Genes of the RAV Family Control Heading Date and Carpel Development in Rice1[OPEN] Michela Osnato,a,b,2,3 Luis Matias-Hernandez,a Andrea Elizabeth Aguilar-Jaramillo ,a Martin M. Kater,b and Soraya Pelaz a,c,2 a Centre for Research in Agricultural Genomics, Centro de Investigaciones Científicas-Institut de Recerca i Tecnologia Agroalimentàries-Universidad Autónoma de Barcelona-Universidad de Barcelona, Campus Universidad Autónoma de Barcelona, 08193 Barcelona, Spain b Department BioSciences, University of Milan, 20133 Milan, Italy c Institució Catalana de Recerca i Estudis Avançats, 08010 Barcelona, Spain ORCID IDs: 0000-0002-2439-8581 (M.O.); 0000-0003-1155-2575 (M.M.K.); 0000-0001-7699-9330 (S.P.). In plants, correct formation of reproductive organs is critical for successful seedset and perpetuation of the species. Plants have evolved different molecular mechanisms to coordinate flower and seed development at the proper time of the year. Among the plant-specific RELATED TO ABI3 AND VP1 (RAV) family of transcription factors, only TEMPRANILLO1 (TEM1) and TEM2 have been shown to affect reproductive development in Arabidopsis (Arabidopsis thaliana). They negatively regulate the floral transition through direct repression of FLOWERING LOCUS T and GIBBERELLIN 3-OXIDASE1/2, encoding major components of the florigen. Here we identify RAV genes from rice (Oryza sativa), and unravel their regulatory roles in key steps of reproductive development. Our data strongly suggest that, like TEMs, OsRAV9/OsTEM1 has a conserved function as a repressor of photoperiodic flowering upstream of the floral activators OsMADS14 and Hd3a, through a mechanism reminiscent of that one underlying floral transition in temperate cereals. Furthermore, OsRAV11 and OsRAV12 may have acquired a new function in the differentiation of the carpel and the control of seed size, acting downstream of floral homeotic factors. Alternatively, this function may have been lost in Arabidopsis. Our data reveal conservation of RAV gene function in the regulation of flowering time in monocotyledonous and dicotyledonous plants, but also unveil roles in the development of rice gynoecium. In plants, the correct formation of reproductive or- to guarantee pollination and maximum survival pos- gans is critical not only for successful seedset but also sibilities for the offspring. Plants that are affected in for the perpetuation of the species. Accordingly, floral their flowering time often have a lower amount of seeds evocation must take place at a favorable time of the year resulting in yield losses. Indeed, precocious flowering is frequently associated with reduced photosynthetic capacity due to a shortened vegetative phase (Endo- 1 This work was supported by the Ministerio de Ciencia, Higashi and Izawa, 2011). Conversely, delayed flow- Tecnología e Innovación Productiva (grant no. PGC2018–095804–B– ering can affect seed maturation due to exposure to I00) the Ministerio de Economía y Competitividad (grant no. unfavorable conditions. A negative correlation also BFU2015–64409–P), Government of Catalonia j Agència de Gestió exists between grain size and grain number (Guo et al., d’Ajuts Universitaris i de Recerca (CERCA Programme, Consolidated 2018; Li et al., 2018), two important agronomic traits Research Group no. 2014 SGR 1406 and Investigator Training Pro- which are controlled by both genetic determinants and gram PhD fellowship to A.E.A.-J.), Flower Power (to M.M.K. and S.P.), EvoRepRice (FIRST; to M.M.K.), the EMBO short-term fellowship environmental conditions. (to M.O.), and the Ministerio de Economía y Competitividad through Plants have evolved different molecular mecha- the “Severo Ochoa Programme for Centres of Excellence in R&D” nisms to coordinate flower and seed development at 2016–2019 (grant no. SEV–2015 0533). the proper time of the year. Actually, the switch from 2 Senior authors. vegetative to reproductive growth is controlled by 3 Author for contact: michela.osnato@cragenomica.es. multiple genetic determinants that integrate the re- The author responsible for distribution of materials integral to the sponses to environmental and physiological conditions findings presented in this article in accordance with the policy de- of the plant. Ultimately, the regulatory pathways un- scribed in the Instructions for Authors (www.plantphysiol.org) is: derlying the floral transition converge on floral inte- Michela (michela.osnato@cragenomica.es). grators that are able to activate genes in the shoot apical M.O., M.M.K., and S.P. designed the experiments, discussed the experiments, and wrote the paper; M.O. performed and analyzed meristem (SAM) that control the initiation and devel- most of the experiments; L.M.-H. and A.E.A.-J. performed analyses opment of the inflorescence meristem (IM), and then of of Arabidopsis transgenic plants expressing OsRAV genes. floral meristems (FM) from which floral organs differ- [OPEN] Articles can be viewed without a subscription. entiate. Upon fertilization, the carpel transforms into a www.plantphysiol.org/cgi/doi/10.1104/pp.20.00562 fruit in which the seeds develop. Plant PhysiologyÒ, August 2020, Vol. 183, pp. 1663–1680, www.plantphysiol.org Ó 2020 American Society of Plant Biologists. All Rights Reserved. 1663 Downloaded from on September 3, 2020 - Published by www.plantphysiol.org Copyright © 2020 American Society of Plant Biologists. All rights reserved. Osnato et al. In the last decade, the molecular basis of the floral 2010). Additional studies on phylogenetic recon- transition has been unveiled in different plant species: the structions revealed the presence of FLC homologs in florigen, a long distance signaling molecule, is first pro- monocots genomes (Ruelens et al., 2013). These FLC- duced in leaves under favorable conditions, and then like factors repress the floral transition in response to transported to the apical meristem to initiate reproductive cold in temperate cereals (Winfield et al., 2009; development (Andrés and Coupland, 2012). In the model Greenup et al., 2010), but appeared to have acquired species Arabidopsis (Arabidopsis thaliana), photoperiodic an opposite function in tropical cereals. Indeed, the flowering is triggered by FLOWERING LOCUS T (FT), a rice FLC homolog OsMADS51 activates the expres- small globular protein of 21 kD (Kardailsky et al., 1999; sion of the floral promoter Ehd1 (Kim et al., 2007). Kobayashi et al., 1999). The expression of FT is activated Upon FT/Hd3a activation, the resulting gene product under inductive long days (LD) in vascular tissues of moves through the phloem from the leaf to the SAM leaves by the positive regulator CONSTANS (CO; Suárez- (Corbesier et al., 2007; Jaeger and Wigge, 2007; Mathieu López et al., 2001; An et al., 2004), and precocious flow- et al., 2007; Tamaki et al., 2007; Notaguchi et al., 2008; ering is prevented by the counteraction of the RELATED Komiya et al., 2009) where it triggers transcriptional TO ABI3 AND VP1 (RAV) transcription repressors reprogramming able to confer the competence to form TEMPRANILLO1 (TEM1) and TEM2 (Castillejo and flowers (Corbesier et al., 2007; Torti et al., 2012). Intrigu- Pelaz, 2008). Since prevention of precocious flowering is ingly, FT-like proteins do not have DNA binding activity important for reproductive fitness other repressors such as per se and must interact with bZIP transcription factors FLOWERING LOCUS C (FLC), SCHLAFMUTZE (SMZ), (TFs), i.e. FLOWERING LOCUS D (FD) in Arabidopsis SCHNARCHZAPFENZ (SNZ), TARGET OF EARLY AC- and OsFD1 in rice (Abe et al., 2005; Wigge et al., 2005; TIVATION TAGGED 1 (TOE1), TOE2, FLOWERING LO- Taoka et al., 2011; Brambilla et al., 2017; Collani et al., CUS M (FLM) or SHORT VEGETATIVE PHASE (SVP) 2019), to activate the expression of downstream genes. play key roles in the control of FT expression in response The interaction of FT and FD proteins require the 14-3-3 to vernalization, age, photoperiod or ambient temperature adaptor proteins for the formation of an active Floral (Hartmann et al., 2000; Scortecci et al., 2001, 2003; Activation Complex (FAC; Taoka et al., 2011; Collani Aukerman and Sakai, 2003; Schmid et al., 2003; Jung et al., et al., 2019), which in Arabidopsis directly activates the 2007; Lee et al., 2007; Li et al., 2008;; Mathieu et al., 2009; FM identity gene AP1 (Wigge et al., 2005), whereas the Lee et al., 2013; Posé et al., 2013). Once flowering is in- rice FAC complex induces the expression of the AP1-like duced, FM identity genes, such as the MADS-box genes genes OsMADS14/15/18 and the SEPALLATA-like gene SUPPRESSOR OF CONSTANS1 (SOC1) and APETALA1 OsMADS34 that are required for the specification of IM (AP1), are induced which in turn repress TEMs expression identity (Kobayashi et al., 2012; Gómez-Ariza et al., 2019) (Kaufmann et al., 2010; Tao et al., 2012). Under nonin- The florigens also mediate the transcriptional repression ductive short days (SD), CO is not active and there is no of PINE, a gene encoding a Zinc Finger type TF involved CO-dependent FT induction. In this light regime, the ac- in the negative regulation of stem elongation. Therefore, cumulation of the plant hormones gibberellins (GA) trig- the FAC coordinates the formation of reproductive gers floral transition by inducing SOC1 and LEAFY (LFY; structures (by activating IM identity genes) and internode Wilson et al., 1992; Blázquez et al., 1997; Moon et al., 2003; elongation (by repressing PINE), guaranteeing the emer- Eriksson et al., 2006; Hisamatsu and King, 2008). Inter- gence of the panicle from the flag leaf, known as heading, estingly, TEMs also regulate GA accumulation by which occurs when rice inflorescence development is repressing GA-3-OXIDASE 1 (GA3OX1) and GA3OX2 completed (Gómez-Ariza et al., 2019). genes (Osnato et al., 2012). In the rice inflorescence, primary and secondary In the crop species rice (Oryza sativa), two closely re- branches form on the flanks of the IM (rachis) and ter- lated genes have been described as FT orthologs: Heading minate in spikelet meristems that develop floret meri- date 3a (Hd3a), which promotes flowering under induc- stems from which the palea, the lemma, two lodicules, tive SD, and Rice Flowering locus T 1 (RFT1), which does it six stamens and one central carpel differentiate. The under noninductive LD (Kojima et al., 2002; Komiya identity of different floral organs is specified by the et al., 2009). An evolutionarily conserved module de- interaction of MADS domain TFs belonging to the fined by the rice orthologs of Arabidopsis CO and FT SEPALLATA (OsMADS1), APETALA3 (OsMADS16) controls photoperiodic flowering (Shrestha et al., 2014). and AGAMOUS (OsMADS3–OsMADS58) subfamilies The rice homolog of CO, Hd1, functions as activator of (Jeon et al., 2000; Kyozuka et al., 2000; Nagasawa et al., Hd3a in SD, and contrarily as repressor in LD (Yano et al., 2003; Dreni et al., 2011). Eventually, the FM is con- 2000; Nemoto et al., 2016). Nevertheless, additional rice- sumed during gynoecium development, when a single specific regulators have been discovered: the floral acti- ovule primordium forms inside the carpel (Dreni et al., vator Early Heading date 1 (Ehd1, Doi et al., 2004), and its 2007). Recently, OsMADS1 was also shown to be es- repressor Grain number, plant height and heading date 7 sential during seed development, specifically in the (Ghd7; Itoh et al., 2010). The Ehd1-Ghd7 pathway de- regulation of grain size and shape (Liu et al., 2018). termines the critical day-length necessary for the In this study, we identified four members of the RAV induction of the florigen through a double gating family of TF in rice, which share sequence similarity with mechanism dependent on the circadian clock and Arabidopsis TEMs. In silico/coexpression analyses based phytochrome-mediated light perception (Itoh et al., on available transcriptomics data revealed that OsRAVs 1664 Plant Physiol. Vol. 183, 2020 Downloaded from on September 3, 2020 - Published by www.plantphysiol.org Copyright © 2020 American Society of Plant Biologists. All rights reserved. RAVs Regulate Heading Date and Carpel Development interact with genes belonging to the MADS-box super- species (Fig. 1; Supplemental Fig. S3). Although the family at different developmental stages, suggesting that four OsRAV proteins clearly clustered together with RAVs are unknown players in the gene regulatory net- AtRAV1/AtRAV1-like and TEM1/TEM2, OsRAV8 work underlying reproductive development in rice. Spe- and OsRAV9 showed the highest similarity with these cifically, OsRAV8 and OsRAV9 negatively correlate with Arabidopsis RAV factors (Riechmann, 2002). IM identity genes of the AP1 subfamily, whereas OsRAV11 In the rice genome, the regions corresponding to the and OsRAV12 act downstream of MADS-domain floral OsRAV8 and OsRAV9 loci may have originated recently, homeotic factors of the SEP and AG subfamilies. Molecular likely due to tandem duplication events after speciation. and functional studies using knock-down and knock-out Indeed, these genes are physically linked (having a dis- mutant lines indicate a conserved function for OsRAV9/ tance of about 50 Kb) at the tip of the short arm of chro- OsTEM1 as repressor of photoperiodic flowering upstream mosome 1 in a region that is enriched in sequences related of the floral activators OsMADS14 and Hd3a, and reveal a to retro-transposons (Supplemental Fig. S5, A and B). role for OsRAV11 and OsRAV12 in the correct develop- Furthermore, when we searched the Plant Genome Du- ment of female reproductive organs, downstream of plication Database (Lee et al., 2013) using the OsRAV8- OsMADS1 and OsMADS13. OsRAV9 locus identifiers, we found intragenome syntenic relationships with a region on the long arm of chromo- some 1 containing OsRAV11 (Supplemental Fig. S5C), and a region on chromosome 5 containing OsRAV12 RESULTS (Supplemental Fig. S5D). As a result of these phyloge- The Rice Genome Contains Four RAV Genes nomics and phylogenetic analyses, OsRAV11 and the re- lated gene OsRAV12 appeared as within-species paralogs Genes belonging to the RAV subfamily are present in of OsRAV8-OsRAV9 with similar genomic structures. all land plant species and encode for putative TFs, which are characterized by two DNA binding domains, an APETALA2-type at the N terminus and a B3-type at the C terminus (Kagaya et al., 1999). In the model species, Expression Patterns of RAV Genes and Floral MADS-box Arabidopsis, the subfamily of RAV genes is composed Genes Are Correlated of six members (Riechmann, 2002); in addition to the To gain insights into the possible function of OsRAV TEMs, four other genes belong to this family: RAV1 and genes in various biological processes and metabolic RAV1-like, which are phylogenetically close to TEMs, and RAV3 and RAV3-like, which are the most divergent and nothing has been reported about their function. The functional characterization of the closest four indicate their regulatory role in different stages of plant devel- opment (Hu et al., 2004; Feng et al., 2005; Castillejo and Pelaz, 2008; Osnato et al., 2012; Matías-Hernández et al., 2014, 2016; Aguilar-Jaramillo et al., 2019) and in the response to abiotic stresses (Fu et al., 2014). A preliminary phylogenomics analysis indicates a clear separation between AP2-B3 coding genes (RAV) and those encoding only the B3 domain (ARF, NGA, VAL), as reported in Supplemental Figure S1. Specifi- cally in rice, although twelve genes were named RAV (Supplemental Table S1; Swaminathan et al., 2008), only four encode putative proteins containing both DNA binding domains as predicted by gene orthology and paralogy (OsRAV8, OsRAV9, OsRAV11, and OsRAV12; Supplemental Fig. S2A). Further analysis carried out by searching the SALAD (Surveyed conserved motif Alignment diagram and the Associating Dendrogram) database (Mihara et al., 2010) revealed the absence of RAV proteins in green and red algae, and the presence of multiple conserved motifs in addition to the AP2 and B3 Figure 1. Phylogenetic analysis of RAV proteins in model species. Tree represents the most related RAV-like proteins from Arabidopsis (At) and domains (Supplemental Fig. S2B), including the bipartite Oryza sativa (Os) retrieved using TEM1 as query in a BLAST-P search, nuclear localization signal and the B3 repression domain alignment with MUSCLE, and selected with G-blocks. TEM1 and TEM2 (Supplemental Fig. S3) together with features associated are RAV2-like and RAV2, respectively. Phylogenetic analysis with to posttranslational modifications (Supplemental Fig. S4). bootstrapping procedure (n 5 100) as statistical test for branch support We also performed a phylogenetic analysis based on was carried out with PhyML, and tree visualization with TreeDyn. Scale the deduced full-length protein sequences retrieved by a bar indicates the number of substitutions per site. Values in red indicate BLAST-P search against the proteomes of the two model percentage branch support values. Plant Physiol. Vol. 183, 2020 1665 Downloaded from on September 3, 2020 - Published by www.plantphysiol.org Copyright © 2020 American Society of Plant Biologists. All rights reserved. Osnato et al. pathways, we carried out an in silico coexpression revealed a negative correlation between the expression of analysis by using the Rice Functionally Related gene the closely related gene OsRAV8 and IM identity genes Expression Network Database (RiceFREND) platform OsMADS14, OsMADS15, OsMADS18, and OsMADS34 (Sato et al., 2013) and constructed a coexpressed gene at the transition from vegetative to reproductive growth list for OsRAV9 as guide gene (Supplemental Dataset (Gómez-Ariza et al., 2019; Supplemental Fig. S7). Taken S1). Among the top 474 genes displaying a positive together, coexpression and available transcriptomics correlation with OsRAV9 (Pearson’s Correlation Coef- datasets may suggest a possible role for at least OsRAV8 ficient higher than 0.3), we found enrichment for bio- and OsRAV9 in the negative regulation of the transition logical processes gene ontology (GO) categories related from vegetative to reproductive phase, similarly to RAV to reproductive processes, response to chemical stimuli, genes in Arabidopsis. and response to oxidative stress (Supplemental Fig. S6, A and B). Supporting this, we also found overrepre- sentation for molecular function of the GO terms oxi- OsRAV Genes Are Differentially Expressed during doreductase and iron-binding activities (Supplemental Plant Development Fig. S6C). Taken together, these findings suggest a possible role for RAV in reproductive development as Because a role for OsRAV genes in rice plant devel- well as abiotic stress response (similarly to Arabidopsis opment is yet to be elucidated, we first inferred their RAVs). In particular, peroxidases and oxidoreductases expression profiles by searching publicly available are detoxification enzymes activated upon accumula- collections of transcriptomics data (Sato et al., 2011). tion of reactive oxygen species (Choudhury et al., 2013), OsRAV9 and OsRAV12 are expressed in vegetative and ion-binding proteins are able to sequester excess tissues (i.e. leaves and roots), the former at early stages iron to avoid reaction with oxygen and the formation of of plant development (Supplemental Fig. S8A), and the damaging reactive oxygen species (Selote et al., 2015). latter at maturity (Supplemental Fig. S8B), whereas On the other hand, GO analysis of the top 176 genes OsRAV11 is widely expressed in different organs with displaying a negative correlation with OsRAV9 sug- the exception of anthers (Supplemental Fig. S8C). gested a possible function in signal transduction path- Moreover, the expression of OsRAV9 seems to follow a ways, due to the enrichment of the terms related to diurnal oscillation: its transcript levels are almost un- protein dephosphorylation and regulation of transcrip- detectable during the day, then increase at dusk and tion for biological processes (Supplemental Fig. S7A). In reach a peak in the middle of the night (Supplemental particular, we found a significant negative Pearson’s Fig. S8D). Likewise, the expression of OsRAV11 and Correlation Coefficient between OsRAV9 and genes in- OsRAV12 appeared to oscillate during the day with a volved in reproductive development including the flori- peak after dusk (Supplemental Fig. S8, E and F), al- gen Hd3a and the IM identity genes such as OsMADS14, though with a smaller amplitude compared to OsRAV9. OsMADS15, and OsMADS34 (Table 1). Furthermore, To validate these expression data, we designed specific a recent transcriptomics analysis of apical meristems primers for each of the four OsRAV genes (Supplemental Table 1. List of the top 20 genes negatively correlated with OsRAV9 Genes listed in bold indicate involvement in the regulation of reproductive development. ABA, Abscisic acid; PCC, Pearson’s Correlation Coefficient. Weighted PCC Locus Identifier Gene name Description 20.568 LOC_Os03g54160 OsMADS14 MADS-domain containing Transcription Factor 20.542 LOC_Os03g54170 OsMADS34 MADS-domain containing Transcription Factor 20.526 LOC_Os07g01820 OsMADS15 MADS-domain containing Transcription Factor 20.497 LOC_Os04g13150 Cyclin-like F-box domain containing protein 20.491 LOC_Os10g06560 Cyclin-dependent kinase G-1 20.477 LOC_Os09g19500 Protein kinase-like domain containing protein 20.476 LOC_Os01g11940 OsFT-like1 Similar to SP3D 20.464 LOC_Os01g19880 Conserved hypothetical protein 20.463 LOC_Os10g06510 Protein kinase-like domain containing protein 20.458 LOC_Os02g55990 Longin-like domain containing protein. 20.451 LOC_Os07g17230 OsWRKY123 WRKY-domain containing Transcription Factor 20.446 LOC_Os05g43930 OMT Similar to O-methyltransferase ZRP4 (EC 2.1.1.-) 20.434 LOC_Os11g31770 Conserved hypothetical protein 20.433 LOC_Os05g38290 Similar to Protein phosphatase 2C (PP2C) 20.4315 LOC_Os02g52780 bZIP Similar to ABA-responsive element binding protein 2 (AREB2) 20.423 LOC_Os03g04990 Conserved hypothetical protein 20.424 LOC_Os06g06320 Hd3a HEADING DATE 3A 20.422 LOC_Os12g13910 Conserved hypothetical protein 20.411 LOC_Os09g27680 Conserved hypothetical protein 20.407 LOC_Os08g36340 HAK4 Similar to HIGH-AFFINITY POTASSIUM TRANSPORTER 4 1666 Plant Physiol. Vol. 183, 2020 Downloaded from on September 3, 2020 - Published by www.plantphysiol.org Copyright © 2020 American Society of Plant Biologists. All rights reserved. RAVs Regulate Heading Date and Carpel Development Fig. S8G). With respect to OsRAV8, we performed stan- Precisely, in rice, the juvenile phase is limited to the dard reverse transcription (RT)-PCR reactions, and even if second leaf (L2), because the transition to the adult a clear band was amplified using genomic DNA as con- phase occurs during the development of the third to trol template, no amplification was observed when using fifth leaf (L3–L5) when the midrib differentiates (Itoh cDNA obtained from different vegetative and reproduc- et al., 2005). We also analyzed the expression of the tive tissues (Supplemental Fig. S8H), confirming that this Peter Pan Syndrome (PPS) gene (Tanaka et al., 2011), the gene is not transcribed at detectable levels in the samples rice ortholog of the Arabidopsis COP1 (Liu et al., 2008), examined. Only very recently OsRAV8 was found to be as a marker for the transition from the juvenile to adult expressed; it was absent in all previous studies likely be- phase. In accordance with its function, PPS displayed a cause of its very specific expression in the apical meristem peak of expression in the fourth leaf four weeks after only at the time of floral transition (Gómez-Ariza et al., germination. OsRAV9 exhibited the highest transcript 2019). levels in L3 and then decreased in L4 and L5 (Fig. 2D), The expression profiles of the other three OsRAVs indicating that its transcription was drastically reduced were investigated by RT-quantitative PCR (qPCR) in at the transition to the adult phase. Also the expression wild-type plants at different developmental stages. of OsRAV11 was detected in young leaves, although at During the juvenile phase, the mRNAs of OsRAV9 and very low levels as compared with PPS and OsRAV9 OsRAV11 were detected in roots, basal region of the (Fig. 2D). stem comprising the SAM, and young leaves (Fig. 2A), In summary, these molecular analyses suggest di- although the abundance was much higher for OsRAV9 versification of expression patterns for OsRAV9 and than OsRAV11. During the adult phase, the expression OsRAV11, the former being transcribed at higher levels of OsRAV9 became almost undetectable in vegetative in the vegetative phase and specifically in juvenile tissues resembling Arabidopsis TEM genes expression, leaves, and the latter at maturity, in particular in old whereas OsRAV11 and OsRAV12 displayed high tran- leaves and female reproductive structures. script levels in mature leaves (Fig. 2B), resembling the expression behavior of the positive regulator of leaf senescence AtRAV1 in Arabidopsis (Woo et al., 2010). OsRAV9 Negatively Regulates Floral Transition OsRAVs were also expressed in female reproductive organs (Fig. 2C), and their transcript levels decreased To investigate the functional conservation between after pollination, suggesting that their activities might rice and Arabidopsis RAV genes, we used the Pro- be restricted to the gynoecium before anthesis. 35S:OsRAV9 and Pro-35S:OsRAV11 constructs to trans- To investigate in detail a possible role of OsRAVs in form Arabidopsis plants (Supplemental Fig. S9A). phase changes during vegetative growth, we dissected After selection of several independent transgenic differentiating leaves from wild-type plants to monitor lines (Supplemental Fig. S9B), phenotypic analyses of their transcript levels at different developmental stages. selected T2 generations revealed a mild late flowering Figure 2. Patterns of OsRAV genes expression during plant development. A to C, Transcript levels of OsRAVs, relative to OsUBQ, in vegetative and reproductive tissues of wild-type plants. A, RT-qPCR in roots, stems, and L2 dis- sected from 1-week-old seedlings. B, RT-qPCR in mature leaves (L5, L6, L7) dissected from 10-week-old adult plants. C, RT-qPCR in mature pistils and fertilized ovaries 1 and 3 d after polli- nation (DAP). D, Transcript abundance of PPS (white), OsRAV9 and OsRAV11, relative to OsUBQ, in juvenile (L2) and adult (L3-L4-L5) leaves. At week 2, L3 is formed and L4 is emerging. At week 3, L4 expands and L5 is emerging. At week 4, L5 is fully expanded. Expres- sion data are mean values of three biological replicates, and error bars represent SD. Plant Physiol. Vol. 183, 2020 1667 Downloaded from on September 3, 2020 - Published by www.plantphysiol.org Copyright © 2020 American Society of Plant Biologists. All rights reserved. Osnato et al. phenotype of transgenic plants expressing OsRAV9 (Castillejo and Pelaz, 2008). Actually, the expression of (OsRAV9-E), but no phenotypic alterations in plants OsRAV9 was high at early stages and dropped at expressing OsRAV11 (Supplemental Fig. S9C). Fur- around the transition to the adult phase. Conversely, thermore, molecular analyses of three representative T3 the expression of Hd3a is almost undetectable at early lines revealed also a correlation between the late flow- stages of vegetative growth, and increases in the adult ering phenotype of OsRAV9-E plants (Fig. 3, A to D) phase (Fig. 4B; Kojima et al., 2002; Komiya et al., 2009). and the down-regulation of FT and GA3oxidase1 (Fig. 3, The florigen begins to be produced in adult leaves E and F), two downstream targets of the TEM factors in 4 weeks after germination under inductive conditions, Arabidopsis (Castillejo and Pelaz, 2008; Osnato et al., and triggers floral transition in adult plants when it 2012). Taken together, these findings suggest that rice reaches its maximum accumulation around 6 weeks . OsRAV9 and the Arabidopsis TEM genes have an at Because the dynamics of OsRAV9 expression strik- least partial conserved function as repressor of photo- ingly resembled those of TEM genes, we decided periodic flowering in Arabidopsis. to investigate if the function was also conserved in Besides the high sequence identity between OsRAV9 rice. We used an RNA interference (RNAi) silencing and AtTEMs, their expression patterns were similar as strategy due to the absence of insertion mutants in both displayed high transcripts levels in the juvenile public collections for OsRAV9. Wild-type rice calli phase that decreased as the plant aged shortly before were transformed with an RNAi construct carrying the transition from vegetative to reproductive growth a Gene Sequence Tag specific for the 39 end of the (Fig. 2; Castillejo and Pelaz, 2008). Consistently, ex- gene under the control of a constitutive promoter pression analyses in wild-type plants showed a mutual (Supplemental Fig. S10A). We obtained eleven inde- exclusive pattern for OsRAV9 and the florigen Hd3a, pendent transgenic lines for the silencing construct, not only during the day (Fig. 4A) but also throughout and selected T1 OsRAV9-RNAi transformants by plant development (Fig. 4B), similarly to the opposite monitoring its transcript levels at the peak of expres- expression patterns of TEM and FT in Arabidopsis sion (Supplemental Fig. S10B). T2 generation lines were Figure 3. Late flowering phenotype of Arabidopsis plants expressing OsRAV9. A, Representative images of wild-type (wt; left) and OsRAV9-E transgenic (right) plants grown for 4 weeks under LD. Scale bar 5 1 cm. B and C, Flowering time scored as number of rosette leaves and number of days to flower of wild-type (gray) and OsRAV9-E lines (green) grown under LD. D to F, Relative expression levels of OsRAV9 and TEM1 downstream targets in wild-type and representative T3 OsRAV9-E lines grown for 1 week under LD. D, Ectopic expression of OsRAV9 in transgenic Arabidopsis lines. E and F, Down-regulation of FT and AtGA3ox1 in OsRAV9-E lines compared with wild-type. Flowering time data are the average of 25 plants each genotype, with SEM. Three biological replicates gave similar results, and one was chosen as representative. Data were analyzed by one-way ANOVA fol- lowed by Dunnett’s multiple comparisons test between wild-type and transgenic lines. Asterisks indicate statistical significance (*P , 0,05). Expression data are reported as mean values of three biological replicates; error bar represents the SEM. AtUBQ10 was used for normalization. 1668 Plant Physiol. Vol. 183, 2020 Downloaded from on September 3, 2020 - Published by www.plantphysiol.org Copyright © 2020 American Society of Plant Biologists. All rights reserved. RAVs Regulate Heading Date and Carpel Development Figure 4. Role of OsRAV9 in heading date. A, Diurnal oscillation of Hd3a and OsRAV9 expression in leaves of 4-week-old wild- type (wt) plants. Gray block indicates night. B, Mutually exclusive expression patterns of Hd3a and OsRAV9 in leaves throughout wild-type (wt) plant development. Gray block indicates floral transition. C and D, Scatter plots representing heading date as number of days to flower of wild-type (in gray) and OsRAV9-i plants (in light green) grown under inductive photoperiods (8-h light/16-h dark, 12-h light/12-h dark). Lines represent the median with 95% of confidence interval. E, Early flowering phenotype of a selected OsRAV9 silencing line (RAV9-i 3, right) compared with wild-type (left) 100 d after germination (DAG). F, Close-up view showing wild-type panicle at the booting stage and OsRAV9-i panicle at anthesis. For molecular analyses, plants were grown under 12-h light/12-h dark at 28°C, whereas for heading date plants were grown under different daylengths. Bars 5 10 cm. Expression data are reported as mean values of three biological replicates; error bar represents the SEM. Flowering time data are the average of 18 to 20 plants each genotype. Three biological replicates gave similar results, and one was chosen as representative. Data were analyzed by one-way ANOVA followed by Dunnett’s multiple comparisons test between wild-type and transgenic lines. Asterisks indicate statistical significance ( *P , 0.05 and**P , 0.033). obtained by self-pollination, and three lines with the plants were still at the booting stage (Fig. 4, E and F). These highest silencing of OsRAV9 were characterized. The findings suggested that OsRAV9 functions as floral re- RNAi lines (reported as OsRAV9-i) flowered slightly ear- pressor in rice like TEMs do in Arabidopsis, likely up- lier than the wild-type under inductive photoperiods, stream of the florigen Hd3a under inductive conditions. more clearly under SD than under 12 h of light (Fig. 4, C Therefore, hereafter we refer to OsRAV9 as OsTEM1. and D; Supplemental Fig. S10C), but not under nonin- ductive LD (Supplemental Fig. S10C). Under SD, the down-regulation of OsRAV9 resulted in significantly early OsTEM1 Regulates Floral Activators OsMADS14 and Hd3a flowering plants (Fig. 4C). Under 12-h light/12-h dark regime, transgenic lines flowered on average 90 DAG Based on the results obtained, it was tempting to and underwent anthesis 1 week later, whereas wild-type speculate a role for OsTEM1 in the direct repression of Plant Physiol. Vol. 183, 2020 1669 Downloaded from on September 3, 2020 - Published by www.plantphysiol.org Copyright © 2020 American Society of Plant Biologists. All rights reserved. Osnato et al. Hd3a as a noncanonical RAV binding site is present in the florigen, upstream of Hd3a in the leaf and down- its regulatory region (Fig. 5A). However, we could not stream of the Hd3a/14-3-3/OsFD1 complex in repro- exclude an indirect effect on Hd3a via additional tran- ductive meristems (Kobayashi et al., 2012). OsMADS14 scriptional regulators. Actually, a negative correlation is also expressed in adult leaves, and a perfect RAV also exists between OsTEM1 and additional floral acti- binding site was found in its promoter (Fig. 5B). vators including OsFT-like1 and IM identity genes (Ta- Therefore, we used a transient expression system based ble 1), previously shown to act in a regulatory loop with on a dual Renilla-Luciferase assay to investigate the Figure 5. Interaction between OsRAV9/OsTEM1 and floral activators. A, Noncanonical RAV binding site in the promoter of Hd3a, 785 bp upstream of the transcription starting site. B, Perfect RAV binding site in the promoter of OsMADS14, 2250 bp upstream of the transcription starting site, and mutated version (right). Arrows represent oligonucleotides used to amplify frag- ments of ProHd3a (H) and ProOsMADS14 (M) without RAV binding sites (Ha and Ma, in gray) and with RAV binding sites (Hb and Mb, in green). C to E, Transactivation activity of OsTEM1 in transiently transformed protoplasts, reported as ratio of transcript levels of LUC and REN reporter genes relative to UBQ. C, RT-qPCR of protoplasts cotransformed with Pro-35S:OsTEM1 and reporter vectors containing sequences of ProHd3a (Ha in gray, Hb in green). D, RT-qPCR of protoplasts cotransformed with Pro- 35S:OsTEM1 and reporter vectors containing sequences of ProOsMADS14 (Ma in gray, Mb in green). E, Analysis of protoplasts cotransformed with Pro-35S-OsTEM1 and reporter vectors containing sequences of ProOsMADS14 (Mb in green, mutated Mb in dark green). Values are the mean of three independent replicates. F to H, Expression analysis of genes involved in heading date in independent T2 lines (green) compared with wild-type plants grown for 28, 35, and 42 d under inductive conditions. F, Down- regulation of OsRAV9/OsTEM1 in silencing lines (green). G and H, Up-regulation of the floral activators OsMADS14 and Hd3a in silencing lines (green lines) compared with wild-type (black line). Expression data are mean value of three biological replicates with three technical replicates each, and error bars represent SD. 1670 Plant Physiol. Vol. 183, 2020 Downloaded from on September 3, 2020 - Published by www.plantphysiol.org Copyright © 2020 American Society of Plant Biologists. All rights reserved. RAVs Regulate Heading Date and Carpel Development direct interaction between OsTEM1 and potential target repressor and up-regulation of different floral activators genes. The effector vector Pro-35S:OsTEM1 was tran- in transgenic lines resulted in early flowering phenotype. siently coexpressed with reporter vectors containing different regulatory regions of Hd3a and OsMADS14 (Fig. 5, C and D; Supplemental Fig. S11). We evaluated OsRAV11 and OsRAV12 Regulate Carpel Development transactivation ability of the floral repressor on target promoters by measuring the relative expression of the The fact that OsRAV11/OsRAV12 have diversified LUCIFERASE reporter genes from firefly (LUC) and from OsTEM1 in their expression patterns and coding from renilla (REN) in cell lysates. Despite the biological sequences prompted us to hypothesize that these genes variability between independent replicates, a clear re- might have acquired different roles in plant develop- duction of LUC/REN relative transcript levels was al- ment, likely after floral induction based on their ex- ways observed when OsTEM1 was cotransformed with pression pattern in the reproductive phase (Fig. 2C). A a reporter vector carrying promoter sequences of preliminary spatio-temporal expression analysis indi- OsMADS14 containing the RAV binding site but not cated that OsRAV11 was expressed at early stages of Hd3a (Fig. 5, C and D; Supplemental Fig. S11, B and C). flower development before organ primordia differen- However, this reduction was abolished when we tiation because its mRNA was detected in the spikelet cotransfected the effector vector with a mutated version meristem (Supplemental Fig. S13A). Later on, tran- of the reporter vector carrying the RAV binding site of scripts became first restricted to the carpel primordia the OsMADS14 promoter. For statistical analyses pur- similar to carpel identity genes, and afterward specifi- poses, the values of these three replicates including cally limited to the apical part of the developing gy- intact and mutated RAV binding sites are shown as noecium (Supplemental Fig. S13B). logarithmic values (Fig. 5E; Supplemental Fig. S11D). The analysis of available transcriptomics data sets Therefore, the transient cotransformation assays performed on floral homeotic mutants that revealed soundly suggest that transcription repression mediated OsRAV11 down-regulation in OsMADS1-RNAi inflo- by OsTEM1 is stronger on DNA regulatory sequences rescences (Supplemental Table S2; Khanday et al., of OsMADS14 and is likely mediated by the RAV 2013), and conversely up-regulation in osmads13 mu- binding site. tant (M. Osnato and M.M. Kater, unpublished data), Finally, we monitored the expression levels of OsTEM1 suggested an interaction between RAV and MADS-box and floral activators shortly before and around the genes controlling floral organ development. This could floral transition in wild-type and OsTEM1-i lines grown be a direct effect since we found CArG-boxes, consensus under inductive conditions. As expected, transcripts sequences recognized and bound by MADS-domain levels of OsTEM1 were confirmed to be higher at week 4 TFs, in OsRAV11 and also in OsRAV12 regulatory se- in wild-type plants, and strong down-regulation was quences (Supplemental Fig. S13D). The regulation by observed in the transgenic lines (Fig. 5F). Conversely, OsMADS1 and OsMADS13 is further supported by the the expression of OsMADS14 increased from week 4 to fact that both appear as regulators of OsRAV genes in the 6 in wild-type plants, and a clear up-regulation was Environmental Gene Regulatory Influence Networks detected in OsTEM1-i lines 2 and 3 at 35 DAG (Fig. 5G). (Wilkins et al., 2016). Therefore, OsRAV11 might have a The related IM genes OsMADS15 and OsMADS34 role in the development of the pistil, likely downstream were expressed at extremely low levels in leaves at of class-D and class-E MADS-domain floral homeotic floral transition (Supplemental Fig. S12A); however, factors. OsMADS18 was transcribed at higher levels in vege- Consistently, we found down-regulation of OsRAV11 tative tissues, and alteration of its mRNA abundance in developing panicles of the osmads1 mutant (Fig. 6A), was found in the transgenic line with highest OsRAV9 characterized by the conversion of floral organs into silencing (Supplemental Fig. S12B). Furthermore, con- glume-like structures (Agrawal et al., 2005; Khanday siderable increase in Hd3a mRNA levels was also et al., 2013), and up-regulation in young panicles of the detected in silencing lines around floral transition floral homeotic mutants osmads16 and osmads13 (Fig. 6B), (Fig. 5H). Taken together, transient cotransformation in which stamens and ovules are homeotically converted of protoplasts and comparative expression analysis into extra carpels respectively (Nagasawa et al., 2003; of wild-type and transgenic RNAi lines suggest that Dreni et al., 2007) perhaps because the ectopic activatio OsTEM1 controls heading date via direct repres- of OsRAV11. Accordingly, we observed strong upre- sion of OsMADS14, although it also modulates Hd3a gulation of OsRAV11 specifically in osmads13 ovule expression. primordia (Fig. 6C; M. Osnato and M.M. Kater, un- We also tested the effect of the down-regulation of published data), which later develop as carpels. OsTEM1 on other genetic pathways involved in the Therefore, we explored the function of OsRAV11 control of heading date (Supplemental Fig. S12C), using a knock-out mutant characterized by the inser- and, although variable, we found up-regulation of the tion of T-DNA in the 59 untranslated region of the gene flowering inductors OsMADS50, OsMADS51, and Ehd1 (Fig. 6, D and E). The loss of OsRAV11 function did not in silencing lines (Supplemental Fig. S12, D and E), cause alteration of heading date, but elongated carpels perhaps suggesting additional roles in parallel path- (Fig. 6F). A detailed phenotypic analysis by SEM upon ways. As a consequence, down-regulation of the OsTEM1 fertilization indicated alteration in size and shape of the Plant Physiol. Vol. 183, 2020 1671 Downloaded from on September 3, 2020 - Published by www.plantphysiol.org Copyright © 2020 American Society of Plant Biologists. All rights reserved. Osnato et al. Figure 6. Molecular and functional characterization of OsRAV11. A to C, Misregulation of OsRAV11 in floral homeotic mutants. A, Down-regulation of OsRAV11 in osmads1 developing inflorescences. B, Up-regulation of OsRAV11 in osmads16 and osmads13 developing inflorescences. C, Strong activation of OsRAV11 in specific cell types (floral meristem and ovule primordia) isolated from osmads13 mutant flowers. D, Schematic representation of the T-DNA insertion in the 59 untranslated region of OsRAV11. Arrows indicate primers used for genotyping. E, Down-regulation of OsRAV11 in pistils dissected from osrav11 mutant flowers at maturity. F to H, Morphological analyses of female reproductive structures at maturity. F, Representative images of wild-type and osrav11 carpels dissected from mature flowers at anthesis obtained by optical microscopy. G and H, Repre- sentative images of reproductive structures obtained by scanning electron microscopy (SEM). G, Wild-type and osrav11 carpels upon fertilization. Glumes were partly removed to show female reproductive organs. H, Apical tissues of wild-type and osrav11 gynoecia after pollination. Bars 5 1 mm (F), 500 mm (G), and 100 mm (H). I, Bar plots representing the weight of seed produced by wild-type (in gray) and osrav11 (in white) plants grown in the greenhouse under inductive conditions. Expression data are reported as mean value of three biological replicates; error bar represents the SEM. Phenotypic data are the average of three bi- ological replicates of the weight of 100 seeds each genotype, with SEM. Statistical significance was examined by Student’s two- tailed unpaired t test (*P , 0.05). 1672 Plant Physiol. Vol. 183, 2020 Downloaded from on September 3, 2020 - Published by www.plantphysiol.org Copyright © 2020 American Society of Plant Biologists. All rights reserved. RAVs Regulate Heading Date and Carpel Development pistil, with an enlarged ovary compared to wild-type (Fig. 7C), indicating that these two genes redundantly pistils (Fig. 6G). Specifically, differentiation of apical regulate the basal-apical patterning of the gynoecium. tissues of the gynoecium were altered in the osrav11 Interestingly, flowers of these transgenic lines pro- mutant as carpel tissues did not fuse (Fig. 6H). Like- duced viable pollen, but most of the ovules were not wise, we observed alteration of seed morphology fertilized, resulting in very poor seedset. Consequently, (Supplemental Fig. S13): mutant plants produced seeds most of the transgenic lines displayed severe fertility with increased seed length and decreased seed width defects (Fig. 7D). (Supplemental Fig. S3G), resulting in statistically sig- To conclude, molecular and functional characteriza- nificant alterations of length-to-width ratio and circu- tion of OsRAV11 and OsRAV12 suggest that these two larity (Supplemental Fig. S3H). Interestingly, we genes might redundantly regulate the differentiation of observed a slight increase in seed weight after de- the female reproductive structures. Intriguingly, a de- husking (Fig. 6I). creased activity of OsRAV11 correlated with an increase Ultimately, to investigate the possible redundancy in seed weight, whereas the loss of OsRAV11 and between OsRAV11 and the closely related OsRAV12 OsRAV12 activity associates with sterility problems. gene in the formation of female reproductive organs, we generated transgenic rice plants in which the ex- pression levels of both genes were reduced by RNA interference (Supplemental Fig. S14). Vegetative growth DISCUSSION of knock-down lines (from now on OsRAV11/12-i) was Arabidopsis and Rice RAV Genes Are Closely Related normal, whereas flowers showed alterations caused by abnormal morphology of female reproductive organs RAV proteins belong to the plant-specific B3 super- (Fig. 7A). Indeed, elongated cylindrical pistils with family of TFs (Swaminathan et al., 2008), and are highly reduced stigmas and enlarged ovaries (Fig. 7B) characterized by an additional AP2 DNA binding do- were observed in transgenic plants with reduced levels main at the N terminus. AP2-B3 type proteins were not of both OsRAV11 and OsRAV12 in the gynoecium found in the green algae Chlamydomonas reinhardtii, but Figure 7. Morphogenetic effects of OsRAV11/OsRAV12 silencing in re- productive phase. A, Representative images of mature flowers dissected from wild-type (wt) and OsRAV11/12-i inflorescences. Arrows indicate the position of the stigmas. B, Representa- tive images of mature carpels dissected from wild-type and one T3 OsRAV11/ 12-I flowers obtained by optical mi- croscopy. Bars 51 mm. C, Down- regulation of OsRAV11 and OsRAV12 in mature pistils of two representative T3 OsRAV11/12-i lines as compared with wild-type. Expression data are reported as mean values of three bio- logical replicates with SEM. Data were analyzed by ordinary one-way ANOVA followed by Dunnett’s multiple com- parisons test between wild-type and transgenic lines. Asterisks indicate sta- tistical significance (*P , 0.05 and **P , 0.033). D, Fertility defects of mature OsRAV11/12-i panicles com- pared with wild-type. Plant Physiol. Vol. 183, 2020 1673 Downloaded from on September 3, 2020 - Published by www.plantphysiol.org Copyright © 2020 American Society of Plant Biologists. All rights reserved. Osnato et al. they appeared early in the evolution of land plant The mechanism of action seems to be different in the species. The presence of two DNA binding domains two species. To avoid precocious flowering, Arabi- suggests that these TFs achieve high affinity by specif- dopsis TEMs directly target the florigens FT (Castillejo ically binding bipartite sequences in regulatory regions and Pelaz, 2008) and AtGA3ox1/2 (Osnato et al., 2012), of downstream targets (Kagaya et al., 1999; Castillejo whereas OsTEM1 might regulate Hd3a indirectly via and Pelaz, 2008; Osnato et al., 2012; Matías-Hernández repression of OsMADS14 as proposed in Figure 8A. et al., 2016). Although this AP1/FUL-like gene is expressed at high In this study, we focused on the four rice RAV genes, levels in reproductive tissues, its mRNA accumulates which display striking similarities with four RAV genes also during the vegetative phase. Accordingly, an ad- of Arabidopsis (Supplemental Fig. S15) that were al- ditional role for OsMADS14 as activator of the florigen ready described as regulators of different aspects of in the leaf has been previously proposed, likely acting plant development and stress responses. Specifically, via a positive regulatory loop with Hd3a (Kobayashi AtRAV1 and AtRAV1-like redundantly control leaf et al., 2012). Actually, knock-down lines silencing senescence (Woo et al., 2010), AtRAV1 and TEM2 OsMADS14, OsMADS15, OsMADS18, and OsMADS34 modulate sensitivity to drought and salinity (Fu et al., display delayed flowering (Kobayashi et al., 2012), 2014), TEM1 and TEM2 repress trichome formation and conversely transgenic rice ectopically expressing (Matías-Hernández et al., 2016), and floral transi- OsMADS14 and OsMADS18 are early flowering (Jeon tion under inductive and noninductive photoperiods et al., 2000; Fornara et al., 2004). A very recent study (Castillejo and Pelaz, 2008; Osnato et al., 2012) as well as also indicates that AP1-like genes could regulate in response to low temperatures (Marín-González et al., drought-escape; indeed, the early flowering phenotype 2015) and to plant age (Aguilar-Jaramillo et al., 2019). under drought conditions correlates with increased Interestingly, preliminary coexpression analyses sug- expression of OsMADS18 (Groen et al., 2020). gested that OsRAV9 and OsRAV11 could act in signal Upon floral transition, the FAC activates the expression transduction pathways activated in response to abiotic of OsMADS14, together with OsMADS15, OsMADS18, stresses. Nevertheless, OsRAV9 and the closely related and OsMADS34, which specify IM identity and trigger OsRAV8 could also act in Gene Regulatory Networks the development of reproductive structures (Kobayashi controlling the transition from vegetative to reproduc- et al., 2010; Kobayashi et al., 2012). Based on tran- tive growth in the leaf and in the apical meristem, scriptomics analysis, we speculate that OsRAV8 could respectively. play a role in the maintenance of the vegetative state of the apical meristem, thus preventing the formation of the rachis under noninductive conditions. OsRAV9/OsTEM1 Is a New Player in Flowering Although phylogenomics and phylogenetic analy- Mechanisms Controlling Floral Transition in Cereals ses revealed that OsRAV genes might have originated from duplication events from a common ancestor It has long been known that AP1/FUL-like proteins after speciation and separation of monocots and di- control seasonal flowering in cereals growing in tem- cots, it is difficult to draw conclusions from orthology perate regions (Fjellheim et al., 2014). Precisely, the with AtRAVs. Furthermore, the expression domains floral transition is triggered by the activation of VER- of the four OsRAV paralogous genes appear to have NALIZATION 1-like (VRN1-like) and FT-like genes in diversified likely due to polymorphisms in their wheat leaves in response to increasing day-length and regulatory regions. Regardless of the remarkable prolonged exposure to low temperatures (Danyluk similarities in the gene structures and coding se- et al., 2003; Shimada et al., 2009; Winfield et al., 2009). quences, OsRAV8 and OsRAV9 are expressed in dif- At least in wheat, VRN1 regulates flowering by directly ferent tissues. Indeed, OsRAV9 mRNA is abundant in binding the promoter of the downstream target FT-like1 juvenile leaves, and its reduction marks the transition (Deng et al., 2015; Tanaka et al., 2018). Surprisingly, the to the adult phase when plants acquire the compe- function of AP1/FUL-like proteins as floral activators tence to flower, whereas the transcripts of OsRAV8 acting in leaves seems to be well conserved in rice (Jeon are detected in the apical meristem and its levels also et al., 2000; Kobayashi et al., 2012), despite the fact that decrease at floral transition when IM identity genes it is a cereal crop of tropical origin that does not require are activated (Gómez-Ariza et al., 2019). Therefore, at vernalization. In this study, we propose a new mecha- least OsRAV9 displayed an expression pattern that nism that is largely independent from previously de- resembles that of TEM genes in Arabidopsis. Sup- scribed molecular networks determining heading date in porting the functional conservation, the ectopic ex- rice (Tsuji et al., 2013). Interestingly, the presence of TEM pression of OsRAV9 in Arabidopsis plants correlated orthologs in the Poacea family (Supplemental Fig. S1) with the repression of the TEM targets FT and opens up the intriguing possibility that the RAV-AP1-FT AtGA30X1 and delayed flowering time. In addition, regulatory module could be conserved among tropical silencing of OsRAV9/OsTEM1 in transgenic rice lines (Oryza spp. tribe) and temperate (Triticeae tribe) cereals. resulted in early flowering due to up-regulation of the Moreover, as mentioned above, AtRAV1 and TEM2 floral activators OsMADS14 and Hd3a. seem to modulate sensitivity to drought and salinity 1674 Plant Physiol. Vol. 183, 2020 Downloaded from on September 3, 2020 - Published by www.plantphysiol.org Copyright © 2020 American Society of Plant Biologists. All rights reserved. RAVs Regulate Heading Date and Carpel Development Figure 8. Model for interaction between RAV and MADS factors during reproduc- tive growth. A, OsRAV9 represses the transcription of OsMADS14, a positive regulator of the florigen Hd3a, in the leaf. Upon floral transition, the complex Hd3a- OsFD activates the expression of IM iden- tity genes OsMADS14-15-18-34 in the apical meristem. B, The expression of OsMADS1 in developing flowers marks the formation of lemma/palea (in green) and central carpel (in pink) from the FM, whereas the presence of OsMADS16 and OsMADS13 prevents the expression of genes involved in carpel development in stamen primordia (in yellow) and in ovule primordium (in violet). (Fu et al., 2014). OsTEM1 has been recently shown to floral homeotic MADS-box genes as well as down- play a role in response to abiotic stresses (OsRAV2; regulation of genes encoding B3-type TFs, including Duan et al., 2016), and OsTEM1 expression was re- OsARFs and OsRAV11 (Supplemental Table S2). Pre- duced in plants growing under drought conditions cisely, the ARF-type TFs OsETTIN1 and - OsETTIN2 (Plessis et al., 2015) where the transcription of control the differentiation of apical tissues of the carpel, OsMADS18 was increased (Groen et al., 2020). There- and at least OsETTIN2 is directly regulated by fore, RAV genes could be involved in adaptive growth OsMADS1 (Khanday et al., 2013). Intriguingly, the by modulating heading date in response to environ- aberrant carpel morphology of loss of OsETTINs mental limitations and fluctuations, by integrating ex- (Khanday et al., 2013) is similar to that of transgenic ternal and internal physiological conditions. lines with reduced level of OsRAV11 and OsRAV12 (Fig. 7B). Mechanisms Controlling Floral Organ Development in Rice OsRAV11 and OsRAV12 Regulate the Development of Gynoecium In the last decade, genetic and functional genomics analyses in different plant species revealed that flower In Arabidopsis, the early flowering TEM loss of development is governed by a complex framework function mutants do not display evident alterations of based on MADS-domain TFs. Rice members of the flower development. Nonetheless, the late flowering grass-specific LOF-SEP clade sequentially regulate dif- TEM overexpressing lines show fertility defects and ferent steps of flower development upon the vegeta- produce shorter siliques containing fewer seeds. This tive to reproductive phase change. First, OsMADS34 phenotype could be related to decreased content of GA, forms tetrameric complex with the AP1-like factors or alternatively to misregulation of genes involved in OsMADS14 and OsMADS15 in the IM to initiate in- the regulation of later organ development. In rice, fur- florescence branch meristem primordia development ther molecular analyses suggest that RAV genes might from which secondary branches and spikelets differ- have acquired additional functions at later stages of entiate (Kobayashi et al., 2012). After the formation of vegetative and reproductive growth. Indeed, OsRAV11 rudimentary glumes and sterile lemmas, the spikelet and OsRAV12 are highly expressed not only in mature meristem is converted into floret meristem, which leaves at ripening, indicating a possible regulatory role produces different floral organs. Another grass-specific in leaf senescence similarly to AtRAV1, but also in the LOF-SEP factor, OsMADS1, plays a central role in the gynoecium before fertilization. Actually, functional determination of floral organ identity, as it interacts characterization of knock-down and knock-out mu- physically and genetically with AP3-like and AG-like tants points at a new function for OsRAV11 and factors, which are involved in the development of male OsRAV12 in the correct formation of female reproduc- and female reproductive organs (Prasad et al., 2001; tive organs. Indeed, the whole basal-apical pattern was Li et al., 2011; Khanday et al., 2013, 2016). Recently, distorted; the ovary was enlarged in osrav11 and mis- transcriptomics analysis performed on OsMADS1 knock- shapen in OsRAV11/12-i plants. Down-regulation of down panicles at very early stages of flower develop- both genes resulted in reduced stigmas and larger car- ment (Khanday et al., 2013) unveiled misregulation of pels than in single osrav11 mutants, which may suggest Plant Physiol. Vol. 183, 2020 1675 Downloaded from on September 3, 2020 - Published by www.plantphysiol.org Copyright © 2020 American Society of Plant Biologists. All rights reserved. Osnato et al. a redundant function of these genes in carpel devel- test for branch support (http://phylogeny.lirmm.fr/phylo_cgi/one_task.cgi? task_type5phyml), and TreeDyn for tree visualization (http://www. opment and differentiation. Therefore, we hypothesize phylogeny.fr/one_task.cgi?task_type5treedyn). Sequence Diversity Diagram a role for OsRAV11 and OsRAV12 in the determination (SeDD; http://vda-lab.github.io/sedd.html) was used to compare two sets of of the basal-apical pattern of the pistil, perhaps in par- RAV protein sequences from Arabidopsis and Oryza sativa and visualize con- allel with ARFs. Furthermore, we propose that at least served versus diversified positions in the two species. Motif clustering analysis OsRAV11 may control the differentiation of the gy- was carried out using SALAD version 3 (https://salad.dna.affrc.go.jp/salad/ en/) with OsRAV9 sequence, and the prediction of functional and structural noecium downstream of MADS-domain TFs (Fig. 8B), motifs in RAV protein sequences via web-based tools of ExPAsY (https:// due to its mis-regulation in the floral homeotic mutants prosite.expasy.org/scanprosite; de Castro et al., 2006). osmads1, osmads13 and osmads16 (Fig. 6, A and C). Besides the specification of the identity of different Expression Profiles floral organs, OsMADS1 has been proposed as a key trait of agronomical interest. During spikelet develop- Expression profiles of rice RAV genes were inferred from a large collection of ment, OsMADS1 also interacts with Gg subunits of GS3 microarray data derived from different tissues at different developmental stages under natural field conditions (http://ricexpro.dna.affrc.go.jp/). Coex- and DEP1 (Fan et al., 2006; Huang et al., 2009; Liu et al., pression analyses were carried out by using OsRAV9 as single guide genes and 2018), which regulate its transcriptional activity on a searching RiceFREND (http://ricefrend.dna.affrc.go.jp/), the Plant Coex- common set of target genes involved in the determi- pression Database (PLANEX; Yim et al., 2013), and the Rice Oligonucleotide nation of seed size and shape. The dominant negative Array Database (Cao et al., 2012). mutation osmads1(lgy3) causes an alternatively spliced protein variant and correlates with more slender grain, Plant Material and Growth Conditions as the mutated protein promotes cell proliferation in Wild-type and transgenic Arabidopsis (Col-0 background) seeds were sown longitudinal direction (Liu et al., 2018). Pyramiding of on soil pots, and plants were grown under LD (16-h light/8-h dark at 22°C) lgy3 and dep1-1 alleles in a japonica cultivar resulted not until maturity. Wild-type and transgenic rice (O. sativa ssp. japonica) seeds were only in a 10% increase in grain yield, but also improved surface sterilized and sown on Murashige Skoog medium with 30 g L21 Suc. grain length-to-width ratio and grain chalkiness (Liu After germination, seedlings were transferred to soil pots and grown under controlled conditions until maturity (SD: 8-h light at 28°C, 16-h dark at 24°C; et al., 2018). We can hypothesize that the elongated 12/12: 12-h light at 28°C, 12-h dark at 24°C or LD; LD:16-h light at 28°C, 8-h seed phenotype associated to down-regulation of dark at 24°C). For functional characterization of OsRAV genes, transgenic lines OsMADS1 (Liu et al., 2018) could be mediated through (cv Nipponbare) and insertion mutant (PFG_2A-10680, cv Hwayoung) were the down-regulation of B3 genes including OsRAV11 used. For expression analysis, segregating progenies of floral homeotic mutants (cv Dongjin) were genotyped (Supplemental Table S3), grown for 10 weeks in since a similar slender phenotype is observed in osrav11 LD and then transferred to inductive conditions. Pools of developing inflores- and OsRAV11/12-i carpels. Further analyses are re- cences were harvested 3 weeks after floral transition from homozygous mutants quired to understand the interactions with putative (osmads1, osmads13, osmads16) and wild-type plants. upstream regulators, interacting proteins and down- stream targets constituting the molecular network that Cloning and Generation of Arabidopsis and Rice regulates the development of the gynoecium in rice. Transgenic Plants The coding sequence of OsRAV9/OsTEM1 was amplified using primer sets SPp538-SPp541, subcloned in pCRII and pENTR-3C by restriction/ligation, MATERIALS AND METHODS and introduced in pALLIGATOR2 vector downstream of Pro-35S by Gateway technology (Supplemental Table S4). Arabidopsis plants (Col-0, tem1-tem2) RAV Sequence Analyses were transformed with the Pro-35S:OsTEM1 construct by floral dip, and GFP- positive seeds were selected by fluorescence microscopy. For the generation of OsRAV8 and AtTEM1 were used as queries in phylogenomics analyses in the RNAi constructs, the Gene Sequence Tags specific for the 39 ends of sequenced land plants species (eudicotyledons, monocotyledones, Ambor- OsRAV9/OsTEM1 and OsRAV11 were amplified using primer sets SPp516- ellales) using gene tree tool of Pan-taxonomic Compara (http://www.gramene. SPp539 and SPp527-SPp537, respectively, then subcloned in pCRII, and after- org/). TEM1 protein sequence was used as query in a BLAST-P (https://blast. ward cloned in pENTR-3C by restriction/ligation (as KpnI-EcoRV fragments). ncbi.nlm.nih.gov/Blast.cgi?LINK_- The resulting constructs were digested with PvuI before LR recombination to LOC5blasthome&PAGE_TYPE5BlastSearch&PROGRAM5blastp) search pBios-378 plant expression vector. Scutellum-derived rice calli (‘Nipponbare’) against the proteomes of Arabidopsis (Arabidopsis thaliana) and rice (Oryza were transformed by Agrobacterium spp. cocultivation. Independent transfor- sativa). mation events were selected, 11 for OsRAV9 and 7 for OsRAV11 constructs, TF binding sites (CArG box for MADS-domain proteins, consensus sequence regenerated and propagated. Plants that underwent regeneration but did not composed of CAACA and CCTG elements at a distance of 3-9 nucleotides for contain the transgenic cassette were used as transformation control. The RAV proteins) were searched in the regulatory regions of genes of interest by primers used for genotyping and cloning are listed in Supplemental Tables S3 using the Promoter Analysis tool of Plant PAN3.0 (http://plantpan.itps.ncku. and S4. edu.tw/index.html). Gene and protein sequences were retrieved from The Arabidopsis Infor- mation Resource (www.arabidopsis.org) and GRAMENE (www.gramene.org). Direct Binding of OsTEM1 to Downstream Genes To generate the set of reporter vectors, different promoter regions of Phylogenetic Analyses OsMADS14 and Hd3a were cloned as SalI-PstI fragments in a modified pGreenII 0800-LUC carrying Pro-35S:LUC and Pro-35S:REN (as internal control to esti- Analysis of phylogenetic relationships between 24 RAV-related full-length mate the proportion of transformed protoplasts). Primers SPp1764- SPp1765 protein sequences and construction of phylogenetic tree were performed using were designed to introduce mutations at the RAV binding site contained in tools available at http://www.phylogeny.fr/ (Dereeper at al., 2008). MUSCLE ProOsMADS14 by PCR-based method. The corresponding reporter vector was was used for protein sequences alignment, and G-blocks for a more stringent used as template for site-directed mutagenesis (15 cycles: 10at 98°C, 20at 66°C, selection (https://www.ebi.ac.uk/Tools/msa/muscle). PhyML was used for 20at 66°C), and the resulting vector used in transactivation assays. Protoplasts phylogenetic analysis with bootstrapping procedure (n 5 100) as statistical were isolated from calli by digesting the cell wall with Macerozyme R-10 and 1676 Plant Physiol. Vol. 183, 2020 Downloaded from on September 3, 2020 - Published by www.plantphysiol.org Copyright © 2020 American Society of Plant Biologists. All rights reserved. RAVs Regulate Heading Date and Carpel Development Cellulase (Yakult Pharmaceuticals), and transfected with different combina- identifiers: LOC_Os03g54160 (OsMADS14), LOC_Os07g01820 (OsMADS15), tions of reporter Pro-35S:OsTEM1 and effector constructs using polyethylene LOC_Os07g41370 (OsMADS18), LOC_Os03g54170 (OsMADS34), LOC_Os03g03100 glycol. After 18 h incubation in darkness at 24°C, transformed protoplasts were (OsMADS50/OsSOC1), LOC_Os01g69850 (OsMADS51). pelleted and resuspended in homogenization buffer for RNA extraction. Transactivation activity of OsTEM1, based on the relative ratio of mRNA a- bundance of LUC and REN reporter genes, was assessed by RT-qPCR. The SUPPLEMENTAL DATA primers used for cloning and expression analyses are listed in Supplemental Tables S3 and S4. The following supplemental materials are available. Supplemental Figure S1. Phylogenomic analysis of TEM homologs in se- quenced land plants. RNA Extraction and Expression Analyses Supplemental Figure S2. Analysis of genes encoding AP2-B3 (RAV) For expression analyses in Arabidopsis, pools of 20 seedlings grown for proteins. 1 week under LD were collected at ZT12. For expression analyses in rice, pools of Supplemental Figure S3. Similarity clustering based on distribution pat- 10 to 15 samples from different tissues and/or developmental stages were terns of known conserved motifs in RAV proteins. collected at ZT13 unless otherwise stated. RNA was extracted with PureLink RNA mini kit (Ambion) and treated with DNaseI RNase free (Ambion). For Supplemental Figure S4. Similarity clustering based on distribution pat- large-scale experiments, RNA was extracted with Maxwell RSC Plant RNA kit terns of conserved features related to posttranslational modification. (Promega), and DNase treatment was performed on-column. Then 1 mg of DNase-treated RNA was retro-transcribed with SuperScript III (Invitrogen), Supplemental Figure S5. Analysis of RAV genes in O. sativa ssp. japonica. and cDNA was used for RT-qPCR with Light Cycler 480 SYBR Green I master Supplemental Figure S6. GO analysis of genes strongly coexpressed with on Light Cycler 480 II (Roche). Three biological replicates and three technical OsRAV9. replicates were performed. In situ hybridization was performed as previously reported (Dreni et al., 2007). The primers used for expression analyses are listed Supplemental Figure S7. GO test of genes negatively correlated with in Supplemental Table S5. OsRAV9 and expression analysis of genes acting in apical meristems at floral transition in rice. Supplemental Figure S8. Inferred expression profiles of OsRAV genes Phenotypic analyses during plant development. Morphological analysis of reproductive structures was performed by Supplemental Figure S9. Analysis of T1 and T2 transgenic lines ectopically Optical Microscopy (Olympus DP71) and SEM. For SEM, flowers at anthesis expressing OsRAV9 and OsRAV11 in Arabidopsis. were fixed in 2.5% (v/v) glutaraldehyde in 0.1 M P-buffer (pH 7.4) overnight at 4°C, washed 4 times for 10 min in 0.1 M P-buffer, postfixed in 1% osmium Supplemental Figure S10. Molecular and phenotypic analysis of T1 trans- tetraoxide with 0.7% ferrocyanide in P-buffer, washed in water, dehydrated genic rice lines silencing OsRAV9. in an ascending ethanol series (50%, 70%, 80%, 90%, and 95% for 10 min each Supplemental Figure S11. Transactivation activity of OsTEM1 on floral and twice with 100% ethanol), and dried by critical-point drying with CO2. activators in rice protoplasts. Alteration on the morphology of the seed were analyzed by using the Smart- grain software (Tanabata et al., 2012). At least 100 seeds per genotype (for Supplemental Figure S12. Expression analyses of genes involved in the three independent biological replicates) were spread uniformly on the glass floral transition in OsRAV9-i rice lines. with a black background, and images were captured with HP Scanner at 300 Supplemental Figure S13. Analyses of OsRAV11 and OsRAV12. dpi. The software determined seed shape parameters such as seed length, width, perimeter, and area, and also calculated length-to-width ratio and Supplemental Figure S14. Molecular characterization of OsRAV11 and circularity. OsRAV12. Supplemental Figure S15. Analysis of RAV proteins from Arabidopsis and Statistical Analyses O. sativa. All statistical analyses are shown in Supplemental Table S6. Statistical sig- Supplemental Table S1. List of RAV genes in O. sativa reported by nificance of each experiment was determined by using GraphPad Prism 7 Swaminathan et al. (2008). (https://www.graphpad.com/scientific-software/prism). For flowering time Supplemental Table S2. List of genes encoding B3-domain TFs down- phenotype, we chose one-way ANOVA; multiple comparisons were then cor- regulated in OsMADS1 knock-down panicles (modified from Khanday rected with Dunnet’s or Dunn’s tests. For other kind of data, we compared two et al., 2013). columns by using unpaired Student’s two-tailed t test, with confidence interval at 95%. Supplemental Table S3. List of primers used for genotyping. Following instructions of GraphPad Prism, we first transformed ratios in log Supplemental Table S4. List of primers used for cloning. of ratios (Y 5 log(Y)), for Figure 5 and Supplemental Figure S11. Then we created a column data table and entered two columns of data (control – Supplemental Table S5. List of primers used for expression analyses. OsTEM1, 1OsTEM1) with matched values on the same row. We chose t tests Supplemental Table S6. Statistical analyses. from the list of column analyses and selected conditions as follows: Experi- mental design: Paired; Assume Gaussian distribution: Yes; Choose test: Ratio Supplemental Dataset S1. Coexpression analyses paired t test. On the second tab of the t test dialog, we chose to compute 1OsTEM1 versus 2OsTEM1. ACKNOWLEDGMENTS Accession Numbers We thank Dr. Marti Bernardo, Dr. Jordi Morata, and Dr. Victor Gonzalez for advice on bioinformatic analyses. Dr. Sebastien Santini for the Phylogeny.fr tool. Sequence data from this article can be found in the GenBank/EMBL data Elia Lacchini for technical help with set-up of genotyping of osmads1 mutants. libraries under the following accession numbers. AtRAV locus identifiers: Dr. Ludovico Dreni for help with rice transgenic plants. Pilar Fontanet for ad- AT1G13260 (AtRAV1), AT3G25730 (AtRAV1-like), At1g25560 (AtRAV2-like/ vice and technical help on protoplast co-transformation assays. Dr. Paula TEM1), AT1G68840 (RAV2/TEM2), At1g50680 (AtRAV3), At1g51120 Suárez-López for critical reading of the manuscript. We also thank the Servei (AtRAV3-like). OsRAV locus identifiers: LOC_Os10g39190 (OsRAV2), de Microscopia at the Universitat Autònoma de Barcelona for help with LOC_Os08g06120 (OsRAV3), LOC_Os03g02900 (OsRAV4), LOC_Os04g49230 SEM. A.E.A.-J. performed this work within the frame of a Ph.D. Program of (OsRAV5), LOC_Os02g45850 (OsRAV6), LOC_Os11g05740 (OsRAV7), the Universitat Autònoma de Barcelona. LOC_Os01g04750 (OsRAV8), LOC_Os01g04800 (OsRAV9/OsTEM1), LOC_Os01g49830 (OsRAV11), LOC_Os05g47650 (OsRAV12). OsMADS locus Received May 5, 2020; accepted June 3, 2020; published June 18, 2020. Plant Physiol. Vol. 183, 2020 1677 Downloaded from on September 3, 2020 - Published by www.plantphysiol.org Copyright © 2020 American Society of Plant Biologists. All rights reserved. Osnato et al. 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