Three TOB1-related YABBY genes are required to maintain proper function of the spikelet and branch meristems in rice

Transcription

1 Research Three TOB1-related YABBY genes are required to maintain proper function of the spikelet and branch meristems in rice Wakana Tanaka, Taiyo Toriba and Hiro-Yuki Hirano Department of Biological Sciences, School of Science, The University of Tokyo, Bunkyo-ku, Tokyo , Japan Authors for correspondence: Hiro-Yuki Hirano Tel: hyhirano@bs.s.u-tokyo.ac.jp Wakana Tanaka Tel: tanaka-wakana@bs.s.u-tokyo.ac.jp Received: 28 February 2017 Accepted: 19 April 2017 doi: /nph Key words: flower development, grass, inflorescence, meristem maintenance, Oryza sativa (rice), spikelet, TONGARI-BOUSHI (TOB), YABBY gene. Summary YABBY genes play important roles in the development of lateral organs such as leaves and floral organs in Angiosperms. However, the function of YABBY genes is poorly understood in monocots. We focused on three rice (Oryza sativa) YABBY genes, TONGARI-BOUSHI (TOB1, TOB2, TOB3), which are closely related to Arabidopsis (Arabidopsis thaliana) FILAMENTOUS FLOWER (FIL). To elucidate the function of these YABBY genes, we employed a reverse genetic approach. TOB genes were expressed in bract and lateral organ primordia, but not in meristems. RNAi knockdown of TOB2 or TOB3 in the tob1 mutant caused abnormal spikelet development. Furthermore, simultaneous knockdown of both TOB2 and TOB3 in tob1 affected not only spikelet, but also inflorescence development. In severe cases, the inflorescences comprised naked branches without spikelets. Analysis of inflorescence development at an early stage showed that the observed phenotypic defects were closely associated with a failure to initiate and maintain reproductive meristems. These results indicate that the TOB genes regulate the maintenance and fate of all reproductive meristems. It is likely that the function of FIL/TOB clade YABBY genes has been conserved between Arabidopsis and rice to maintain the proper function of meristems, even though these genes are expressed in lateral organ primordia. Introduction Plant development depends on the function of the shoot apical meristem (SAM) and root apical meristem, which are established during embryogenesis. Pluripotent stem cells, which are maintained in the apical region of the SAM, are the ultimate source of the aboveground plant body. Cells supplied from the stem cells have specified fates and differentiate into lateral organs, such as leaves and floral organs, at the peripheral region of the meristem. After cell fate is specified for differentiation, communication between the meristem and lateral organ primordia is also required for lateral organ differentiation. For example, the adaxial cell fate of leaf primordia is determined by an unknown signal from the meristem (Sussex, 1951; Reinhardt et al., 2005). In Arabidopsis (Arabidopsis thaliana), YABBY genes are involved in various aspects of development, such as differentiation and patterning of the leaf, and development of floral organs including carpel and ovules (Bowman & Smyth, 1999; Sawa et al., 1999; Siegfried et al., 1999; Villanueva et al., 1999). Early studies suggested that YABBY genes, such as FILAMENTOUS FLOWER (FIL)andYABBY3 (YAB3), are involved in the specification of abaxial cell fates in lateral organs (Sawa et al., 1999; Siegfried et al., 1999). However, recent reports on multiple mutants of FIL-related YABBY genes have revealed that these YABBY genes play a more general role in leaf development, such as specification of both abaxial and adaxial identity, and promotion of lamina expansion, (Stahle et al., 2009; Sarojam et al., 2010). The general involvement of YABBY genes in leaf development is also indicated by studies in Antirrhinum (Antirrhinum majus) (Golz et al., 2004; Navarro et al., 2004). The function of YABBY genes is also associated with meristem activity. Ectopic SAM and axillary meristems are formed in the fil yab3 mutant (Kumaran et al., 2002). Expansion of CLAVATA3 (CLV3) and WUSCHEL (WUS), which are associated with stem cell activity, is observed in the fil or fil yab3 mutant (Goldshmidt et al., 2008). An enlargement of the SAM is observed in the fil mutant of Arabidopsis on a Columbia background (Lugassi et al., 2010). By contrast, the activity of the SAM is not maintained in triple or quadruple mutants of YABBY genes in the FIL clade (Sarojam et al., 2010). YABBY genes are expressed in the lateral organs and not in the meristem per se (Sawa et al., 1999; Siegfried et al., 1999; Goldshmidt et al., 2008; Sarojam et al., 2010). Therefore, the defects in the meristem observed in multiple yabby mutants seem to be the consequence of noncell-autonomous activity of YABBY genes. The noncellautonomous action of YABBY genes has also been suggested in an Antirrhinum mutant that showed defects in the adaxial domain of the leaf, which does not express the YABBY gene (Golz 825

2 826 Research New Phytologist et al., 2004). Furthermore, it also has been demonstrated that the Arabidopsis FIL protein does not move between cells, suggesting that this YABBY gene is involved in producing a signal that communicates between the lateral organs and the meristem (Goldshmidt et al., 2008). In addition, it is likely that the noncell-autonomous action of YABBY genes is mediated by LATERAL SUPPRESSOR in Arabidopsis (Goldshmidt et al., 2008). Thus, bidirectional communication between the meristem and lateral organs is required for proper meristem maintenance and lateral organ development in eudicots. In rice (Oryza sativa), the function of YABBY genes has been studied mainly with respect to reproductive development (Yamaguchi et al., 2004; Tanaka et al., 2012a,b). In rice, the loss-offunction of TONGARI-BOUSHI1 (TOB1), a member of the FIL clade of the YABBY gene family, results in various defects in spikelet development, such as depressed growth of the lemma and palea, and formation of a cone-like seamless organ instead of the lemma and palea (Tanaka et al., 2012b). Unlike Arabidopsis YABBY genes, which show a polar expression pattern localized to the abaxial domain, TOB1 is uniformly expressed in spikelet organs such as the lemma and palea without showing a polarized expression pattern. DROOPING LEAF (DL) plays a crucial role in carpel specification, whereas its Arabidopsis ortholog CRABS CLAW (CRC) does not have such a function, instead being involved in the elaboration of carpel morphology (Bowman & Smyth, 1999; Yamaguchi et al., 2004). DL also shows a uniform expression pattern in the carpel primordia, in contrast to the abaxially localized expression of CRC in developing carpels. In maize, the YABBY gene related to Arabidopsis FIL and rice TOB1 is expressed in the adaxial region of the leaf primordia (Juarez et al., 2004). Taken together, these observations suggest that function of the YABBY genes has seemingly diversified between eudicots and grasses. Despite these apparent differences, the defects observed in the spikelet meristem (SM) in the tob1 mutant raise the possibility that FIL-clade YABBY genes are associated with meristem function in rice as well as in Arabidopsis (Tanaka et al., 2012b). YABBY genes encode nuclear proteins containing a zinc-finger and a helix-loop-helix domain (Bowman & Smyth, 1999; Sawa et al., 1999; Siegfried et al., 1999; Villanueva et al., 1999; Yamaguchi et al., 2004; Toriba et al., 2007; Tanaka et al., 2012b). Our previous study suggested that TOB1 is involved in transcriptional repression rather than activation because overexpression of TOB1 protein and expression of a chimeric protein consisting of TOB1 and a repression domain (SRDX) resulted in similar phenotypes (Tanaka et al., 2012b). Indeed, it has been demonstrated that YABBY proteins act in transcriptional repression in both Antirrhinum and Arabidopsis by interacting physically with GRO/TUP-like transcriptional corepressors, such as STYLOSA (STY), LEUNIG (LUG) and LEUNIG HOMOLOG (LUH) (Navarro et al., 2004; Stahle et al., 2009). FIL-clade YABBY proteins also interact physically with SEUSS (SEU), which functions with LUG as a coregulator (Stahle et al., 2009). Mutations in sty, lug and luh enhance phenotypes resulting from the mutation of YABBY genes (Stahle et al., 2009). Furthermore, LUG and SEU are involved in various developmental processes in Arabidopsis (Liu & Meyerowitz, 1995; Conner & Liu, 2000; Franks et al., 2002; Sridhar et al., 2004; Grigorova et al., 2011; reviewed in Liu & Karmarkar, 2008). In contrast to Arabidopsis and Antirrhinum, the functions of these transcriptional corepressors and coregulators are poorly understood in rice. Rice inflorescence consists of the rachis, primary and secondary branches, and spikelets (Tanaka et al., 2013, 2014; Hirano et al., 2014). The inflorescence meristem (IM) generates primary branch meristems (BMs) and supplies cells to form the rachis. The primary BM generates secondary BMs and SMs as a lateral meristem, and finally converts to a terminal SM at the top of the branch. The SM initiates lateral organs, such as rudimentary glumes and sterile lemmas, and converts to the floret meristem (FM) that produces floral organs, lemma and palea, although the SM and FM are not clearly distinguished in rice. Thus, the inflorescence architecture is complex, and the fates of the meristems change during inflorescence development. This is in contrast to the relatively simple inflorescence in Arabidopsis, where the IM directly produces the FMs. In rice, loss-of-function of TOB1 affects only the SM and the effects seem to be partial (Tanaka et al., 2012b). It is possible that other YABBY genes that are closely related to TOB1 share redundant functions with TOB1 and conceal the defects of TOB1 function. It would be interesting to determine whether TOB1 and its related YABBY genes are involved in regulating the IM and BM during rice inflorescence development, and are also associated with SAM function during the vegetative phase. In the present study, we revealed that downregulation of either TOB2 or TOB3 enhanced the defects in spikelet development in the tob1 mutant. Inflorescence development was markedly disturbed in transgenic tob1 plants in which both TOB2 and TOB3 were simultaneously silenced. These plants generated pole-like branches without any spikelets, suggesting that the activity of the BM was severely compromised. It is likely, therefore, that both TOB2 and TOB3 redundantly regulate inflorescence and spikelet development together with TOB1. The spatial expression patterns of the TOB genes suggest that BM activity is associated with TOB gene activity in bract primordia. Lastly, we found that rice homologs of SEU are involved in spikelet development, probably in association with YABBY gene function. Materials and Methods Plant materials and growth conditions Oryza sativa L. ssp. japonica cultivar was used as a plant material. Taichung 65 (T65) was used as a wild-type (WT) strain for comparing phenotypes and for in situ analysis. The tob1 mutant has been reported previously (Tanaka et al., 2012b). Plants were usually grown in pots containing soil (outdoor). Transgenic plants were grown in a NK System Biotron (model LH-350S, LH- 220S; Nippon Medical & Chemical Instruments, Osaka, Japan). In situ hybridization In order to generate probes for the TOB2, TOB3, OsSEU1 and OsSEU3 transcripts, partial cdna fragments were amplified with

3 New Phytologist Research 827 the primers listed in Supporting Information Table S1, and cloned into a pcrii vector (Invitrogen). RNAs were transcribed with T7 or SP6 RNA polymerase using digoxigenin-labeled UTPs (Roche), to make antisense or sense probe. The TOB1 probe was prepared as described previously (Tanaka et al., 2012b). Plant tissue was fixed and dehydrated by the methods of Itoh et al. (2000), replaced with xylene and then embedded in Paraplast Plus (Oxford Labware, St Louis, MO, USA). Microtome sections (8 10 lm) were mounted on glass slides. In situ hybridization experiment and immunological detection of signals were performed according to the methods of Kouchi & Hata (1993). RNAi knockdown In order to make the constructs for RNA interference, partial sequences of TOB2 and TOB3 were amplified with the primers listed in Table S1, and then cloned into a pentr D-TOPO vector (Invitrogen). The cloned fragment was then transferred by an LR recombination reaction into a modified panda vector, panda-eg1 (Miki & Shimamoto, 2004; Suzaki et al., 2008). In order to make a double RNAi construct for OsSEU1 and OsSEU3, partial cdna fragments were amplified with the primers listed in Table S1. After digestion with EcoRI, the fragments were ligated and cloned into a pentr D-TOPO vector (Invitrogen). A double RNAi construct for TOB2 and TOB3 was generated by a similar method after amplification of a partial sequence of each gene with the primers listed in Table S1. These fragments were then transferred by LR recombination reaction into a modified panda vector, panda-eg1 (Miki & Shimamoto, 2004; Suzaki et al., 2008). According to the method of Hiei et al. (1994), the recombinant plasmids were introduced into calli derived from T65 or tob1 via A. tumefaciens (EHA101). Scanning electron microscopy Young inflorescences, mature branches and spikelets were fixed in 0.1 M sodium phosphate buffer containing 4% paraformaldehyde and 0.25% glutaraldehyde, ph 7.2, at 4 C overnight. They were then dehydrated in a graded ethanol series, and ethanol was replaced with 3-methylbutyl acetate. The samples were dried at their critical point, sputter-coated with platinum, and then observed with a scanning electron microscope (model JSM-820S; Jeol, Tokyo, Japan) at an accelerating voltage of 5 kv. RT-PCR analysis In order to examine expression levels of TOB and OsSEU genes in the transgenic RNAi plants, developing panicles with branch (BM) and spikelet meristems (SMs) were collected from the axillary shoots. Total RNA was isolated using TRIsure (Bioline, London, UK) in accordance with the manufacturer s instructions. First-strand cdna was then synthesized from 3 to 5 lg of total RNA by using the SuperScript III First-Strand Synthesis System for reverse transcription polymerase chain reaction (RT-PCR) (Invitrogen) and the oligo (dt) 15 primer. In total, 2 ll of each reverse transcription product was used for 27 cycles of PCR with the primers listed in Table S1. Yeast two-hybrid assay The full-length coding regions of TOB1, TOB2, TOB3, OsSEU2 and OsSEU3 were amplified with the primers listed in Table S1, and then cloned into the pentr D-TOPO vector (Invitrogen). The cloned fragments were transferred by LR recombination reaction into a modified pgbkt7 vector (Clontech, Palo Alto, CA, USA), in which the Gateway reading frame cassette B (Invitrogen), containing the GAL4 DNA-binding domain (BD), was cloned into the SmaI site. These fragments were also cloned into a modified pad-gal4-2.1 vector (Yamaguchi et al., 2008), containing the GAL4 activation domain (AD). Protein interaction was examined by co-transforming the plasmids into the yeast reporter strain AH109. Transformed yeast cells were plated onto medium lacking leucine and tryptophan and grown at 30 C for 3 d. Colonies were then transferred to medium lacking leucine, tryptophan, histidine and adenine. Results The expression patterns of TOB2 and TOB3 are similar to that of TOB1 The rice genome has three YABBY genes in the FIL clade: TOB1, TOB2 (formerly reported as OsYABBY4) andtob3 (OsYABBY3) (Toriba et al., 2007; Tanaka et al., 2012b). Analysis of in situ hybridization revealed that these three TOB genes have very similar expression patterns (Fig. 1). In developing inflorescences, these genes were expressed in the primordia of lateral organs such as rudimentary glume, sterile lemma, lemma and palea, which are initiated from the SM (Fig. 1a c,f h,k m). The expression in the lemma and palea was relatively strong and uniform. By contrast, no expression was observed in the SM (Fig. 1a,b,f,g,k,l). In the vegetative shoot, the three TOB genes were expressed in the leaf primordia (Fig. 1d,e,i,j,n,o). In particular, strong TOB signals were detected in their marginal region and in the developing vasculature (Fig. 1e,j,o). None of the three TOB genes was expressed in the shoot apical meristem (SAM) or in the SM (Fig. 1b,d,g,i,l,n). Taken together, these similarities in spatial expression patterns suggest that the three TOB genes have related functions in rice development. RNAi suppression of TOB2 or TOB3 enhances tob1 phenotypes In order to elucidate the function of TOB2 and TOB3, we tried to obtain transposon insertion knockout lines of these genes, but failed to isolate any. We therefore suppressed the endogenous activity of TOB2 and TOB3 by the RNAi method using a genespecific region for each gene to make RNAi constructs. First, WT plants were transformed with either the TOB2-RNAi or

4 828 Research New Phytologist (a) (b) (c) (d) (e) (f) (g) (h) (i) (j) (k) (l) (m) (n) (o) Fig. 1 Temporal and spatial expression patterns of the three TONGARI-BOUSHI (TOB) genes during spikelet and leaf development in rice (Oryza sativa). (a, f, k) Longitudinal section of the inflorescence at an early stage when lateral organ primordia emerge from the spikelet meristem (SM). (b, g, l) Longitudinal section of the spikelet at an early stage when the lemma (le) initiates. (c, h, m) Longitudinal section of the spikelet at a late stage when carpels initiate. (d, i, n) Longitudinal section of the shoot apex of a 1-month-old plant. (e, j, o) Cross-section of the shoot apex of a 1-month-old plant. Arrowhead indicates expression of the TOB genes at the margin of the leaf primordium. fm, floral meristem; le, lemma; sl, sterile lemma; rg, rudimentary glume; lo, lodicule; st, stamen; ca, carpel; pa, palea; P1, plastochron one leaf primordium; sam, shoot apical meristem; P2, plastochron two leaf primordium; P3, plastochron three leaf primordium. Bars, 50 lm. TOB3-RNAi construct; however, no abnormalities were observed in > 20 transgenic plants assessed for each construct (Fig. S1). A similar result was obtained for WT plants in which both TOB2 and TOB3 were simultaneously suppressed (Fig. S1). Next, therefore, we suppressed TOB2 or TOB3 expression in the tob1 mutant. In our previous study (Tanaka et al., 2012b), we categorized tob1 spikelets into five phenotypic classes from weak to strong: namely, elongation of the awn (class I), reduction of the palea (class II), reduction of the lemma (class III), formation of a cone-shaped seamless organ without margin (class IV) and loss of the floret (class V). TOB2-RNAi;tob1 plants showed all classes of spikelet phenotypes found in the tob1 mutant (Fig.2a g). As compared with the tob1 single mutant, however, the proportion of each phenotypic class was changed in TOB2-RNAi;tob1 plants: spikelets with the weakest phenotype (class I) were decreased, whereas those with more severe phenotypes (class IV and V) were increased (Fig. 3a). In particular, clusters of class V spikelets were frequently found in TOB2-RNAi;tob1 plants (Fig. 2h,i), whereas no such clusters were observed in the tob1 mutant. Similar results were obtained in the analyses of TOB3-RNAi; tob1 plants (Figs 3b, S2). Thus, the downregulation of TOB2 or TOB3 expression enhanced the phenotype of the tob1 mutant. Simultaneous RNAi suppression of TOB2 and TOB3 in the tob1 mutant gives rise to naked branches without spikelets In order to obtain further insight into TOB gene function, we simultaneously knocked down TOB2 and TOB3 expression in the tob1 mutant. TOB2-TOB3-RNAi;tob1 plants exhibited severe defects in panicle development in addition to spikelet development (Figs 2a,b, 4). The TOB2-TOB3-RNAi;tob1 plants could be roughly classified into two groups. As compared with TOB2-RNAi;tob1 or TOB3-RNAi;tob1 plants, one group of TOB2-TOB3-RNAi;tob1 plants displayed much more enhanced phenotypes of tob1 with respect to spikelet phenotypes (group A). For example, the proportion of the most severe tob1 phenotype, class V, was markedly increased in TOB2-TOB3-RNAi;tob1 plants (Fig. 3c). The transgenic plants in group A also generated abnormal spikelets, which were not observed in the tob1 mutant, such as those completely lacking the lemma and palea (Fig. 4a,b), and those forming unidentified organs or malformed carpels and stamens (Fig. 4b d). We sometimes observed a small spikelet or branches within the spikelets (Fig. 4c f). These phenotypes suggest that the SM that differentiates the floral organs produces a new SM or reverts

5 New Phytologist Research 829 (a) (b) (c) (d) Fig. 2 Phenotypes of spikelets in wild-type (WT) and TONGARI-BOUSHI (TOB)2-RNAi; tob1 plants of rice (Oryza sativa). (a, b, h) WT. (c g, i) TOB2-RNAi;tob1. (a, b) A WT spikelet. (c) A spikelet with an elongated awn (class I). (d) A spikelet with a reduced size palea (class II). (e) A spikelet with an unidentified organ (arrowhead) on the opposite side of the palea (class III). (f) A spikelet forming a cone-shaped organ with an awn (class IV). (g) A spikelet without organs inside the sterile lemma (class V). (h) A cluster of normal spikelets in WT. (i) A cluster of class V spikelets (arrowheads) in TOB2-RNAi;tob1. le, lemma; pa, palea; sl, sterile lemma; st, stamen; ca, carpel; rpa, reduced palea; co, cone-shaped organ. Bars, 1 mm. (e) (f) (g) (h) (i) to the BM. In a few cases, branches were terminated without forming spikelets (Fig. 4g). Taken together, these findings indicate that downregulation of the three TOB genes causes more severe defects both in the activity of the SM and in the maintenance of its fate. In the second group (B), simultaneous knockdown of TOB2 and TOB3 in tob1 led to the production of naked branches lacking spikelets. In WT, the BM initiates SMs that develop into lateral spikelets on its flanks, and the BM itself ultimately converts to the terminal SM, which in turn develops into the terminal spikelet (Fig. 4h,i,x) (Itoh et al., 2005; Tanaka et al., 2013; Hirano et al., 2014). However, TOB2- TOB3-RNAi;tob1 plants in group B generated no or very few spikelets on branches, resulting in the formation of naked branches (Fig. 4h m). Thus, the panicles in these transgenic plants looked like a cluster of rods/poles (Fig. 4j,k). The branches were terminated bluntly and were sometimes accompanied by unidentified structures (Fig. 4l,m,o). A close-up view showed that the apical region of the branches lacked meristem-like dome structures (Fig. 4p). In some cases, tob1 class V spikelets were formed on the top of the branches (Fig. 4q). In other cases, branch-like structures were formed inside the rudimentary glumes, probably due to a reversion of meristem fate (Fig. 4r). Taken together, these observations suggest that the function and maintenance of the BM are profoundly compromised: in other words, the BM cannot generate any SMs and ultimately terminates without transition to the SM. Therefore, the three TOB genes seem to be required for proper function of the BMs in addition to the SM. We examined the expression levels of TOB2 and TOB3 in TOB2-TOB3-RNAi;tob1 plants by RT-PCR (Fig. S3). The reduced levels of TOB2 and TOB3 were roughly correlated with the extent of spikelet defects in the TOB2-TOB3-RNAi;tob1 lines (Fig. 3c). In particular, expression of TOB2 and TOB3 was significantly suppressed in TOB2-TOB3-RNAi;tob1 #17, which produced naked branches without spikelets (Fig. 4). The expression of YABBY genes other than those in the FIL clade was not affected in these knockdown lines except for plant #17 (Fig. S3). The expressivity of the tob1 phenotype differed slightly among experiments, suggesting that TOB gene activity is affected by growth conditions, as described previously (Tanaka et al., 2012b). The proportion of class III V spikelets, especially class V, was increased in TOB2-TOB3-RNAi;tob1, suggesting that double knockdown of TOB genes had a more severe effect than growth conditions. The WT rice panicle has a trace of the bract at its base, as observed in other grasses such as maize (Whipple et al., 2010), suggesting that bract development is strongly suppressed (Fig. 4s). A similarly suppressed bract was also observed in the tob1 mutant (Fig. 4t). By contrast, in TOB2-TOB3-RNAi;tob1 plants, the bract was highly elongated and formed tendril-like

6 830 Research New Phytologist (a) (b) (c) Fig. 3 Proportion of spikelet phenotypes in the tongari-boushi1 (tob1) mutant and knockdown lines of TOB2 and/or TOB3 in rice (Oryza sativa). (a) Comparison of spikelet phenotypes between tob1 and TOB2-RNAi;tob1. (b) Comparison of spikelet phenotypes between tob1 and TOB3-RNAi;tob1. (c) Comparison of spikelet phenotypes between tob1 and TOB2-TOB3-RNAi;tob1. The number below the bar indicates the number of each transgenic plant, whereas the number above the bar indicates the number of spikelets examined. No spikelet was formed in #17 plants. For each plant, the number of each class of spikelets was counted in a primary panicle. structures, suggesting that the three TOB genes function redundantly to suppress growth of the bract (Fig. 4u w). In group B, all TOB2-TOB3-RNAi;tob1 plants exhibited abnormalities in the vegetative phase. The plants were very small and failed to produce axillary shoots (tillers), raising the possibility that the axillary meristem is defective (Fig. 4n). Initiation and maintenance of the BM and SM are compromised in the TOB2-TOB3-RNAi;tob1 plant In order to examine developmental defects in TOB2-TOB3- RNAi;tob1 plants, we analyzed young inflorescencea by scanning electron microscopy (SEM). Fig. 4 Phenotypes of spikelets and inflorescences in TONGARI-BOUSHI (TOB)2-TOB3-RNAi;tob1 plants of rice (Oryza sativa). (a, b) A spikelet lacking the lemma and palea. Malformed (a) carpels and (b) stamens are formed inside the sterile lemma. (c, d) A spikelet forming an extra secondary spikelet (2sp in c) and branches (e-br in d) within the primary spikelet. Malformed carpels and stamens are observed. (e, f) Close-up view of (c) an extra spikelet and (d) branches. (g) A branch that terminated before forming a spikelet. (h) Inflorescences of wild-type (WT), tob1 mutant, and TOB2-TOB3-RNAi;tob1 plants (#21, #10 and #17). The inflorescences of TOB2-TOB3-RNAi;tob1 plants have almost naked branches. (i) A primary branch with a secondary branch, lateral spikelets, and a terminal spikelet in WT. (j, k) Naked branches generated in TOB2-TOB3-RNAi;tob1. (j, k) Branches of #10 and #17 plants, respectively. (l, m) Apical part of a naked branch with an unidentified structure (us). (n) Shoot phenotypes of WT (left) and TOB2-TOB3-RNAi;tob1 #17 (right). (o, p) A scanning electron microscope (SEM) image of the apical end of a naked branch. An unidentified structure (us) is associated with the branch at the top in (o). (q) An abnormal spikelet with only one rudimentary glume and one sterile lemma. (r) A branch-like structure within the rudimentary glume. (s w) Phenotype of the bract of the rachis. Degenerated bracts are seen in (s) WT and (t) tob1, but abnormally elongated bracts are present in TOB2-TOB3-RNAi;tob1 (u w). (x) Schematic view of the generation and conversion of the reproductive meristems. Solid and dashed lines indicate production and conversion, respectively. m-ca, malformed carpel; cm, cell mass; sl, sterile lemma; m-st, malformed stamen; e-br, ectopic branch; br, branch; rg, rudimentary glume; ts, terminal spikelet; ls, lateral spikelet; 2br, secondary branch; brt, bract; e-brt, elongated bract; IM, inflorescence meristem; pbm, primary branch meristem; sbm, secondary branch meristem; SM, spikelet meristem. Bars: (a g, k m, q w) 1 mm; (h j, n) 2 cm; (o, p) 500 lm.

7 New Phytologist Research 831 In WT rice, after transition from the SAM, the IM (inflorescence or rachis meristem) forms a bract (rachis bract) and sequentially initiates the primary BMs (Fig. 5a,c; reviewed in Itoh et al., 2005; Tanaka et al., 2013; Hirano et al., 2014). The primary and secondary BMs are subtended by the bract (branch bract), which has hair-like cells on the apical region (Fig. 5a,c; Itoh et al., 2005). The primary BM initiates the secondary branch meristems and the SMs, which are also subtended by the bracts (branch and spikelet bracts). (a) (b) (c) (h) (d) (e) (f) (g) (i) (j) (k) (l) (m) (n) (o) (p) (q) (r) (s) (u) (t) (v) (w) (x)

8 832 Research New Phytologist (a) (b) (c) (d) (e) (f) (g) (h) (i) (j) (k) Fig. 5 Inflorescence phenotypes at early developmental stages in TONGARI-BOUSHI (TOB)2-TOB3-RNAi;tob1 plants of rice (Oryza sativa). (a, b) Scanning electron microscope (SEM) image of an inflorescence at the stage of primary branch meristem (BM) initiation in (a) wild-type (WT) and (b) TOB2-TOB3-RNAi;tob1. Arrowheads indicate the primary BM; arrow indicates the degenerated inflorescence meristem. The TOB2-TOB3-RNAi;tob1 plant is abbreviated as tob1/2/3. (c, d) SEM image of developing primary BMs at the stage of secondary BM initiation in (c) WT and (d) TOB2-TOB3- RNAi;tob1. Arrow indicates a prematurely terminated primary BM; bracket indicates the position where the secondary BM should initiate. (e, f) SEM image of a developing primary branch at the stage of spikelet meristem (SM) initiation in (e) WT and (f) TOB2-TOB3-RNAi;tob1. Arrow in (f) indicates the arrested primary BM. (g, h) SEM image of developing spikelets in (g) WT and (h) TOB2-TOB3-RNAi;tob1. Yellow arrow in (h) indicates an abnormal SM with a ring-like primordium; white arrows in (h) indicate arrested SMs with abnormal primordia. (i k) Longitudinal section of a developing inflorescence in (i) WT and (j, k) TOB2-TOB3-RNAi;tob1. Arrowheads in (j, k) indicate the empty bracts; arrows indicate the positions where (j) the axillary meristems or (k) branches should initiate. IM, inflorescence meristem; pbb, bract primordia of the primary branch meristem; rb, rachis bract; sbb, bract primordia of the secondary branch meristem; sbm, secondary branch meristem; pbm, primary branch meristem; tsm, terminal spikelet meristem; sb, bract primordia of the spikelet meristem. Bars, 100 lm. In contrast to WT, the number of the primary BMs was reduced in TOB2-TOB3-RNAi;tob1 plants, probably because of early inactivation of the IM (Fig. 5b). The morphology of the developing primary branch was also abnormal. For example, the primary BM failed to initiate the axillary meristems in a regular manner and then terminated prematurely (Fig. 5d, bracket). In

9 New Phytologist Research 833 another case, the primary BM terminated immediately after its initiation without producing any meristems (Fig. 5d, arrow). In WT, the primary BM ultimately converts into the terminal SM after generating secondary BMs and SMs (Fig. 5e). In the TOB2- TOB3-RNAi;tob1 plant, by contrast, the primary BM showed arrested development without conversion into the terminal SM (Fig. 5f). The SM initiates spikelets and floral organs in a regular manner in WT (Fig. 5g). However, we frequently observed a cluster of spikelets that had an arrested meristem with a ring-like primordium or abnormal organ primordia in the TOB2-TOB3- RNAi;tob1 plant (Fig. 5h). These morphological defects suggest that the activity and maintenance of all reproductive meristems is impaired in the TOB2-TOB3-RNAi;tob1 plant. Next, we examined the developing inflorescences by making longitudinal sections. In these sections, the bracts were recognized as hair-like structures and were necessarily associated with the axillary meristems, developing primary branches or spikelets (Fig. 5i). In the TOB2-TOB3-RNAi;tob1 plant, however, we failed to observe the BM in the axil of the bract during the branch differentiation stage (Fig. 5j). We called these bracts that subtended no obvious structure empty bracts. Such empty bracts were also found at the positions where the primary branches should be initiated (Fig. 5k). Thus, the initiation of meristems was often compromised in the TOB2-TOB3-RNAi;tob1 plant. These defects are likely to be related to the formation of naked branches, as described earlier (Fig. 4). Taken together, these results suggest that all three TOB genes are required for initiation and proper maintenance of the BMs and SMs. (a) (c) (e) (b) (d) (f) TOB genes are expressed in the bract primordia at early stages of inflorescence development We examined the temporal and spatial expression patterns of the three TOB genes during early inflorescence development. The TOB genes were expressed in the apical and adaxial region of the rachis bract primordia at the primary branch initiation stage, although TOB2 expression was weak (Fig. 6a,c,e). In addition, TOB signals were detected in the small appendages at the basal region of the primary branch meristems, that is, the bract primordia of the primary branches. At a subsequent stage, TOB1 expression was detected at the bract primordia of the secondary branches. Similar expression of TOB2 and TOB3 was detected (Fig. 6b,d,f). At all inflorescence stages, no transcript of the three TOB genes was detected in the rachis, branch or SM. It is therefore likely that the failure in SM initiation that causes the naked branches is a consequence of a putative noncell-autonomous activity of the TOB genes expressed in the branch bract primordium. SEU-like genes in rice Our previous study suggested that TOB1 may be involved in transcriptional repression, as deduced from the phenotypic similarity between TOB1-SRDX-expressing and TOB1- overexpressing plants (Tanaka et al., 2012b). In reference to Fig. 6 Expression patterns of the TONGARI-BOUSHI (TOB) genes at early stages of inflorescence development in rice (Oryza sativa). (a f) Spatial and temporal expression patterns of (a, b) TOB1, (c, d) TOB2 and (e, f) TOB3 at the stage of (a, c, e) primary branch meristem (BM) initiation and (b, d, f) secondary BM initiation. rb, rachis bract; pbb, bract primordia of the primary branch meristem; pbm, primary branch meristem; sbb, bract primordia of the secondary branch meristem; sbm, secondary branch meristem. Bars, 50 lm. research in Arabidopsis and Antirrhinum (Navarro et al., 2004; Stahle et al., 2009), we focused on homologs of SEU as factors involved in transcriptional repression. Rice contains three SEU-like genes (OsSEU) in its genome, and all three OsSEU proteins share the conserved central domain and at least one Q-rich region (Fig. S4) as in Arabidopsis SEU (Franks et al., 2002; Bao et al., 2010). OsSEU1 and OsSEU2 are very similar to each other (94% identity), and share 47% identity overall with Arabidopsis SEU. Phylogenetic analysis revealed that OsSEU1 and OsSEU2 are co-orthologs of Arabidopsis SEU, whereas OsSEU3 is an ortholog of AtSLK proteins (Fig. S4). RT-PCR analysis showed that the OsSEU genes were expressed in various organs, suggesting that they are likely to be involved in multiple aspects of gene regulation (Fig. S5). Next, we examined the spatial expression patterns of these genes by in situ hybridization. Owing to the high sequence

10 834 Research New Phytologist (a) (b) (c) (d) (e) (f) (g) (h) Fig. 7 Temporal and spatial expression patterns of OsSEUSS (OsSEU) 1/2 and OsSEU3 in spikelet development in rice (Oryza sativa). (a d) Expression of OsSEU1 and OsSEU2 detected by (a c) antisense and (d) sense probes. (e h) Expression of OsSEU3 detected by (e g) antisense and (h) sense probes. pa, palea; fm, floral meristem; le, lemma; sl, sterile lemma; rg, rudimentary glume; st, stamen; ca, carpel. Bars, 50 lm. similarity between OsSEU1 and OsSEU2, it would be difficult to distinguish their expression. We therefore performed the experiment using a probe for OsSEU1, which is likely to detect the transcripts of both OsSEU1 and OsSEU2 (OsSEU1/2). No signal was detected when a sense probe for OsSEU1/2 or OsSEU3 was used (Fig. 7d,h). Expression of OsSEU1/2 was detected in the SM and lateral floral organs (Fig. 7a c). OsSEU1/2 expression persisted to a later developmental stage when the lemma and palea primordia were fused at the top of the spikelet (Fig. 7c). Similar broad expression of OsSEU3 was observed (Fig. 7e g). In order to investigate whether the TOB and OsSEU proteins interact physically with each other, we performed a yeast twohybrid assay. Owing to the high similarity between OsSEU1 and OsSEU2 (94% identity), we examined the physical interaction of OsSEU2 and OsSEU3 with the three TOB proteins. OsSEU3 was found to interact with all three TOB proteins in yeast (Fig. S6). By contrast, OsSEU2 interacted weakly with TOB1 and TOB2, and showed no interaction with TOB3. RNAi knockdown of the OsSEU genes enhanced tob1 mutation In order to examine the function of OsSEU, we aimed to knock down the endogenous activities of OsSEU genes by the RNAi method. For this purpose, we used a segment of OsSEU1 cdna that is conserved in both OsSEU1 and OsSEU2, and a sequence specific to OsSEU3 cdna. The RNAi construct (OsSEU1-3-RNAi) was made by fusing these sequences head to tail to knock down the three OsSEU genes simultaneously. We first introduced the OsSEU1-3-RNAi construct into WT, but the transgenic plants did not exhibit obvious phenotypes (Fig. S1). We therefore then transformed the tob1 mutant with the OsSEU1-3-RNAi construct. All five phenotypic classes in tob1 were also observed in the OsSEU1-3-RNAi;tob1 plants (Fig. 8a d). The proportion of spikelets showing severe phenotypes was higher in OsSEU1-3-RNAi;tob1 plants than in the tob1 mutant (Fig. 8i). In particular, transgenic plants #1, #12 and #14 produced a number of class V spikelets, and the proportion of this class in these plants was larger than that observed in any TOB2- TOB3-RNAi;tob1 plants. Accordingly, these results indicated that suppression of the three OsSEU genes enhanced the tob1 phenotype. The OsSEU1-3-RNAi;tob1 plants also exhibited more abnormal phenotypes of the spikelet, which were not observed in the tob1 single mutant (Fig. 8e h). Malformed lemma/palea-like organs were observed, and the phyllotaxy of these organs and other organs such as the sterile lemma was disturbed (Fig. 8e). In some plants, the development of lemma/palea-like organs was incomplete (Fig. 8f); and in others, no lemma/palea-like organs developed at all (Fig. 8g). Branch-like structures and a few spikelets were formed inside the sterile lemma (Fig. 8h).

11 New Phytologist Research 835 (a) (b) (c) (d) (e) (f) (g) (h) Fig. 8 Spikelet phenotypes of wild-type (WT) and OsSEUSS (OsSEU) 1-3-RNAi;tongariboushi1 (tob1) plants of rice (Oryza sativa). (a, b) WT. (c h) OsSEU1-3-RNAi;tob1. (a, b) A WT spikelet. (c) A spikelet forming a coneshaped seamless organ (class IV). (d) A spikelet with no floret inside the sterile lemma (class V). (e) A spikelet with malformed lemma/palea-like organs. (f) A spikelet with an incomplete lemma/palea-like organ (arrowhead). (g) A spikelet without a lemma or palea. (h) A spikelet generating secondary spikelets (arrowheads) and branches within the sterile lemma. (i) Proportion of spikelet phenotypes in the tob1 mutant and OsSEU1-3-RNAi;tob1 plant. The number below the bar indicates the number of each transgenic plant, whereas the number above the bar indicates the number of spikelets examined. For each plant, the number of each class of spikelets was counted in a primary panicle. le, lemma; pa, palea; sl, sterile lemma; st, stamen; ca, carpel; co, cone-shaped organ; rle, reduced lemma size; br, branch; m-ca, malformed carpels. Bars, 1 mm. (i) We examined expression levels of OsSEU genes in the OsSEU1-3-RNAi;tob1 plants by RT-PCR analysis (Fig. S3). The reduced level of OsSEU expression was roughly correlated with the extent of spikelet defects in the OsSEU1-OsSEU3-RNAi;tob1 lines (Fig. 8i). In addition, we noticed that severity of tob1 spikelet phenotypes is affected by growth conditions, even when the plants are cultivated in a growth chamber under sunlight (Figs 3c, 8i). Taken together, these observations suggest that the activity and fate of the SM are strongly compromised and that OsSEU proteins interact with a factor other than TOB proteins that is closely associated with the function of the SM. Discussion In this article, we revealed that three TONGARI-BOUSHI (TOB)-related YABBY genes control spikelet and inflorescence development in rice. The TOB genes, which are expressed in organ primordia, are likely to act noncell-autonomously on the meristems and regulate the maintenance and identity of the spikelet (SM) and branch (BM) meristems. TOB proteins seem to work together with OsSEU proteins co-regulators involved in transcriptional repression to ensure the proper development of spikelets.

12 836 Research New Phytologist Three TOB genes seem to regulate the maintenance and identity of the SM In our previous study, we showed that TOB1 function is necessary for the robust organization and maintenance of the SM (Tanaka et al., 2012b). In this study, single or double knockdown of TOB2 and TOB3 enhanced spikelet phenotypes in the tob1 mutant, suggesting that TOB2 and TOB3 also have a role in maintaining the SM. In addition, TOB genes are also likely to be required for SM identity, because a secondary spikelet and a branch-like structure were observed within the spikelets in TOB2-TOB3-RNAi;tob1. Interestingly, similar organ reiteration is observed in the Arabidopsis crc flower when combined with other mutations (Alvarez & Smyth, 1999; Prunet et al., 2008). The results of phenotypic analysis and spatiotemporal expression patterns indicated that the three TOB genes act redundantly in spikelet development. However, there seems to be a difference among them in their contribution to spikelet development. Most TOB2-RNAi;WT and TOB3-RNAi;WT plants were indistinguishable from wild-type (WT), whereas TOB1-RNAi;WT plants exhibit pleiotropic spikelet phenotypes similar to those of the tob1 mutant, which has a severe mutation (Tanaka et al., 2012b). Here, we used an RNA interference method to examine the function of the TOB genes. It is possible that knockout tob mutants generated by the recently developed CRISPR/Cas9 system might show more severe phenotypes than those observed here, and triple knockout lines would provide important information on the function of these TOB genes in the vegetative phase (see later). The severity of tob1 phenotype seemed to be affected by growth conditions when the plants were cultivated in the field (Tanaka et al., 2012b). In this study, we noted that the severity of the tob1 phenotype was also affected even under a relatively constant temperature (cultivation in an outdoor-type growth chamber using sunlight). It is possible that the activities of TOB2 and TOB3 vary depending on environmental factors, and probably mask the defect in tob1 to different extents in accordance with their activity levels. TOB gene activity is required for proper function of the BM in inflorescence development The simultaneous downregulation of TOB2 and TOB3 in tob1 also affected inflorescence development. In severe cases, the inflorescences were composed of naked branches without any spikelets in TOB2-TOB3-RNAi;tob1 lines. Therefore, all primary BMs in these lines seem to lack the activity necessary to form axillary meristems and to develop into the terminal spikelet. This inference was confirmed by the observation that SMs and secondary BMs often failed to form in the axil of the bract, as indicated by empty bracts, during early developmental stages of the inflorescence. The number of naked primary branches was reduced in the most severe lines (#10, #17). This result is consistent with the SEM observation that the number of BMs was reduced at the stage of primary branch differentiation. It is therefore likely that the function of the inflorescence meristem (IM) and BM in forming the axillary meristems is strongly compromised in TOB2- TOB3-RNAi;tob1 lines. The three TOB genes were expressed in the bract primordia of the inflorescence and branches, but not in the IMs and BMs per se. Thus, defects in the function of these meristems in TOB2- TOB3-RNAi;tob1 are likely to result from loss or reduced activity of these three TOB genes in the bract primordia. This finding is similar to that in our previous study where TOB1, which is expressed in lateral organs, was shown to act noncellautonomously to promote the activity of the SM (Tanaka et al., 2012b). Therefore, the three TOB genes seem to act in a similar way to maintain the activity of the IMs and BMs. In addition to the meristem defects, the rachis bract was highly elongated and changed into a tendril-like structure in TOB2- TOB3-RNAi;tob1 plants. The expression of TOB genes was localized to the adaxial region of the rachis bract primordia. It is possible that the tendril-like bract is radialized by a defect in the adaxial abaxial polarity. Likewise, this bract growth has also been observed in multiple yabby mutants in Arabidopsis (Siegfried et al., 1999; Sarojam et al., 2010). Unlike Arabidopsis leaves, however, foliage leaves developed normally in the TOB2-TOB3- RNAi;tob1 lines, and the genes were expressed in a nonpolar manner in the vegetative leaf primordia. Therefore, TOB function in leaf development remains to be elucidated in future studies. In general, elongation of the bract is suppressed in grasses, and a few genes responsible for this suppression have been identified (Wang et al., 2009; Whipple et al., 2010; reviewed in Whipple, 2017). It is possible, therefore, that TOB gene function may be associated with bract suppression genes. In the vegetative phase, the severe TOB2-TOB3-RNAi;tob1 lines also failed to form axillary shoots. Because leaf initiation appeared to be normal in the severe lines, the reduction in the activity of three TOB genes might affect initiation or maintenance of the axillary meristem also in the vegetative phase. The axillary meristem develops via the premeristem, which is a specific and transient developmental stage in rice (Tanaka et al., 2015). It will be of great interest to determine whether TOB gene activity is also required for maintenance of the premeristem. Mutations in LAX PANICLE1 (LAX1 and LAX2) strongly affect initiation of the spikelet in rice, producing sparse inflorescences (Komatsu et al., 2003; Tabuchi et al., 2011). Unlike in TOB2-TOB3-RNAi;tob1 plants, terminal spikelets are produced and the primary branch length is normal in these mutants. Thus, the TOB and LAX genes seem to have partially overlapping roles in inflorescence development in rice. LAX1 encodes a transcription factor with a basic helix-loop-helix (bhlh) domain, similar to maize BARREN STALK1, mutation of which also causes sparse inflorescences (Komatsu et al., 2003; Gallavotti et al., 2004). Both LAX1 and BA1 are expressed at the boundary of the BM and SM, and not in the meristems per se. It has been shown that LAX1 protein moves from the boundary to the axillary meristem (Oikawa & Kyozuka, 2009). Considering that the TOB genes act noncell-autonomously, communication between the meristem and the other parts of the developing inflorescence must be

13 New Phytologist Research 837 important for both BM and SM activity in grass inflorescence development. In Arabidopsis, the boundary gene LATERAL SUPPRESSOR (LAS) mediates a putative FIL-derived signal (Goldshmidt et al., 2008). A mutation in MONOCULM1 (MOC1), a LAS ortholog in rice, causes a reduction in the number of spikelets in the inflorescence, as observed in TOB2-TOB3-RNAi;tob1 (Li et al., 2003). As a result, it is possible that MOC1 is also involved in the function of TOB genes in rice. Naked inflorescences are found in mutants such as Arabidopsis pin-formed1 and monopteros/arf1 and maize barren inflorescence2, which are defective in polar transport and signaling of auxin (Okada et al., 1991; Przemeck et al., 1996; McSteen et al., 2007). Interestingly, local auxin depletion is reportedly associated with axillary meristem formation in Arabidopsis and tomato (Wang et al., 2014a,b). It will be interesting to know whether, and if so how, TOB genes are involved with auxin action in axillary meristem formation in rice inflorescences. TOB genes act together with OsSEU genes encoding transcriptional coregulators during spikelet development Previously, we suggested that TOB1 was involved in transcriptional repression on the basis of the observation that overexpression of TOB1 and TOB1-SRDX showed similar phenotypes (Tanaka et al., 2012b). In the present study, we found that TOB proteins interact physically with OsSEU proteins, although TOB proteins interacted more strongly with OsSEU3 than OsSEU2. In Arabidopsis and Antirrhinum, YABBY proteins interact physically with LUG/STY or SEU to form repressor complexes (Navarro et al., 2004; Stahle et al., 2009). Thus, the physical interaction between TOB and OsSEU proteins supports the idea that TOBs proteins are involved in transcriptional repression in rice. When all three OsSEU genes were suppressed simultaneously in the tob1 mutant background, the phenotype of tob1 was markedly enhanced. Similar phenotypic enhancement was observed when TOB2 and/or TOB3 were knocked down in the tob1 mutant. It is, therefore, possible that OsSEU genes are required for the action of the TOB2 and TOB3 genes. Evolutionary insight into YABBY gene function in angiosperm development YABBY genes belonging to the FIL clade have been identified and their spatial expression patterns have been analyzed in various angiosperms (Sawa et al., 1999; Siegfried et al., 1999; Golz et al., 2004; Juarez et al., 2004; Yamada et al., 2004, 2011; Tanaka et al., 2012b). Although all FIL-related YABBY genes are expressed in the primordia of lateral organs, their spatial expression patterns within these organs vary considerably. For example, abaxial expression patterns are observed for YABBY genes in eudicots such as Arabidopsis and Antirrhinum (Sawa et al., 1999; Siegfried et al., 1999; Golz et al., 2004; Yamada et al., 2011), whereas adaxial expression of the related YABBY genes has been reported in maize and Amborella trichopoda (Juarez et al., 2004; Yamada et al., 2004). In rice, TOB1 is expressed throughout the primordia of spikelet organs without specific localization (Tanaka et al., 2012b). These various expression patterns suggest that FILclade YABBY genes play seemingly diverse roles in plant development depending on the species. Indeed, the phenotypes of single yabby mutants differ between eudicots and rice (Sawa et al., 1999; Siegfried et al., 1999; Golz et al., 2004; Tanaka et al., 2012b). Consistent with their expression in lateral organs, YABBY genes are involved in the differentiation of lateral organs (Sawa et al., 1999; Siegfried et al., 1999; Golz et al., 2004; Tanaka et al., 2012b). Recent analysis using multiple yabby mutants in Arabidopsis revealed that YABBY genes are required for the maintenance of meristem activity, in addition to the development of lateral organs (Goldshmidt et al., 2008; Stahle et al., 2009; Sarojam et al., 2010). Because YABBY genes are not expressed in the meristem, they seem to act noncell-autonomously on the meristem. In rice, we reported previously that TOB1 is responsible for maintaining the activity of the SM, because the tob1 mutant displayed defects in the SM and in spikelet development (Tanaka et al., 2012b). In the present study, we clearly showed that three TOB genes are required to maintain meristem activity not only in the SM, but also in the BM and IM, by making knockdown lines of TOB2 and TOB3 in the tob1 mutant. In addition, axillary meristem formation or maintenance was found to be compromised in the vegetative phase of these plants, as discussed earlier. Thus, the TOB genes seem to be required for the activity of all aerial meristems in a similar manner. Taken altogether, the function of FIL/TOB clade YABBY genes seems to be conserved between Arabidopsis and rice in the sense that these genes are involved in maintaining the proper function of meristems. It is possible that the ancestral and fundamental role of these YABBY genes was associated with communication between a lateral organ (or organ subtending the meristem) and the meristem. It is worth noting that YABBY genes are specific to the genome of seed plants (Floyd & Bowman, 2007; Toriba et al., 2007), the apical meristems of which comprise a group of cells, and that they are not found in the genome of ferns and mosses, which have a single apical cell as the initial cell (Evert, 2006). Recently, it was revealed that a CLAVATA3/Endosperm surrounding region-related peptide, ZmFCP1, which is presumably produced in organ primordia, negatively regulates meristem size in maize through its receptor FEA3 (Je et al., 2016). It is therefore possible that signaling from lateral organs may be a more general mechanism underlying the regulation of meristem activity. Apart from their essential function, the YABBY genes seem to be involved in various developmental processes in angiosperms. Inflorescence architecture is diverse in angiosperms. Unlike the Arabidopsis flower, the meristem of which is produced directly from the IM, the rice flower is formed through various types of meristem, such as the IM, primary and secondary BMs, SMs and FMs. In the present paper, we showed that three TOB genes are required to initiate and maintain each of these meristems, suggesting that TOB gene function is associated with proper development of the inflorescence in rice. This finding also suggests the possibility that YABBY genes are involved in the construction of