopsis. Our data reveal conser- 69 vation of RAV gene function in the regulation of flowering time in monocotyledonous 70 and dicotyledonous plants, but also unveil roles in the development of rice gynoecium. 71 72 INTRODUCTION 73 In plants, the correct formation of reproductive organs is critical not only for successful 74 seed-set but also for the perpetuation of the species. Accordingly, floral evocation must 2 Downloaded from on June 22, 2020 - Published by www.plantphysiol.org Copyright © 2020 American Society of Plant Biologists. All rights reserved. 75 take place at a favorable time of the year to guarantee pollination, and maximum sur- 76 vival possibilities for the offspring. Plants that are affected in their flowering time often 77 have a lower amount of seeds resulting in yield losses. Indeed, precocious flowering is 78 frequently associated with reduced photosynthetic capacity due to a shortened vegeta- 79 tive phase (Endo-Higashi and Izawa, 2011). Conversely, delayed flowering can affect 80 seed maturation due to exposure to unfavorable conditions. A negative correlation also 81 exists between grain size and grain number (Guo et al., 2018; Li et al., 2018), two im- 82 portant agronomic traits which are controlled by both genetic determinants and envi- 83 ronmental conditions. 84 Plants have evolved different molecular mechanisms to coordinate flower and seed 85 development at the proper time of the year. Actually, the switch from vegetative to re- 86 productive growth is controlled by multiple genetic determinants that integrate the re- 87 sponses to environmental and physiological conditions of the plant. Ultimately, the reg- 88 ulatory pathways underlying the floral transition converge on floral integrators that are 89 able to activate genes in the shoot apical meristem (SAM) that control the initiation and 90 development of the inflorescence meristem (IM), and then of floral meristems (FM) 91 from which floral organs differentiate. Upon fertilization, the carpel transforms into a 92 fruit in which the seeds develop. 93 In the last decade, the molecular basis of the floral transition has been unveiled in dif- 94 ferent plant species: the florigen, a long distance signaling molecule, is first produced 95 in leaves under favorable conditions, and then transported to the apical meristem to 96 initiate reproductive development (Andrés and Coupland, 2012). In the model species 97 Arabidopsis (Arabidopsis thaliana), photoperiodic flowering is triggered by FLOWER- 98 ING LOCUS T (FT), a small globular protein of 21 kDa (Kardailsky et al., 1999; Koba- 99 yashi et al., 1999). The expression of FT is activated under inductive long days (LD) in 100 vascular tissues of leaves by the positive regulator CONSTANS (CO, Suárez-López et 101 al., 2001; An et al., 2004), and precocious flowering is prevented by the counteraction 102 of the RAV (RELATED TO ABI3 AND VP1) transcription repressors TEMPRANILLO1 103 (TEM1) and TEMPRANILLO2 (TEM2) (Castillejo and Pelaz, 2008). Since prevention of 104 precocious flowering is important for reproductive fitness other repressors such as 105 FLOWERING LOCUS C (FLC), SCHLAFMUTZE (SMZ), SCHNARCHZAPFENZ (SNZ), 106 TARGET OF EARLY ACTIVATION TAGGED 1 (TOE1), TOE2, FLOWERING LOCUS 107 M (FLM) or SHORT VEGETATIVE PHASE (SVP) play key roles in the control of FT 108 expression in response to vernalization, age, photoperiod or ambient temperature 109 (Hartmann et al., 2000; Scortecci et al., 2001, 2003; Aukerman and Sakai, 2003; 110 Schmid et al., 2003; Jung et al., 2007; Li et al., 2008; Lee et al., 2009; Mathieu et al., 3 Downloaded from on June 22, 2020 - Published by www.plantphysiol.org Copyright © 2020 American Society of Plant Biologists. All rights reserved. 111 2009; Lee et al., 2013; Posé et al., 2013). Once flowering is induced, FM identity 112 genes, such as the MADS-box genes SUPPRESSOR OF CONSTANS1 (SOC1) and 113 APETALA1 (AP1), are induced which in turn repress TEMs expression (Tao et al., 114 2012; Kaufmann et al., 2010). Under non-inductive Short Days (SD), CO is not active 115 and there is no CO-dependent FT induction. In this light regime, the accumulation of 116 the plant hormones Gibberellins (GA) triggers floral transition by inducing SOC1 and 117 LEAFY (LFY) (Wilson et al., 1992; Blázquez et al., 1997; Moon et al., 2003; Eriksson et 118 al., 2006; Hisamatsu and King, 2008). Interestingly, TEMs also regulate GA accumula- 119 tion by repressing GA-3-OXIDASE 1 (GA3OX1) and GA3OX2 genes (Osnato et al., 120 2012). 121 In the crop species rice (Oryza sativa), two closely related genes have been described 122 as FT orthologs: Heading date 3a (Hd3a), which promotes flowering under inductive 123 SD, and Rice Flowering locus T 1 (RFT1), which does it under non-inductive LD (Koji- 124 ma et al., 2002; Komiya et al., 2009). An evolutionarily conserved module defined by 125 the rice orthologs of Arabidopsis CO and FT controls photoperiodic flowering (Shrestha 126 et al., 2014). The rice homolog of CO, Heading date 1 (Hd1), functions as activator of 127 Hd3a in SD, and contrarily as repressor in LD (Yano et al., 2000; Nemoto et al., 2016). 128 Nevertheless, additional rice-specific regulators have been discovered: the floral acti- 129 vator Early Heading date 1 (Ehd1, Doi et al., 2004), and its repressor Grain number, 130 plant height and heading date 7 (Ghd7, Itoh et al., 2010). The Ehd1-Ghd7 pathway 131 determines the critical day-length necessary for the induction of the florigen through a 132 double gating mechanism dependent on the circadian clock and phytochrome- 133 mediated light perception (Itoh et al., 2010). Additional studies on phylogenetic recon- 134 structions revealed the presence of FLC homologs in monocots genomes (Ruelens et 135 al., 2013). These FLC-like factors repress the floral transition in response to cold in 136 temperate cereals (Winfield et al., 2009; Greenup et al., 2020), but appeared to have 137 acquired an opposite function in tropical cereals. Indeed, the rice FLC homolog Os- 138 MADS51 activates the expression of the floral promoter Ehd1 (Kim et al., 2007). 139 Upon FT/Hd3a activation, the resulting gene product moves through the phloem from 140 the leaf to the SAM (Corbesier et al., 2007; Jäger and Wigge, 2007; Mathieu et al., 141 2007; Tamaki et al., 2007; Notaguchi et al., 2008; Komiya et al., 2009) where it triggers 142 transcriptional reprogramming able to confer the competence to form flowers (Corbesi- 143 er et al., 2007; Torti et al., 2012). Intriguingly, FT-like proteins do not have DNA binding 144 activity per se and must interact with bZIP Transcription Factors (TFs), i.e. FLOWER- 145 ING LOCUS D (FD) in Arabidopsis and OsFD1 in rice (Abe et al., 2005; Wigge et al., 146 2005; Taoka et al., 2011; Brambilla et al., 2017; Collani et al., 2019), to activate the 4 Downloaded from on June 22, 2020 - Published by www.plantphysiol.org Copyright © 2020 American Society of Plant Biologists. All rights reserved. 147 expression of downstream genes. The interaction of FT and FD proteins require the 148 14-3-3 adaptor proteins for the formation of an active Floral Activation Complex (FAC) 149 (Taoka et al., 2011; Collani et al., 2019), which in Arabidopsis directly activates the FM 150 identity gene AP1 (Wigge et al., 2005), whereas the rice FAC complex induces the ex- 151 pression of the AP1-like genes OsMADS14/15/18 and the SEPALLATA-like gene Os- 152 MADS34 that are required for the specification of IM identity (Kobayashi et al., 2012; 153 Gomez-Ariza et al., 2019) The florigens also mediate the transcriptional repression of 154 PINE, a gene encoding a Zinc Finger type TF involved in the negative regulation of 155 stem elongation. Therefore, the FAC coordinates the formation of reproductive struc- 156 tures (by activating IM identity genes) and internode elongation (by repressing PINE), 157 guaranteeing the emergence of the panicle from the flag leaf, known as heading, which 158 occurs when rice inflorescence development is completed (Gomez-Ariza et al., 2019). 159 In the rice inflorescence, primary and secondary branches form on the flanks of the IM 160 (rachis) and terminate in spikelet meristems that develop floret meristems from which 161 the palea, the lemma, two lodicules, six stamens and one central carpel differentiate. 162 The identity of different floral organs is specified by the interaction of MADS domain 163 TFs belonging to the SEPALLATA (OsMADS1), APETALA3 (OsMADS16) and AGA- 164 MOUS (OsMADS3-58) subfamilies (Jeon et al., 2000; Nagasawa et al., 2003; Dreni et 165 al., 2011). Eventually, the FM is consumed during gynoecium development, when a 166 single ovule primordium forms inside the carpel (Dreni et al., 2007). Recently, Os- 167 MADS1 was also shown to be essential during seed development, specifically in the 168 regulation of grain size and shape (Liu et al., 2018). 169 In this study, we identified four members of the RAV family of TF in rice, which share 170 sequence similarity with Arabidopsis TEMs. In silico/co-expression analyses based on 171 available transcriptomics data revealed that OsRAVs interact with genes belonging to 172 the MADS-box superfamily at different developmental stages, suggesting that RAVs 173 are unknown players in the gene regulatory network underlying reproductive develop- 174 ment in rice. Specifically, OsRAV8 and OsRAV9 negatively correlate with IM identity 175 genes of the AP1 subfamily, whereas OsRAV11 and OsRAV12 act downstream of 176 MADS-domain floral homeotic factors of the SEP and AG subfamilies. Molecular and 177 functional studies using knock-down and knock-out mutant lines indicate a conserved 178 function for OsRAV9/OsTEM1 as repressor of photoperiodic flowering upstream of the 179 floral activators OsMADS14 and Hd3a, and reveal a role for OsRAV11 and OsRAV12 180 in the correct development of female reproductive organs, downstream of OsMADS1 181 and OsMADS13. 182 5 Downloaded from on June 22, 2020 - Published by www.plantphysiol.org Copyright © 2020 American Society of Plant Biologists. All rights reserved. 183 RESULTS 184 The rice genome contains four RAV genes 185 Genes belonging to the RAV subfamily are present in all land plant species and en- 186 code for putative TFs which are characterized by two DNA binding domains, an 187 APETALA2-type at the N-terminus and a B3-type at the C-terminus (Kagaya et al., 188 1999). In the model species Arabidopsis thaliana, the subfamily of RAV genes is com- 189 posed of six members (Riechmann, 2002), in addition to the TEMs other four genes 190 belong to this family; RAV1 and RAV1-like that are phylogenetically close to TEMs, and 191 RAV3 and RAV3-like which are the most divergent and nothing has been reported 192 about their function. The functional characterization of the closest four indicate their 193 regulatory role in different stages of plant development (Hu et al,. 2004; Castillejo and 194 Pelaz, 2008; Osnato et al., 2012; Feng et al., 2014; Matías-Hernández et al., 2016; 195 Aguilar-Jaramillo et al., 2019) and in the response to abiotic stresses (Fowler et al., 196 2005; Fu et al., 2014). 197 A preliminary phylogenomics analysis indicates a clear separation between AP2-B3 198 coding genes (RAV) and those encoding only the B3 domain (ARF, NGA, VAL), as 199 reported in Supplemental Fig. S1. Specifically in rice, although twelve genes were 200 named RAV (Supplemental Table S1; Swaminathan et al., 2008), only four encode 201 putative proteins containing both DNA binding domains as predicted by gene orthology 202 and paralogy: OsRAV8, OsRAV9, OsRAV11 and OsRAV12 (Supplemental Fig. S2A). 203 Further analysis carried out by searching the SALAD (Surveyed conserved motif 204 ALignment diagram and the Associating Dendrogram) database (Mihara et al., 2009) 205 revealed the absence of RAV proteins in green and red algae, and the presence of 206 multiple conserved motifs in addition to the AP2 and B3 domains (Supplemental Fig. 207 S2B), including the bipartite nuclear localization signal and the B3 repression domain 208 (Supplemental Fig. S3) together with features associated to post-translational modifica- 209 tions (Supplemental Fig. S4). We also performed a phylogenetic analysis based on the 210 deduced full-length protein sequences retrieved by a BLAST-P search against the pro- 211 teomes of the two model species (Supplemental Fig. S3; Fig. 1). Although the four 212 OsRAV proteins clearly clustered together with AtRAV1/AtRAV1-like and TEM1/TEM2, 213 OsRAV8 and OsRAV9 showed the highest similarity with these Arabidopsis RAV fac- 214 tors (Riechmann et al., 2002). 215 In the rice genome, the regions corresponding to the OsRAV8 and OsRAV9 loci may 216 have originated recently, likely due to tandem duplication events after speciation. In- 217 deed, these genes are physically linked (having a distance of about 50 Kb) at the tip of 6 Downloaded from on June 22, 2020 - Published by www.plantphysiol.org Copyright © 2020 American Society of Plant Biologists. All rights reserved. 218 the short arm of chromosome 1 in a region that is enriched in sequences related to 219 retro-transposons (Supplemental Fig. S5, A and B). Furthermore, when we searched 220 the Plant Genome Duplication Database (PGDD, Lee et al., 2013) using the OsRAV8- 221 OsRAV9 locus identifiers, we found intra-genome syntenic relationships with a region 222 on the long arm of chromosome 1 containing OsRAV11 (Supplemental Fig. S5C), and 223 a region on chromosome 5 containing OsRAV12 (Supplemental Fig. S5D). As a result 224 of these phylogenomics and phylogenetic analyses, OsRAV11 and the related gene 225 OsRAV12 appeared as within-species paralogs of OsRAV8-OsRAV9 with similar ge- 226 nomic structures. 227 228 Expression patterns of RAV genes and floral MADS-box genes are correlated 229 To gain insights into the possible function of OsRAV genes in various biological pro- 230 cesses and metabolic pathways, we carried out an in silico co-expression analysis by 231 using the RiceFREND platform (Sato et al., 2013) and constructed a coexpressed gene 232 list for OsRAV9 as guide gene (Supplemental Dataset S1). Among the top 474 genes 233 displaying a positive correlation with OsRAV9 (PCC higher than 0,3), we found enrich- 234 ment for biological processes GO categories related to reproductive processes, re- 235 sponse to chemical stimuli and response to oxidative stress (Supplemental Fig. S6, A 236 and B). Supporting this, we also found overrepresentation for molecular function of the 237 GO terms oxidoreductase and iron binding activities (Supplemental Fig. S6C). Taken 238 together, these findings suggest a possible role for RAV in reproductive development 239 as well as abiotic stress response (similarly to Arabidopsis RAVs). In particular, peroxi- 240 dases and oxidoreductases are detoxification enzymes activated upon accumulation of 241 reactive oxygen species (ROS) (Choudhury et al., 2013), and ion binding proteins are 242 able to sequester excess iron to avoid reaction with oxygen and the formation of dam- 243 aging ROS (Selote et al., 2015). On the other hand, GO analysis of the top 176 genes 244 displaying a negative correlation with OsRAV9 suggested a possible function in signal 245 transduction pathways, due to the enrichment of the terms related to protein 246 dephosphorylation and regulation of transcription for biological processes (Supple- 247 mental Fig. S7A). In particular we found a significant negative Pearson’s Correlation 248 Coefficient (PCC) between OsRAV9 and genes involved in reproductive development 249 including the florigen Hd3a and the IM identity genes such as OsMADS14, OsMADS15 250 and OsMADS34 (Table 1). Furthermore, a recent transcriptomics analysis of apical 251 meristems revealed a negative correlation between the expression of the closely relat- 252 ed gene OsRAV8 and IM identity genes OsMADS14, OsMADS15, OsMADS18 and 253 OsMADS34 at the transition from vegetative to reproductive growth (Gomez-Ariza et 7 Downloaded from on June 22, 2020 - Published by www.plantphysiol.org Copyright © 2020 American Society of Plant Biologists. All rights reserved. 254 al., 2019; Supplemental Fig. S7). Taken together, co-expression and available tran- 255 scriptomics datasets may suggest a possible role for at least OsRAV8 and OsRAV9 in 256 the negative regulation of the transition from vegetative to reproductive phase, similarly 257 to RAV genes in Arabidopsis. 258 259 OsRAV genes are differentially expressed during plant development 260 As a role for OsRAV genes in rice plant development is yet to be elucidated, we first 261 inferred their expression profiles by searching publicly available collections of tran- 262 scriptomics data (Sato et al., 2011). OsRAV9 and OsRAV12 are expressed in vegeta- 263 tive tissues (i.e. leaves and roots), the former at early stages of plant development 264 (Supplemental Fig. S8A), and the latter at maturity (Supplemental Fig. S8B), whereas 265 OsRAV11 is widely expressed in different organs with the exception of anthers (Sup- 266 plemental Fig. S8C). Moreover, the expression of OsRAV9 seems to follow a diurnal 267 oscillation: its transcript levels are almost undetectable during the day, then increase at 268 dusk and reach a peak in the middle of the night (Supplemental Fig. S8D). Likewise, 269 the expression of OsRAV11 and OsRAV12 appeared to oscillate during the day with a 270 peak after dusk (Supplemental Fig. S8, E and F), although with a smaller amplitude 271 compared to OsRAV9. 272 In order to validate these expression data, we designed specific primers for each of the 273 four OsRAV genes (Supplemental Fig. S8G). With respect to OsRAV8, we performed 274 standard RT-PCR reactions, and even if a clear band was amplified using genomic 275 DNA as control template, no amplification was observed when using cDNA obtained 276 from different vegetative and reproductive tissues (Supplemental Fig. S8H), confirming 277 that this gene is not transcribed at detectable levels in the samples examined. Only 278 very recently OsRAV8 was found to be expressed, it was absent in all previous studies 279 likely because of its very specific expression in the apical meristem only at the time of 280 floral transition (Gomez-Ariza et al., 2019). 281 The expression profiles of the other three OsRAVs were investigated by RT-qPCR in 282 wild-type plants at different developmental stages. During the juvenile phase, the 283 mRNAs of OsRAV9 and OsRAV11 were detected in roots, basal region of the stem 284 comprising the SAM, and young leaves (Fig. 2A), although the abundance was much 285 higher for OsRAV9 than OsRAV11. During the adult phase, the expression of OsRAV9 286 became almost undetectable in vegetative tissues resembling Arabidopsis TEM genes 287 expression, whereas OsRAV11 and OsRAV12 displayed high transcript levels in ma- 288 ture leaves (Fig. 2B), resembling the expression behavior of the positive regulator of 8 Downloaded from on June 22, 2020 - Published by www.plantphysiol.org Copyright © 2020 American Society of Plant Biologists. All rights reserved. 289 leaf senescence AtRAV1 in Arabidopsis (Woo et al., 2010). OsRAVs were also ex- 290 pressed in female reproductive organs (Fig. 2C), and their transcript levels decreased 291 after pollination, suggesting that their activities might be restricted to the gynoecium 292 before anthesis. 293 To investigate in detail a possible role of OsRAVs in phase changes during vegetative 294 growth, we dissected differentiating leaves from wild-type plants to monitor their tran- 295 script levels at different developmental stages. Precisely, in rice the juvenile phase is 296 limited to the second leaf (L2), since the transition to the adult phase occurs during the 297 development of the third to fifth leaf (L3-L4-L5) when the midrib differentiates (Itoh et 298 al., 2005). We also analyzed the expression of the Peter Pan Syndrome (PPS) gene 299 (Tanaka et al., 2011), the rice ortholog of the Arabidopsis COP1 (Liu et al., 2008), as a 300 marker for the transition from the juvenile to adult phase. In accordance with its func- 301 tion, PPS displayed a peak of expression in the fourth leaf four weeks after germina- 302 tion. OsRAV9 exhibited the highest transcript levels in L3 and then decreased in L4 303 and L5 (Fig. 2D), indicating that its transcription was drastically reduced at the transi- 304 tion to the adult phase. Also the expression of OsRAV11 was detected in young 305 leaves, although at very low levels as compared to PPS and OsRAV9 (Fig. 2D). 306 In summary, these molecular analyses suggest diversification of expression patterns 307 for OsRAV9 and OsRAV11, the former being transcribed at higher levels in the vegeta- 308 tive phase and specifically in juvenile leaves, and the latter at maturity, in particular in 309 old leaves and female reproductive structures. 310 311 OsRAV9 negatively regulates floral transition 312 In order to investigate the functional conservation between rice and Arabidopsis RAV 313 genes, we used the Pro35S:OsRAV9 and Pro35S:OsRAV11 constructs to transform 314 Arabidopsis plants (Supplemental Fig. S9A). After selection of several independent 315 transgenic lines (Supplemental Fig. S9B), phenotypic analyses of selected T2 genera- 316 tions revealed a mild late flowering phenotype of transgenic plants expressing OsRAV9 317 (OsRAV9-E), but no phenotypic alterations in plants expressing OsRAV11 (Supple- 318 mental Fig. S9C). Furthermore, molecular analyses of three representative T3 lines 319 revealed also a correlation between the late flowering phenotype of OsRAV9-E plants 320 (Fig. 3, A and D) and the down-regulation of FT and GA3oxidase1 (Fig. 3, E and F), 321 two downstream targets of the TEM factors in Arabidopsis (Castillejo and Pelaz, 2008; 322 Osnato et al., 2012). Taken together, these findings suggest that rice OsRAV9 and the 9 Downloaded from on June 22, 2020 - Published by www.plantphysiol.org Copyright © 2020 American Society of Plant Biologists. All rights reserved. 323 Arabidopsis TEM genes have an at least partial conserved function as repressor of 324 photoperiodic flowering in Arabidopsis. 325 Besides the high sequence identity between OsRAV9 and AtTEMs, also their expres- 326 sion patterns were similar as both displayed high transcripts levels in the juvenile 327 phase that decreased as the plant aged shortly before the transition from vegetative to 328 reproductive growth (Fig. 2; Castillejo and Pelaz, 2008). Consistently, expression anal- 329 yses in wild-type plants showed a mutual exclusive pattern for OsRAV9 and the flori- 330 gen Hd3a, not only during the day (Fig. 4A) but also throughout plant development 331 (Fig. 4B), similarly to the opposite expression patterns of TEM and FT in Arabidopsis 332 (Castillejo and Pelaz, 2008). Actually, the expression of OsRAV9 was high at early 333 stages and dropped at around the transition to the adult phase. Conversely, the ex- 334 pression of Hd3a is almost undetectable at early stages of vegetative growth, and in- 335 creases in the adult phase (Fig. 4B, Kojima et al., 2002; Komiya et al., 2009). The flori- 336 gen begins to be produced in adult leaves four weeks after germination under inductive 337 conditions, and triggers floral transition in adult plants when it reaches its maximum 338 accumulation around weeks 6. 339 Since the dynamics of OsRAV9 expression strikingly resembled those of TEM genes, 340 we decided to investigate if the function was also conserved in rice. We used an RNAi 341 silencing strategy due to the absence of insertion mutants in public collections for 342 OsRAV9. Wild-type rice calli were transformed with an RNAi construct carrying a Gene 343 Sequence Tag specific for the 3’ end of the gene under the control of a constitutive 344 promoter (Supplemental Fig. S10A). We obtained eleven independent transgenic lines 345 for the silencing construct, and selected T1 OsRAV9-RNAi transformants by monitoring 346 its transcript levels at the peak of expression (Supplemental Fig. S10B). T2 generation 347 lines were obtained by self-pollination, and three lines with the highest silencing of 348 OsRAV9 were characterized. The RNAi lines (reported as OsRAV9-i) flowered slightly 349 earlier than the wild-type under inductive photoperiods, more clearly under SD than 350 under 12 hours of light (Fig. 4, C and D; Supplemental Fig. S10C), but not under non 351 inductive LD (Supplemental Fig. S10C). Under SD the down-regulation of OsRAV9 352 resulted in significantly early flowering plants (Fig. 4C). Under 12h light/12h dark re- 353 gime, transgenic lines flowered on average 90 days after germination (DAG) and un- 354 derwent anthesis one week later, whereas wild-type plants were still at the booting 355 stage (Fig. 4, E and F). These findings suggested that OsRAV9 functions as floral re- 356 pressor in rice like TEMs do in Arabidopsis, likely upstream of the florigen Hd3a under 357 inductive conditions. Therefore, hereafter we refer to OsRAV9 as OsTEM1. 358 10 Downloaded from on June 22, 2020 - Published by www.plantphysiol.org Copyright © 2020 American Society of Plant Biologists. All rights reserved. 359 OsTEM1 regulates floral activators OsMADS14 and Hd3a 360 Based on the results obtained it was tempting to speculate a role for OsTEM1 in the 361 direct repression of Hd3a as a non-canonical RAV binding site is present in its regula- 362 tory region (Fig. 5A). However, we could not exclude an indirect effect on Hd3a via 363 additional transcriptional regulators. Actually, a negative correlation also exists be- 364 tween OsTEM1 and additional floral activators including OsFT-like1 and IM identity 365 genes (Table 1), previously shown to act in a regulatory loop with the florigen, up- 366 stream of Hd3a in the leaf and downstream of the Hd3a/14-3-3/OsFD1 complex in re- 367 productive meristems (Kobayashi et al., 2012). OsMADS14 is also expressed in adult 368 leaves and a perfect RAV binding site was found in its promoter (Fig. 5B). Therefore, 369 we used a transient expression system based on a dual Renilla-Luciferase assay to 370 investigate the direct interaction between OsTEM1 and potential target genes. The 371 effector vector Pro35S:OsTEM1 was transiently co-expressed with reporter vectors 372 containing different regulatory regions of Hd3a and OsMADS14 (Fig. 5, C and D; Sup- 373 plemental Fig. S11). We evaluated transactivation ability of the floral repressor on tar- 374 get promoters by measuring the relative expression of LUC and REN reporter genes in 375 cell lysates. Despite the biological variability between independent replicates, a clear 376 reduction of LUC/REN relative transcript levels was always observed when OsTEM1 377 was co-transformed with a reporter vector carrying promoter sequences of OsMADS14 378 containing the RAV binding site but not Hd3a (Fig. 5, C and D; Supplemental Fig. S11, 379 B and C). However, this reduction was abolished when we co-transfected the effector 380 vector with a mutated version of the reporter vector carrying the RAV binding site of the 381 OsMADS14 promoter. For statistical analyses purposes, the values of these three rep- 382 licates including intact and mutated RAV binding sites, are shown as logarithmic values 383 (Fig. 5E; Supplemental Fig. S11D). Therefore, the transient co-transformation assays 384 soundly suggest that transcription repression mediated by OsTEM1 is stronger on DNA 385 regulatory sequences of OsMADS14 and is likely mediated by the RAV binding site. 386 Finally, we monitored the expression levels of OsTEM1 and floral activators shortly 387 before and around the floral transition in wild-type and OsTEM1-i lines grown under 388 inductive conditions. As expected, transcripts levels of OsTEM1 were confirmed to be 389 higher at week 4 in wild-type plants, and strong down-regulation was observed in the 390 transgenic lines (Fig. 5F). Conversely, the expression of OsMADS14 increased from 391 week 4 to week 6 in wild-type plants, and a clear up-regulation was detected in 392 OsTEM1-i lines #2 and #3 at 35 DAG (Fig. 5G). The related IM genes OsMADS15 and 393 OsMADS34 were expressed at extremely low levels in leaves at floral transition (Sup- 394 plemental Fig. 12A); however, OsMADS18 was transcribed at higher levels in vegeta- 11 Downloaded from on June 22, 2020 - Published by www.plantphysiol.org Copyright © 2020 American Society of Plant Biologists. All rights reserved. 395 tive tissues, and alteration of its mRNA abundance was found in the transgenic line 396 with highest OsRAV9 silencing (Supplemental Fig. 12B). Furthermore, considerable 397 increase in Hd3a mRNA levels was also detected in silencing lines around floral transi- 398 tion (Fig. 5H). Taken together, transient co-transformation of protoplasts and compara- 399 tive expression analysis of wild-type and transgenic RNAi lines suggest that OsTEM1 400 controls heading date via direct repression of OsMADS14, although it also modulates 401 Hd3a expression. 402 We also tested the effect of the down-regulation of OsTEM1 on other genetic pathways 403 involved in the control of heading date (Supplemental Fig. 12C), and, although varia- 404 ble, we found up-regulation of the flowering inductors OsMADS50, OsMADS51 and 405 Ehd1 in silencing lines (Supplemental Fig. S12, D and E), perhaps suggesting addi- 406 tional roles in parallel pathways. As a consequence, down-regulation of the OsTEM1 407 repressor and up-regulation of different floral activators in transgenic lines resulted in 408 early flowering phenotype. 409 410 OsRAV11 and OsRAV12 regulate carpel development 411 The fact that OsRAV11/OsRAV12 have diversified from OsTEM1 in their expression 412 patterns and coding sequences, prompted us to hypothesize that these genes might 413 have acquired different roles in plant development, likely after floral induction based on 414 their expression pattern in the reproductive phase (Fig. 2C). A preliminary spatio- 415 temporal expression analysis indicated that OsRAV11 was expressed at early stages 416 of flower development prior organ primordia differentiation as its mRNA was detected 417 in the spikelet meristem (Supplemental Fig. S13A). Later on, transcripts became first 418 restricted to the carpel primordia similar to carpel identity genes, and afterwards specif- 419 ically limited to the apical part of the developing gynoecium (Supplemental Fig. S13B). 420 The analysis of available transcriptomics data-sets performed on floral homeotic mu- 421 tants that revealed OsRAV11 down-regulation in OsMADS1-RNAi inflorescences 422 (Khanday et al., 2013; Supplemental Table S2), and conversely up-regulation in os- 423 mads13 mutant (M. Osnato and M.M. Kater, unpublished), suggested an interaction 424 between RAV and MADS-box genes controlling floral organ development. This could 425 be a direct effect since we found CArG-boxes, consensus sequences recognized and 426 bound by MADS-domain TFs, in OsRAV11 and also in OsRAV12 regulatory sequenc- 427 es (Supplemental Fig. S13D). The regulation by OsMADS1 and OsMADS13 is further 428 supported by the fact that both appear as regulators of OsRAV genes in the Environ- 429 mental Gene Regulatory Influence Networks (EGRIN, Wilkins et al., 2016). Therefore, 12 Downloaded from on June 22, 2020 - Published by www.plantphysiol.org Copyright © 2020 American Society of Plant Biologists. All rights reserved. 430 OsRAV11 might have a role in the development of the pistil, likely downstream of 431 class-D and class-E MADS-domain floral homeotic factors. 432 Consistently, we found down-regulation of OsRAV11 in developing panicles of the os- 433 mads1 mutant (Fig. 6A), characterized by the conversion of floral organs into glume- 434 like structures (Agrawal et al., 2005; Khanday et al., 2013), and up-regulation in young 435 panicles of the floral homeotic mutants osmads16 and osmads13 (Fig. 6B), in which 436 stamens and ovules are homeotically converted into extra carpels respectively (Naga- 437 sawa et al., 2003; Dreni et al., 2007) perhaps because the ectopic activation of 438 OsRAV11. Accordingly, we observed strong upregulation of OsRAV11 specifically in 439 osmads13 ovule primordia (Fig. 6C; M. Osnato and M.M. Kater, unpublished), which 440 later develop as carpels. 441 Therefore, we explored the function of OsRAV11 using a knock-out mutant character- 442 ized by the insertion of T-DNA in the 5’ UTR of the gene (Fig. 6, D and E). The loss of 443 OsRAV11 function did not cause alteration of heading date, but elongated carpels (Fig. 444 6F). A detailed phenotypic analysis by Scanning Electron Microscopy (SEM) upon ferti- 445 lization indicated alteration in size and shape of the pistil, with an enlarged ovary com- 446 pared to wild-type pistils (Fig. 6G). Specifically, differentiation of apical tissues of the 447 gynoecium were altered in the osrav11 mutant as carpel tissues did not fuse (Fig. 6H). 448 Likewise, we observed alteration of seed morphology (Supplemental Fig. S13): mutant 449 plants produced seeds with increased seed length and decreased seed width (Sup- 450 plemental Fig. S3G), resulting in statistically significant alterations of length-to-width 451 ratio and circularity (Supplemental Fig. S3H). Interestingly, we observed a slight in- 452 crease in seed weight after de-husking (Fig. 6I). 453 Ultimately, to investigate the possible redundancy between OsRAV11 and the closely 454 related OsRAV12 gene in the formation of female reproductive organs, we generated 455 transgenic rice plants in which the expression levels of both genes were reduced by 456 RNA interference (Supplemental Fig. S14). Vegetative growth of knock-down lines 457 (from now on OsRAV11/12-i) was normal, whereas flowers showed alterations caused 458 by abnormal morphology of female reproductive organs (Fig. 7A). Indeed, elongated 459 cylindrical pistils with highly reduced stigmas and enlarged ovaries (Fig. 7B) were ob- 460 served in transgenic plants with reduced levels of both OsRAV11 and OsRAV12 in the 461 gynoecium (Fig. 7C), indicating that these two genes redundantly regulate the basal- 462 apical patterning of the gynoecium. Interestingly, flowers of these transgenic lines pro- 463 duced viable pollen, but most of the ovules were not fertilized, resulting in very poor 464 seedset. Consequently, most of the transgenic lines displayed severe fertility defects 465 (Fig. 7D). 13 Downloaded from on June 22, 2020 - Published by www.plantphysiol.org Copyright © 2020 American Society of Plant Biologists. All rights reserved. 466 To conclude, molecular and functional characterization of OsRAV11 and OsRAV12 467 suggest that these two genes might redundantly regulate the differentiation of the fe- 468 male reproductive structures. Intriguingly, a decreased activity of OsRAV11 correlated 469 with an increase in seed weight, whereas the loss of OsRAV11 and OsRAV12 activity 470 associates with sterility problems. 471 472 DISCUSSION 473 Arabidopsis and rice RAV genes are closely related 474 RAV proteins belong to the plant-specific B3 super-family of TFs (Swaminathan et al., 475 2008), and are characterized by an additional AP2 DNA binding domain at the N- 476 terminus. AP2-B3 type proteins were not found in the green algae Chlamydomonas 477 reinhardtii, but they appeared early in the evolution of land plant species. The presence 478 of two DNA binding domains suggests that these TFs achieve high affinity by specifi- 479 cally binding bipartite sequences in regulatory regions of downstream targets (Kagaya 480 et al., 1999; Castillejo and Pelaz, 2008; Osnato et al., 2012; Matías-Hernández et al., 481 2016). 482 In this study, we focused on the four rice RAV genes which display striking similarities 483 with four RAV genes of Arabidopsis (Supplemental Fig. S15) that were already de- 484 scribed as regulators of different aspects of plant development and stress responses. 485 Specifically, AtRAV1 and AtRAV1-like redundantly control leaf senescence (Woo et al., 486 2010), AtRAV1 and TEM2 modulate sensitivity to drought and salinity (Fu et al., 2014), 487 TEM1 and TEM2 repress trichome formation (Matías-Hernández et al., 2016) and floral 488 transition under inductive and non-inductive photoperiods (Castillejo and Pelaz, 2008; 489 Osnato et al., 2012) as well as in response to low temperatures (Marín-González et al., 490 2015) and to plant age (Aguilar-Jaramillo et al., 2019). Interestingly, preliminary co- 491 expression analyses suggested that OsRAV9 and OsRAV11 could act in signal trans- 492 duction pathways activated in response to abiotic stresses. Nevertheless, OsRAV9 and 493 the closely related OsRAV8 could also act in Gene Regulatory Networks controlling the 494 transition from vegetative to reproductive growth in the leaf and in the apical meristem, 495 respectively. 496 497 OsRAV9/OsTEM1 is a novel player in flowering 498 Although phylogenomics and phylogenetic analyses revealed that OsRAV genes might 499 have originated from duplication events from a common ancestor after speciation and 14 Downloaded from on June 22, 2020 - Published by www.plantphysiol.org Copyright © 2020 American Society of Plant Biologists. All rights reserved. 500 separation of monocots and dicots, it is difficult to draw conclusions from orthology with 501 AtRAVs. Furthermore, the expression domains of the four OsRAV paralogous genes 502 appear to have diversified likely due to polymorphisms in their regulatory regions. Re- 503 gardless of the remarkable similarities in the gene structures and coding sequences, 504 OsRAV8 and OsRAV9 are expressed in different tissues. Indeed, OsRAV9 mRNA is 505 abundant in juvenile leaves, and its reduction marks the transition to the adult phase 506 when plants acquire the competence to flower, whereas the transcripts of OsRAV8 are 507 detected in the apical meristem and its levels also decrease at floral transition when IM 508 identity genes are activated (Gomez-Ariza et al., 2019). Therefore, at least OsRAV9 509 displayed an expression pattern that resembles that of TEM genes in Arabidopsis. 510 Supporting the functional conservation, the ectopic expression of OsRAV9 in Ara- 511 bidopsis plants correlated with the repression of the TEM targets FT and AtGA30X1 512 and delayed flowering time. In addition, silencing of OsRAV9/OsTEM1 in transgenic 513 rice lines resulted in early flowering due to up-regulation of the floral activators Os- 514 MADS14 and Hd3a. 515 The mechanism of action seems to be different in the two species. To avoid precocious 516 flowering, Arabidopsis TEMs directly target the florigens FT (Castillejo and Pelaz, 517 2008) and AtGA3ox1/2 (Osnato et al., 2012), whereas OsTEM1 might regulate Hd3a 518 indirectly via repression of OsMADS14 as proposed in Fig. 8A. Although this AP1/FUL- 519 like gene is expressed at high levels in reproductive tissues, its mRNA accumulates 520 also during the vegetative phase. Accordingly, an additional role for OsMADS14 as 521 activator of the florigen in the leaf has been previously proposed, likely acting via a 522 positive regulatory loop with Hd3a (Kobayashi et al., 2012). Actually, knock-down lines 523 silencing OsMADS14, OsMADS15, OsMADS18 and OsMADS34 display delayed flow- 524 ering (Kobayashi et al., 2012), and conversely transgenic rice ectopically expressing 525 OsMADS14 and OsMADS18 are early flowering (Jeon et al., 2000; Fornara et al., 526 2004). A very recent study also indicates that AP1-like genes could regulate drought- 527 escape; indeed, the early flowering phenotype under drought conditions correlates with 528 increased expression of OsMADS18 (Groen et al., 2020). 529 Upon floral transition, the FAC activates the expression of OsMADS14, together with 530 OsMADS15, OsMADS18 and OsMADS34, which specify IM identity and trigger the 531 development of reproductive structures (Kobayashi et al., 2012; Kobayashi et al., 532 2010). Based on transcriptomics analysis, we speculate that OsRAV8 could play a role 533 in the maintenance of the vegetative state of the apical meristem, thus preventing the 534 formation of the rachis under non inductive conditions. 535 15 Downloaded from on June 22, 2020 - Published by www.plantphysiol.org Copyright © 2020 American Society of Plant Biologists. All rights reserved. 536 Mechanisms controlling floral transition in cereals 537 It has long been known that AP1/FUL-like proteins control seasonal flowering in cere- 538 als growing in temperate regions (Fjellheim et al., 2014). Precisely, the floral transition 539 is triggered by the activation of VERNALIZATION 1-like (VRN1-like) and FT-like genes 540 in wheat leaves in response to increasing day-length and prolonged exposure to low 541 temperatures (Danyluk et al., 2003; Shimada et al., 2009). At least in wheat, VRN1 542 regulates flowering by directly binding the promoter of the downstream target FT-like1 543 (Deng et al., 2015; Tanaka et al., 2018). Surprisingly, the function of AP1/FUL-like pro- 544 teins as floral activators acting in leaves seems to be well conserved in rice (Jeon et 545 al., 2000; Kobayashi et al. 2012), despite the fact that it is a cereal crop of tropical 546 origin that does not require vernalization. Currently, we propose a novel mechanism 547 that is largely independent from previously described molecular networks determining 548 heading date in rice (Tsuji et al., 2013). Interestingly, the presence of TEM orthologs in 549 the Poacea family (Supplemental Fig. S1) opens up the intriguing possibility that the 550 RAV-AP1-FT regulatory module could be conserved among tropical (Oryza tribe) and 551 temperate (Triticeae tribe) cereals. 552 Moreover, as mentioned above AtRAV1 and TEM2 seem to modulate sensitivity to 553 drought and salinity (Fu et al., 2014), OsTEM1 has been recently shown to play a role 554 in response to abiotic stresses (OsRAV2, Duan et al., 2016), and OsTEM1 expression 555 was reduced in plants growing under drought conditions (Plessis et al., 2015) where 556 the transcription of OsMADS18 was increased (Groen et al., 2020). Therefore, RAV 557 genes could be involved in adaptive growth by modulating heading date in response to 558 environmental limitations and fluctuations, by integrating external and internal physio- 559 logical conditions. 560 561 Mechanisms controlling floral organ development in rice 562 In the last decade, genetic and functional genomics analyses in different plant species 563 revealed that flower development is governed by a complex framework based on 564 MADS-domain TFs. Rice members of the grass-specific LOF-SEP clade sequentially 565 regulate different steps of flower development upon the vegetative to reproductive 566 phase change. First, OsMADS34 forms tetrameric complex with the AP1-like factors 567 OsMADS14 and OsMADS15 in the IM to initiate inflorescence branch meristem pri- 568 mordia development from which secondary branches and spikelets differentiate (Koba- 569 yashi et al., 2012). After the formation of rudimentary glumes and sterile lemmas, the 570 spikelet meristem is converted into floret meristem which produces different floral or- 16 Downloaded from on June 22, 2020 - Published by www.plantphysiol.org Copyright © 2020 American Society of Plant Biologists. All rights reserved. 571 gans. Another grass-specific LOF-SEP factor, OsMADS1, plays a central role in the 572 determination of floral organ identity, as it interacts physically and genetically with AP3- 573 like and AG-like factors which are involved in the development of male and female re- 574 productive organs (Li et al., 2011; Khanday et al., 2013; Khanday et al., 2016). Recent- 575 ly, transcriptomics analysis performed on OsMADS1 knock-down panicles at very early 576 stages of flower development (Khanday et al., 2013) unveiled mis-regulation of floral 577 homeotic MADS-box genes as well as down-regulation of genes encoding B3-type 578 TFs, including OsARFs and OsRAV11 (Supplemental Table S2). Precisely, the ARF- 579 type TFs OsETTIN1 and 2 control the differentiation of apical tissues of the carpel, and 580 at least OsETTIN2 is directly regulated by OsMADS1 (Khanday et al., 2013). Intriguing- 581 ly, the aberrant carpel morphology of loss of OsETTINs (Khanday et al., 2013) is simi- 582 lar to that of transgenic lines with reduced level of OsRAV11 and OsRAV12 (Fig. 7B). 583 584 OsRAV11 and OsRAV12 regulate the development of gynoecium 585 In Arabidopsis, the early flowering TEM loss of function mutants do not display evident 586 alterations of flower development. Nonetheless, the late flowering TEM overexpressing 587 lines show fertility defects and produce shorter siliques containing fewer seeds. This 588 phenotype could be related to decreased content of GA, or alternatively to misregula- 589 tion of genes involved in the regulation of later organ development. In rice, Further mo- 590 lecular analyses suggest that RAV genes might have acquired additional functions at 591 later stages of vegetative and reproductive growth. Indeed, OsRAV11 and OsRAV12 592 are highly expressed not only in mature leaves at ripening, indicating a possible regula- 593 tory role in leaf senescence similarly to AtRAV1, but also in the gynoecium before ferti- 594 lization. Actually, functional characterization of knock-down and knock-out mutants 595 points at a novel function for OsRAV11 and OsRAV12 in the correct formation of fe- 596 male reproductive organs. Indeed, the whole basal-apical pattern was distorted; the 597 ovary was enlarged in osrav11 and misshapen in OsRAV11/12-i plants. Down- 598 regulation of both genes resulted in reduced stigmas and larger carpels than in single 599 osrav11 mutants, which may suggest a redundant function of these genes in carpel 600 development and differentiation. Therefore, we hypothesize a role for OsRAV11 and 601 OsRAV12 in the determination of the basal-apical pattern of the pistil, perhaps in paral- 602 lel with ARFs. Furthermore, we propose that at least OsRAV11 may control the differ- 603 entiation of the gynoecium downstream of MADS-domain TFs (Fig. 8B), due to its mis- 604 regulation in the floral homeotic mutants osmads1, osmads13 and osmads16 (Fig. 6, A 605 and C). 17 Downloaded from on June 22, 2020 - Published by www.plantphysiol.org Copyright © 2020 American Society of Plant Biologists. All rights reserved. 606 Besides the specification of the identity of different floral organs, OsMADS1 has been 607 proposed as a key trait of agronomical interest During spikelet development, Os- 608 MADS1 also interacts with Gγ subunits of GS3 and DEP1 (Liu et al., 2018), which 609 regulate its transcriptional activity on a common set of target genes involved in the de- 610 termination of seed size and shape. The dominant negative mutation osmads1(lgy3) 611 causes an alternatively spliced protein variant and correlates with more slender grain, 612 as the mutated protein promotes cell proliferation in longitudinal direction (Liu et al. 613 2018). Pyramiding of lgy3 and dep1-1 alleles in a japonica cultivar resulted not only in 614 a 10% increase in grain yield, but also improved grain length-to-width ratio and grain 615 chalkiness (Liu et al. 2018). We can hypothesize that the elongated seed phenotype 616 associated to down-regulation of OsMADS1 (Liu et al., 2018) could be mediated 617 through the down-regulation of B3 genes including OsRAV11 since a similar slender 618 phenotype is observed in osrav11 and OsRAV11/12-i carpels. Further analyses are 619 required to understand the interactions with putative upstream regulators, interacting 620 proteins and downstream targets constituting the molecular network that regulates the 621 development of the gynoecium in rice. 622 623 624 MATERIALS AND METHODS 625 RAV sequence analyses 626 OsRAV8 and AtTEM1 were used as queries in phylogenomics analyses in sequenced 627 land plants species (eudicotyledons, monocotyledones, Amborellales) using gene tree 628 tool of Pan-taxonomic Compara (http://www.gramene.org/). TEM1 protein sequence 629 was used as query in a BLAST-P search against the proteomes of Arabidopsis (Ara- 630 bidopsis thaliana) and rice (Oryza sativa). 631 TF binding sites (CArG box for MADS-domain proteins, consensus sequence com- 632 posed of CAACA and CCTG elements at a distance of 3-9 nucleotides for RAV pro- 633 teins) were searched in the regulatory regions of genes of interest by using the Pro- 634 moter Analysis tool of Plant PAN3.0 (http://plantpan.itps.ncku.edu.tw/index.html). 635 Gene and protein sequences were retrieved from TAIR (www.arabidopsis.org) and 636 GRAMENE (www.gramene.org). 637 638 Phylogenetic analyses 18 Downloaded from on June 22, 2020 - Published by www.plantphysiol.org Copyright © 2020 American Society of Plant Biologists. All rights reserved. 639 Analysis of phylogenetic relationships between 24 RAV-related full length protein se- 640 quences and construction of phylogenetic tree were performed using tools available at 641 http://www.phylogeny.fr/ (Dereeper at al., 2008). MUSCLE was used for protein se- 642 quences alignment, and G-blocks for a more stringent selection. PhyML was used for 643 phylogenetic analysis with bootstrapping procedure (N=100) as statistical test for 644 branch support, and TreeDyn for tree visualization. Sequence Diversity Diagram 645 (SeDD) was used to compare two sets of RAV protein sequences from Arabidopsis 646 thaliana and Oryza sativa and visualize conserved versus diversified positions in the 647 two species. Motif clustering analysis was carried out using Surveyed conserved motif 648 ALignment diagram and the Associating Dendrogram (SALAD version 3, 649 https://salad.dna.affrc.go.jp/salad/en/) with OsRAV9 sequence, and the prediction of 650 functional and structural motifs in RAV protein sequences via web-based tools of 651 ExPAsY (https://prosite.expasy.org/scanprosite; Castro et al., 2006). 652 653 Expression profiles 654 Expression profiles of rice RAV genes were inferred from a large collection of microar- 655 ray data derived from different tissues at different developmental stages under natural 656 field conditions (http://ricexpro.dna.affrc.go.jp/). Co-Expression Analyses were carried 657 out by using OsRAV9 as single guide genes and searching the Rice Functionally Re- 658 lated gene Expression Network Database (RiceFREND, 659 http://ricefrend.dna.affrc.go.jp/), the Plant Co-expression Database (PLANEX, Yim et 660 al., 2013) and the Rice Oligonucleotide Array Database (ROAD, Cao et al., 2012). 661 662 663 Plant material and growth conditions 664 Wild-type and transgenic Arabidopsis thaliana (Col-0 background) seeds were sown on 665 soil pots and plants were grown under LD (16 hours light/8 hours dark at 22°C) until 666 maturity. Wild-type and transgenic rice (O. sativa subspecies Japonica) seeds were 667 surface sterilized and sown on Murashige Skoog medium with 30g/l Sucrose. After 668 germination, seedlings were transferred to soil pots and grown under controlled condi- 669 tions until maturity (SD: 8 hours light at 28°C, 16 hours dark at 24°C; 12/12: 12 hours 670 light at 28°C, 12 hours dark at 24°C or LD; LD:16 hours light at 28°C, 8 hours dark at 671 24°C). For functional characterization of OsRAV genes, transgenic lines (cv. Nippon- 672 bare) and insertion mutant (PFG_2A-10680, cv. Hwayoung) were used. For expression 19 Downloaded from on June 22, 2020 - Published by www.plantphysiol.org Copyright © 2020 American Society of Plant Biologists. All rights reserved. 673 analysis, segregating progenies of floral homeotic mutants (cv. Dongjin) were geno- 674 typed (Supplemental Tables S3), grown for 10 weeks in LD and then transferred to 675 inductive conditions. Pools of developing inflorescences were harvested 3 weeks after 676 floral transition from homozygous mutants (osmads1, osmads13, osmads16) and wild- 677 type plants. 678 679 Cloning and generation of Arabidopsis and rice transgenic plants 680 The coding sequence of OsRAV9/OsTEM1 was amplified using primer sets SPp538- 681 SPp541, sub-cloned in pCRII and pENTR-3C by restriction/ligation, and introduced in 682 pALLIGATOR2 vector downstream of Pro35S by Gateway technology (Supplemental 683 Table S4). Arabidopsis plants (Col-0, tem1-tem2) were transformed with the 684 Pro35S:OsTEM1 construct by floral dip, and GFP-positive seeds were selected by fluo- 685 rescence microscopy. For the generation of the RNAi constructs, the Gene Sequence 686 Tags (GSTs) specific for the 3’ ends of OsRAV9/OsTEM1 and OsRAV11 were ampli- 687 fied using primer sets SPp516-SPp539 and SPp527-SPp537 respectively, then sub- 688 cloned in pCRII, and afterwards cloned in pENTR-3C by restriction/ligation (as KpnI- 689 EcoRV fragments). The resulting constructs were digested with PvuI prior to LR re- 690 combination to pBios-378 plant expression vector. Scutellum-derived rice calli (cv Nip- 691 ponbare) were transformed by Agrobacterium co-cultivation. Independent transfor- 692 mation events were selected, 11 for OsRAV9 and 7 for OsRAV11 constructs, regener- 693 ated and propagated. Plants that underwent regeneration but did not contain the trans- 694 genic cassette were used as transformation control. The primers used for genotyping 695 and cloning are listed in Supplemental Tables S3 and S4. 696 697 Direct binding of OsTEM1 to downstream genes 698 To generate the set of reporter vectors, different promoter regions of OsMADS14 and 699 Hd3a were cloned as SalI-PstI fragments in a modified pGreenII 0800-LUC carrying 700 Pro35S:LUC and Pro35S:REN (as internal control to estimate the proportion of trans- 701 formed protoplasts). Primers SPp1764- SPp1765 were designed to introduce muta- 702 tions at the RAV binding site contained in ProOsMADS14 by PCR-based method. The 703 corresponding reporter vector was used as template for site-directed mutagenesis (15 704 cycles: 10´´ at 98ºC, 20´´ at 66ºC, 20´´ at 66ºC), and the resulting vector employed in 705 transactivation assays. Protoplasts were isolated from calli by digesting the cell-wall 706 with Macerozyme R-10 and Cellulase (Yakult Pharmaceuticals), and transfected with 707 different combinations of reporter Pro35S:OsTEM1 and effector constructs using PEG. 20 Downloaded from on June 22, 2020 - Published by www.plantphysiol.org Copyright © 2020 American Society of Plant Biologists. All rights reserved. 708 After 18 hours incubation in darkness at 24°C, transformed protoplasts were pelleted 709 and resuspended in homogenization buffer for RNA extraction. Transactivation activity 710 of OsTEM1, based on the relative ratio of mRNA abundance of Luciferase and Renilla 711 reporter genes, was assessed by RT-qPCR. The primers used for cloning and expres- 712 sion analyses are listed in Supplemental Tables S4 and S5. 713 714 RNA extraction and expression analyses 715 For expression analyses in Arabidopsis, pools of 20 seedlings grown for one week un- 716 der LD were collected at ZT12. For expression analyses in rice, pools of 10-15 sam- 717 ples from different tissues and/or developmental stages were collected at ZT13 unless 718 otherwise stated. RNA was extracted with PureLink RNA mini kit (Ambion) and treated 719 with DNaseI RNase free (Ambion). For large scale experiments, RNA was extracted 720 with Maxwell RSC Plant RNA kit (Promega) and DNase treatment was performed on- 721 column. 1 μg of DNase-treated RNA was retro-transcribed with SuperScript III (Invitro- 722 gen), and cDNA was used for RT-qPCR with Light Cycler 480 SYBR Green I master on 723 Light Cycler 480 II (Roche). Three biological replicates and three technical replicates 724 were performed. In situ hybridization was performed as previously reported (Dreni et 725 al., 2007). The primers used for expression analyses are listed in Supplemental Table 726 S5. 727 728 Phenotypic Analyses 729 Morphological analysis of reproductive structures was performed by Optical Microscopy 730 (Olympus DP71) and Scanning Electron Microscopy. For SEM, flowers at anthesis 731 were fixed in 2.5% v/v glutaraldehyde in 0.1 M P-buffer (pH 7.4) overnight at 4°C, 732 washed 4 times for 10 minutes in 0.1 M P-buffer, post-fixed in 1% osmium tetraoxide 733 with 0.7% ferrocyanide in P-buffer, washed in water, dehydrated in an ascending etha- 734 nol series (50, 70, 80, 90, and 95% for 10 min each and twice with 100% ethanol), and 735 dried by critical-point drying with CO2. Alteration on the morphology of the seed were 736 analysed by using the Smart-grain software (Tanabata et al., 2012). At least 100 seeds 737 per genotype (for three independent biological replicates) were spread uniformly on the 738 glass with a black background, and images were captured with HP Scanner at 300 dpi. 739 The software determined seed shape parameters such as seed length, width, perime- 740 ter and area, and also calculated length-to-width ratio and circularity. 741 21 Downloaded from on June 22, 2020 - Published by www.plantphysiol.org Copyright © 2020 American Society of Plant Biologists. All rights reserved. 742 Statistical analyses 743 All statistical analyses are shown in Supplemental Table S6. Statistical significance of 744 each experiment was determined by using GraphPad Prism 7. For flowering time phe- 745 notype, we chose one-way ANOVA, multiple comparison were then corrected with 746 Dunnet's or Dunn's tests. For other kind of data, we compared two columns by using 747 unpaired t-test (two-tailed options), confidence Interval 95%. 748 Following instructions of GraphPad Prism, we first transformed ratios in log of ratios 749 (Y=log(Y)), for Fig. 5 and Supplemental Fig. S11. Then we created a column data table 750 and enter two columns of data (control – OsTEM1, +OsTEM1) with matched values on 751 the same row. We chose t tests from the list of column analyses, and selected condi- 752 tions as follows: Experimental design: Paired; Assume Gaussian distribution: Yes; 753 Choose test: Ratio paired t test. On the second tab of the t test dialog, we chose to 754 compute +OsTEM1 vs – OsTEM1. 755 756 Accession numbers 757 Sequence data from this article can be found in the GenBank/EMBL data libraries un- 758 der the following accession numbers. 759 AtRAV locus identifiers: AT1G13260 (AtRAV1), AT3G25730 (AtRAV1-like), At1g25560 760 (AtRAV2-like/TEM1), AT1G68840 (RAV2/TEM2), At1g50680 (AtRAV3), At1g51120 761 (AtRAV3-like). 762 OsRAV locus identifiers: LOC_Os10g39190 (OsRAV2), LOC_Os08g06120 (OsRAV3), 763 LOC_Os03g02900 (OsRAV4), LOC_Os04g49230 (OsRAV5), LOC_Os02g45850 764 (OsRAV6), LOC_Os11g05740 (OsRAV7), LOC_Os01g04750 (OsRAV8), 765 LOC_Os01g04800 (OsRAV9/OsTEM1), LOC_Os01g49830 (OsRAV11), 766 LOC_Os05g47650 (OsRAV12). 767 OsMADS locus identifiers: LOC_Os03g54160 (OsMADS14); LOC_Os07g01820 (Os- 768 MADS15); LOC_Os07g41370 (OsMADS18); LOC_Os03g54170 (OsMADS34); 769 LOC_Os03g03100 (OsMADS50/OsSOC1); LOC_Os01g69850 (OsMADS51) 770 771 SUPPLEMENTAL DATA 772 Supplemental Figure S1. Phylogenomic analysis of TEMPRANILLO (TEM) homologs 773 in sequenced land plants. 774 Supplemental Figure S2. Analysis of genes encoding AP2-B3 (RAV) proteins. 22 Downloaded from on June 22, 2020 - Published by www.plantphysiol.org Copyright © 2020 American Society of Plant Biologists. All rights reserved. 775 Supplemental Figure S3. Similarity clustering based on distribution patterns of known 776 conserved motifs in RAV proteins. 777 Supplemental Figure S4. Similarity clustering based on distribution patterns of con- 778 served features related to post-translational modification. 779 Supplemental Figure S5. Analysis of RAV genes in Oryza sativa ssp Japonica. A, 780 Distribution of RAV genes in the rice reference genome (Nipponbare ecotype). 781 Supplemental Figure S6. GO analysis of genes strongly coexpressed with OsRAV9. 782 Supplemental Figure S7. GO test of genes negatively correlated with OsRAV9 and 783 expression analysis of genes acting in apical meristems at floral transition in rice. 784 Supplemental Figure S8. Inferred expression profiles of OsRAV genes during plant 785 development. 786 Supplemental Figure S9. Analysis of T1 and T2 transgenic lines ectopically expressing 787 OsRAV9 and OsRAV11 in Arabidopsis. 788 Supplemental Figure S10. Molecular and phenotypic analysis of T1 transgenic rice 789 lines silencing OsRAV9. 790 Supplemental Figure S11. Transactivation activity of OsTEM1 on floral activators in 791 rice protoplasts. 792 Supplemental Figure S12. Expression analyses of genes involved in the floral transi- 793 tion in OsRAV9-i rice lines. 794 Supplemental Figure S13. Analyses of OsRAV11-OsRAV12. 795 Supplemental Figure S14. Molecular characterization of OsRAV11-OsRAV12. 796 Supplemental Figure S15. Analysis of RAV proteins from Arabidopsis thaliana and 797 Oryza sativa. 798 Supplemental Table S1. List of RAV genes in Oryza sativa reported by Swaminathan 799 et al., 2008. 800 Supplemental Table S2. List of genes encoding B3-domain TFs down-regulated in 801 OsMADS1 knock-down panicles (modified from Khanday et al., 2013). 802 Supplemental Table S3. List of primers used for genotyping. 803 Supplemental Table S4. List of primers used for cloning. 804 Supplemental Table S5. List of primers used for expression analyses. 805 Supplemental Table S6. Statistical analyses. 806 Supplemental Dataset S1. Co-expression analyses 23 Downloaded from on June 22, 2020 - Published by www.plantphysiol.org Copyright © 2020 American Society of Plant Biologists. All rights reserved. 807 808 ACKNOWLEDGMENTS 809 Dr. Marti Bernardo, Dr. Jordi Morata and Dr. Victor Gonzalez for advice on bioinformat- 810 ic analyses; Dr. Sebastien Santini for Phylogeny.fr tool; Elia Lacchini for technical help 811 with set-up of genotyping of osmads1 mutants, Dr. Ludovico Dreni for help with rice 812 transgenic plants, and Pilar Fontanet for advice and technical help on protoplast co- 813 transformation assays. We thank the Servei de Microscopia at the Universitat Autòno- 814 ma de Barcelona for help with SEM. Dr. Paula Suárez-López for critical reading of the 815 manuscript. 816 817 Tables 818 Table 1. List of the top 20 genes negatively correlated with OsRAV9. In bold, 819 genes involved in the regulation of reproductive development. Weighted Locus Identifier Gene name Description PCC MADS-domain containing -0.568 LOC_Os03g54160 OsMADS14 Transcription Factor MADS-domain containing -0.542 LOC_Os03g54170 OsMADS34 Transcription Factor MADS-domain containing -0.526 LOC_Os07g01820 OsMADS15 Transcription Factor Cyclin-like F-box domain -0.497 LOC_Os04g13150 containing protein cyclin-dependent kinase G-1 -0.491 LOC_Os10g06560 Protein kinase-like domain -0.477 LOC_Os09g19500 containing protein Similar to SP3D -0.476 LOC_Os01g11940 OsFT-like1 Conserved hypothetical protein -0.464 LOC_Os01g19880 Protein kinase-like domain -0.463 LOC_Os10g06510 containing protein Longin-like domain -0.458 LOC_Os02g55990 containing protein. WRKY-domain containing -0.451 LOC_Os07g17230 OsWRKY123 Transcription Factor Similar to O-methyltransferase ZRP4 -0.446 LOC_Os05g43930 OMT (EC 2.1.1.-) Conserved hypothetical protein -0.434 LOC_Os11g31770 Similar to Protein phosphatase 2C -0.433 LOC_Os05g38290 (PP2C) Similar to ABA-responsive element -0.4315 LOC_Os02g52780 bZIP binding protein 2 (AREB2). 24 Downloaded from on June 22, 2020 - Published by www.plantphysiol.org Copyright © 2020 American Society of Plant Biologists. All rights reserved. Conserved hypothetical protein -0.423 LOC_Os03g04990 HEADING DATE 3A -0.424 LOC_Os06g06320 Hd3a Conserved hypothetical protein -0.422 LOC_Os12g13910 Conserved hypothetical protein -0.411 LOC_Os09g27680 Similar to HIGH-AFFINITY -0.407 LOC_Os08g36340 HAK4 POTASSIUM TRANSPORTER 4 820 821 FIGURE LEGENDS 822 Figure 1. Phylogenetic analysis of RELATED TO ABI3 AND VP1 (RAV) proteins in 823 model species. Tree representing the most related RAV-like proteins from Arabidopsis 824 thaliana (At) and Oryza sativa (Os) retrieved using TEMPRANILLO1 (TEM1) as query 825 in a BLAST-P search, alignment with MUSCLE, and selected with G-blocks. TEM1 and 826 TEM2 are RAV2-like and RAV2, respectively. Phylogenetic analysis with bootstrapping 827 procedure (N=100) as statistical test for branch support was carried out with PhyML, 828 and tree visualization with TreeDyn. Scale bar indicates the number of substitutions per 829 site, values in red indicate percentage branch support values. 1 830 Figure 2. Patterns of OsRAV genes expression during plant development. A to C, 831 Transcript levels of OsRAVs, relative to OsUBQ, in vegetative and reproductive tissues 832 of wild-type plants. OsRAV9 in light green, OsRAV11 in light lilac and OsRAV12 in dark 833 lilac. A, RT-qPCR in roots, stems, and second leaf (L2) dissected from 1-week-old 834 seedlings. B, RT-qPCR in mature leaves (L5, L6, L7) dissected from 10-week-old adult 835 plants. C, RT-qPCR in mature pistils and fertilized ovaries 1 and 3 days after pollination 836 (DAP). D, Transcript abundance of PPS (white), OsRAV9 and OsRAV11, relative to 837 OsUBQ, in juvenile (L2) and adult (L3-L4-L5) leaves. At week 2, L3 is formed and L4 is 838 emerging. At week 3, L4 expands and L5 is emerging. At week 4, L5 is fully expanded. 839 Expression data are mean value of three biological replicates, and error bars represent 840 SD. 841 842 Figure 3. Late flowering phenotype of Arabidopsis (Arabidopsis thaliana) plants ex- 843 pressing OsRAV9. A, Representative images of wild-type (wt, left) and OsRAV9-E 844 transgenic (right) plants grown for 4 weeks under long days (LD). Scale bar indicates 1 845 cm. B and C, Flowering time scored as number of rosette leaves and number of days 846 to flower of wild-type (grey) and OsRAV9-E lines (green) grown under LD. D to F, Rela- 847 tive expression levels of OsRAV9 and TEM1 downstream targets in wild-type and rep- 25 Downloaded from on June 22, 2020 - Published by www.plantphysiol.org Copyright © 2020 American Society of Plant Biologists. All rights reserved. 848 resentative T3 OsRAV9-E lines grown for 1 week under LD. D, Ectopic expression of 849 OsRAV9 in transgenic Arabidopsis lines. E and F, Down-regulation of FT and 850 AtGA3ox1 in OsRAV9-E lines compared to wild-type. 851 Flowering time data are the average of 25 plants each genotype, with standard error of 852 the mean. Three biological replicates gave similar results, and one was chosen as rep- 853 resentative. Data were analyzed by one-way ANOVA followed by Dunnett's multiple 854 comparisons test between wild-type and transgenic lines. Asterisks indicate statistical 855 significance, with * P-value<0,05. Expression data are reported as mean value of three 856 biological replicates; error bar represents the standard error of the mean. AtUBQ10 857 was used for normalization. 858 859 Figure 4. Role of OsRAV9 in heading date. A, Diurnal oscillation of Hd3a and OsRAV9 860 expression in leaves of 4-week-old wild-type (wt) plants. Grey block indicates night. B, 861 Mutually exclusive expression patterns of Hd3a and OsRAV9 in leaves throughout 862 wild-type (wt) plant development. Grey block indicates floral transition. C to D, Scatter 863 plots representing heading date as number of days to flower of wild-type (in grey) and 864 OsRAV9-i plants (in light green) grown under inductive photoperiods (8h light/16h dark, 865 12h light/12h dark). Lines represent the median with 95% of CI. E, Early flowering phe- 866 notype of a selected OsRAV9 silencing line (RAV9-i #3, right) compared to wild-type 867 (left) 100 DAG. Bar represent 10 cm. F, Close-up view showing wild-type panicle at the 868 booting stage and OsRAV9-i panicle at anthesis. For molecular analyses, plants were 869 grown under 12 hours light/12 hours dark at 28ºC, whereas for heading date plants 870 were grown under different daylengths. Expression data are reported as mean value of 871 three biological replicates; error bar represents the standard error of the mean. Flower- 872 ing time data are the average of 18–20 plants each genotype. Three biological repli- 873 cates gave similar results, and one was chosen as representative. Data were analyzed 874 by one-way ANOVA followed by Dunnett's multiple comparisons test between wild-type 875 and transgenic lines. Asterisks indicate statistical significance, with * P-value<0,05, ** 876 P-value<0,033. 877 878 Figure 5. Interaction between OsRAV9/OsTEM1 and floral activators. A, Non canoni- 879 cal RAV binding site in the promoter of Hd3a, 785 bp upstream of the TSS. B, Perfect 880 RAV binding site in the promoter of OsMADS14, 2250 bp upstream of the TSS, and 881 mutated version (right). Arrows represent oligonucleotides used to amplify fragments of 882 ProHd3a and ProOsMADS14 without RAV binding sites (Ha and Ma, in orange) and 883 with RAV binding sites (Hb and Mb, in green). C and E, Transactivation activity of 884 OsTEM1 in transiently transformed protoplasts, reported as ratio of transcript levels of 26 Downloaded from on June 22, 2020 - Published by www.plantphysiol.org Copyright © 2020 American Society of Plant Biologists. All rights reserved. 885 LUC and REN reporter genes relative to UBQ. C, RT-qPCR of protoplasts co- 886 transformed with Pro35S:OsTEM1 and reporter vectors containing sequences of 887 ProHd3a (Ha in grey, Hb in green). D, RT-qPCR of protoplasts co-transformed with 888 Pro35S:OsTEM1 and reporter vectors containing sequences of ProOsMADS14 (Ma in 889 grey, Mb in green). E, Analysis of protoplasts co-transformed with Pro35S-OsTEM1 890 and reporter vectors containing sequences of ProOsMADS14 (Mb in green, mutated 891 Mb in dark green). Values are the mean of three independent replicates. F to H, ex- 892 pression analysis of genes involved in heading date in independent T2 lines (green) 893 compared to wild-type plants grown for 28, 35 and 42 days under inductive conditions. 894 F, Down-regulation of OsRAV9/OsTEM1 in silencing lines (green). G and H, Up- 895 regulation of the floral activators OsMADS14 and Hd3a in silencing lines (green lines) 896 compared to wild-type (black line). Expression data are mean value of three biological 897 replicates with three technical replicates each, and error bars represent SD. 898 899 Figure 6. Molecular and functional characterization of OsRAV11. A to C, Mis- 900 regulation of OsRAV11 in floral homeotic mutants. A, Down-regulation of OsRAV11 in 901 osmads1 developing inflorescences. B, Up-regulation of OsRAV11 in osmads16 and 902 osmads13 developing inflorescences. C, Strong activation of OsRAV11 in specific cell 903 types (floral meristem and ovule primordia) isolated from osmads13 mutant flowers. D, 904 Schematic representation of the T-DNA insertion in the 5’ UTR of OsRAV11. Arrows, 905 primers used for genotyping. E, Down-regulation of OsRAV11 in pistils dissected from 906 osrav11 mutant flowers at maturity. F to H, Morphological analyses of female reproduc- 907 tive structures at maturity. F, Representative images of wild-type and osrav11 carpels 908 dissected from mature flowers at anthesis obtained by optical Microscopy. Bar, 1 mm. 909 G and H, Representative images of reproductive structures obtained by Scanning Elec- 910 tron Microscopy (SEM). G, Wild-type and osrav11 carpels upon fertilization. Glumes 911 were partly removed to show female reproductive organs. Bar, 500 m. H, Apical tis- 912 sues of wild-type and osrav11 gynoecia after pollination. Bar, 100 m. I, Bar-plots rep- 913 resenting the weight of seed produced by wild-type (in grey) and osrav11 (in white) 914 plants grown in the greenhouse under inductive conditions. Expression data are re- 915 ported as mean value of three biological replicates; error bar represents the standard 916 error of the mean. Phenotypic data are the average of three biological replicates of the 917 weight of 100 seeds each genotype, with standard error of the mean. Statistical signifi- 918 cance was examined by two tailed unpaired t-test, with * P-value<0,05. 919 27 Downloaded from on June 22, 2020 - Published by www.plantphysiol.org Copyright © 2020 American Society of Plant Biologists. All rights reserved. 920 Figure 7. Morphogenetic effects of OsRAV11/OsRAV12 silencing in reproductive 921 phase. A, epresentative images of mature flowers dissected from wild-type (wt) and 922 OsRAV11/12-i inflorescences. Arrows indicate the position of the stigmas. B, repre- 923 sentative images of mature carpels dissected from wild-type and one T3 OsRAV11/12-I 924 flowers obtained by optical microscopy. Bar, 1mm. C, Down-regulation of OsRAV11 925 and OsRAV12 in mature pistils of two representative T3 OsRAV11/12-i lines as com- 926 pared to wild-type. Expression data are reported as mean value of three biological rep- 927 licates with standard error of the mean. Data were analyzed by ordinary one-way 928 ANOVA followed by Dunnett's multiple comparisons test between wild-type and trans- 929 genic lines. Asterisks indicate statistical significance, with * P-value<0,05 and ** P- 930 value<0,033. D, fertility defects of mature OsRAV11/12-i panicles compared to wild- 931 type. 932 933 Figure 8. Model for interaction between RAV and MADS factors during reproductive 934 growth. A, OsRAV9 represses the transcription of OsMADS14, a positive regulator of 935 the florigen Hd3a, in the leaf. 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