transcriptional corepressor asp1 and clv-likeance between stem cell self-renewal and organ/ meristem...

Transcriptional Corepressor ASP1 and CLV-LikeSignaling Regulate Meristem Maintenance in Rice1[OPEN]

Chie Suzuki, Wakana Tanaka, and Hiro-Yuki Hirano2,3

Department of Biological Sciences, School of Science, The University of Tokyo, Bunkyo-ku, Tokyo 113-8654,Japan

ORCID IDs: 0000-0003-0503-4608 (C.S.); 0000-0002-2635-5462 (W.T.); 0000-0001-7364-8893 (H.-Y.H.).

Stem cell homeostasis is maintained by the WUSCHEL-CLAVATA (WUS-CLV) negative feedback loop in Arabidopsis(Arabidopsis thaliana). In rice (Oryza sativa), FLORAL ORGAN NUMBER2 (FON2) functions in the negative regulation of stemcell proliferation, similar to Arabidopsis CLV3. In this study, through genetic enhancer analysis, we found that loss of function ofABERRANT SPIKELET AND PANICLE1 (ASP1), encoding an Arabidopsis TOPLESS (TPL)-like transcriptional corepressor,enhances the fon2 flower phenotype, which displayed an increase in floral organ number. In the fon2 asp1 double mutant, theinflorescence was severely affected, resulting in bifurcation of the main axis (rachis), a phenotype that has not previously beenreported. The stem cells showed marked overproliferation in fon2 asp1, resulting in extreme enlargement and splitting of theinflorescence meristem. These results suggest that ASP1 and FON2 synergistically regulate stem cell maintenance in rice.Unexpectedly, genetic analysis indicated that TILLERS ABSENT1, the rice ortholog of WUS, is not involved in promotingstem cell proliferation in this meristem. Transcriptome analysis suggested that ASP1 and FON signaling negatively regulate aset of genes with similar functions, and they act on these genes in concert. Taken together, our results suggest that TPL-likecorepressor activity plays a crucial role in meristem maintenance, and that stem cell proliferation is properly maintained via thecooperation of ASP1 and FON2.

Plant development depends on the activity of theshoot apical meristem (SAM), which harbors a group ofstem cells at the tip. These stem cells proliferate tomaintain self-renewal and to supply cells for organdifferentiation (for review, see Ha et al., 2010; Aichingeret al., 2012; Somssich et al., 2016). The vegetative SAMdifferentiates leaf primordia and turns into the inflo-rescence meristem when it enters the reproductivephase after perceiving a flowering signal. The inflores-cence meristem directly initiates either flower meri-stems that differentiate floral organs or intermediate

types of meristems such as the branch and spikeletmeristems (reviewed in Tanaka et al., 2013; Hiranoet al., 2014). In each meristem, the appropriate bal-ance between stem cell self-renewal and organ/meristem differentiation is essential for proper plantdevelopment.

In Arabidopsis (Arabidopsis thaliana), the WUSCHEL-CLAVATA (WUS-CLV) feedback loop is a fundamen-tal mechanism underlying stem cell maintenance in theSAM (Brand et al., 2000; Schoof et al., 2000; reviewedin Ha et al., 2010; Aichinger et al., 2012; Somssich et al.,2016). The CLV3 peptide is secreted from stem cellsand perceived by multiple receptors, including CLV1,CLV2/CORYNE, and RECEPTOR-LIKE PROTEINKINASE2 (Clark et al., 1997; Fletcher et al., 1999;Müller et al., 2008; Kinoshita et al., 2010), whichtransmit the signal to negatively regulate expression ofthe WUS gene, encoding a homeodomain transcrip-tion factor (Mayer et al., 1998).WUS is expressed in theorganizing center located under the stem cell regionand acts non-cell-autonomously in stem cells throughmovement of the WUS protein itself (Mayer et al.,1998; Yadav et al., 2011). WUS positively regulatesstem cell proliferation partly in association with cy-tokinin action and activates CLV3 expression(Leibfried et al., 2005; Gordon et al., 2009; Yadav et al.,2011; Chickarmane et al., 2012). This negative feed-back loop between CLV signaling and WUS actionregulates stem cell homeostasis in the meristem.

Genetic regulation similar to Arabidopsis WUS-CLVis conserved in other plants, such as rice (Oryza sativa)

1This work was supported in part by Grants-in-Aid for ScientificResearch from the Ministry of Education, Culture, Sports, Science,and Technology (MEXT) (grant nos 23248001 and 25113008 to H.-Y.H.), the Japan Society for the Promotion of Science (grant 16J04197and a Research Fellowship for Young Scientists to C.S.), and NIG-JOINT (2016-A1-64).

2Author for contact: [email protected] author.The author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Hiro-Yuki Hirano ([email protected]).

C.S. and H.-Y.H. conceived the original screening and researchplans; W.T. and H.-Y.H. supervised the experiments; C.S. performedmost of the experiments; C.S. designed the experiments and analyzedthe data; C.S. and H.-Y.H. conceived the project and wrote the articlewith contributions of all the authors. H.-Y.H. agrees to serve as theauthor responsible for contact and ensures communication.

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andmaize (Zea mays; for review, see Tanaka et al., 2014;Somssich et al., 2016). In rice, FLORAL ORGAN NUM-BER1 (FON1) and FON2, respective orthologs of Ara-bidopsis CLV1 and CLV3, negatively regulate meristemmaintenance in the reproductive phase (Suzaki et al.,2004, 2006). The loss of function of either FON1 or FON2causes enlargement of the floral meristem, resulting inincreased numbers of floral organs such as pistils andstamens. FON2 is expressed in the apical region of thefloral meristem, putative stem cells, and its expressiondomain is expanded in the enlarged floral meristem inthe fon1 mutant. Overexpression of FON2 causes afailure in maintenance of the floral meristem in wildtype; by contrast, fon1 is insensitive to this over-expression (Suzaki et al., 2006). Thus, FON2 acts as anegative regulator of stem cell proliferation throughFON1, similar to CLV3 action in Arabidopsis. FON2-LIKE CLE PROTEIN1 (FCP1) and FCP2, close paralogsof FON2, play a role in maintenance of the SAM in thevegetative phase by repressing the expression ofWOX4, a member of WUSCHEL-RELATED HOMEO-BOX (WOX) gene family and an ortholog of Arabi-dopsis WOX4 (Ohmori et al., 2013). By contrast,TILLERS ABSENT1 (TAB1; also known as OsWUS), anortholog ofWUS, plays an important role in initiation ofthe axillary meristem in rice but is not involved inmaintenance of the established meristem (Tanaka et al.,2015).In maize, THICK TASSEL DWARF1 and FASCIATED

EAR2 (FEA2), respective orthologs of CLV1 and CLV2,negatively regulate meristem maintenance (Taguchi-Shiobara et al., 2001; Bommert et al., 2005; reviewedin Somssich et al., 2016). Recent studies revealed thatZmFCP1, a maize FCP1 ortholog, also functions inmeristemmaintenance (Je et al., 2016, 2018). ZmFCP1 isexpressed in organ primordia and is thought to beperceived by FEA3, a novel CLV-related LRR receptor-like protein. Je et al. (2016) also showed that this FCP1/FEA3-like pathway is conserved in Arabidopsis.Rice produces an inflorescence, known as a panicle,

consisting of a main axis (rachis), primary and sec-ondary branches, and spikelets. During inflorescencedevelopment, the inflorescence meristem initiates theprimary branch meristems, which in turn initiatethe secondary branch and spikelet meristems. Floralorgans including lodicules, stamens, and a pistilare differentiated from the flower meristem, whoseidentity is changed from the spikelet meristem. Loss-or gain-of-function mutations in genes such asABERRANT SPIKELET AND PANICLE1 (ASP1), AB-ERRANT PANICLE ORGANIZATION1 (APO1)/STRONG CULM2, RFL/APO2, DENSE AND ERECTPANICLE1 (DEP1)/DENSE PANICLE1, TONGARI-BOUSH1-3, LAX PANICLE1 (LAX1), LAX2, OsCKX2,OsSPL14, and TAWAWA1 lead to small or large in-florescences (Komatsu et al., 2003; Ashikari et al., 2005;Ikeda et al., 2007; Rao et al., 2008; Huang et al., 2009;Jiao et al., 2010; Miura et al., 2010; Ookawa et al., 2010;Tabuchi et al., 2011; Ikeda-Kawakatsu et al., 2012;Yoshida et al., 2012, 2013; Tanaka et al., 2017). The

functions of most of these genes have been shown to beassociated with meristem activity and fate. Unlikethese genes, loss-of-function mutations in FON1 orFON2 do not cause obvious defects in inflorescencearchitecture.TOPLESS (TPL) and four TOPLESS-RELATED genes

(TPR1, TPR2, TPR3, and TPR4) encode transcriptionalcorepressors that play a pivotal role in development,such as embryo and flower patterning in Arabidopsis;for example, RNA silencing of TPR2 in the tpl tpr1 tpr3tpr4 quadruple mutant results in transformation of theshoot pole into a root pole (Long et al., 2002, 2006;Krogan et al., 2012). These genes are also required forsignaling of hormones such as auxin, jasmonate, andbrassinosteroid (Szemenyei et al., 2008; Pauwels et al.,2010; Oh et al., 2014; Ryu et al., 2014). The TPL and TPRcorepressors, which have no DNA-binding motif,physically interact with transcriptional factors thatrecognize a specific DNA sequence and silence a largenumber of target genes by recruiting histone deacety-lases (Long et al., 2006; Causier et al., 2012). TPL(reported as WSIP1) and TPR4 (WSIP2) physically in-teract with WUS, and this interaction is necessary formeristem maintenance (Kieffer et al., 2006). Further-more, WUS regulates the expression of TPL and someTPRs in the meristem (Busch et al., 2010). Despite theseobservations, it is not clear how individual TPL/TPRgenes function in stem cell maintenance, probably be-cause of their high genetic redundancy in Arabidopsis.Rice has three TPL-like genes including ASP1, sug-

gesting that this type of transcriptional corepressor hasrelatively low functional redundancy in rice (Yoshidaet al., 2012). Indeed, using an asp1 single mutant thatdisplays pleiotropic defects in inflorescence architec-ture and flower development, we previously revealedthat ASP1 plays a critical role in the regulation ofmeristem fate in the reproductive phase (Yoshida et al.,2012). In maize, a mutation in RAMOSA1 ENHANCERLOCUS2 (REL2), a TPL-like gene, enhances the highlybranching phenotype of ramosa1 (ra1) inflorescence(Gallavotti et al., 2010). REL2 has been shown to regu-late axillary meristem fate by physically interactingwith RA1, a transcription factor with a C2H2-type zinc-finger motif.Although several genes involved in the WUS-CLV-

like pathway in rice have been identified (Suzaki et al.,2004, 2006, 2008, 2009; Ohmori et al., 2013), ourknowledge of the genetic mechanisms underlyingmeristem maintenance in this plant remains limited. Toexplore these mechanisms further, in this study weused a genetic approach to identify a gene whose mu-tation enhances the fon2 flower phenotype. As a result,we found that a loss-of-function mutation in ASP1 ledto an enhanced floral phenotype in fon2. In addition,fon2 asp1 double mutants showed a marked inflores-cence phenotype—namely, bifurcation of the rachis—due to massive enlargement of the inflorescence meri-stem, suggesting that FON2 and ASP1 are required forthe negative regulation of stem cell proliferation in bothflower and inflorescence meristems. Transcriptome

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analysis indicated that FON2 signaling and ASP1 ac-tion are likely to negatively regulate a common set ofgenes, such as those related to transcriptional regula-tion. Lastly, genetic analysis indicated that, unexpect-edly, TAB1 is not associated with the overproliferationof stem cells in the fon2 asp1 double mutant.

RESULTS

Phenotypes of Strain 1B-280, in Which the fon2 MutationIs Enhanced

To gain new insight into the FON signaling pathway,we initially carried out a genetic screen as describedpreviously (Yasui et al., 2017). In brief, wemutagenizedthe fon2-3 mutant with N-methyl-N-nitrosourea andscreened about 3000 M2 plants for genetic enhancers orsuppressors of the fon2 mutation. Among the mutantsobtained, we focused on strain 1B-280, which showedan enhanced floral phenotype of fon2-3.

Whereas wild-type rice flowers had one pistil, about60% of the fon2-3 flowers had two or three pistils (Fig. 1,A, B, and G), and the pistil number in 1B-280 was fur-ther increased (Fig. 1, C andG). In particular, about 20%of 1B-280 flowers produced more than four pistils, afeature that was rarely observed in the fon2-3 singlemutant (Fig. 1G).

In addition to the flower, 1B-280 showed an abnor-mality in the inflorescence. Wild-type rice inflores-cences consist of a rachis, primary and secondarybranches, and spikelets (Fig. 2, A and B). The inflores-cence in the fon2-3 single mutant was more or lessnormal (Fig. 2C). By contrast, 1B-280 exhibited amarked inflorescence phenotype in which the rachiswas bifurcated (Fig. 2D), and each bifurcated rachisproduced shorter primary branches.

We observed that the flower and inflorescence phe-notypes in 1B-280 plants were heritable through severalgenerations. These results suggest that a second sitemutation was responsible for enhancing the flowerphenotype of fon2 and for disturbing the inflorescencearchitecture in 1B-280.

Identification of the Gene Responsible for Enhancement ofthe fon2 Floral Phenotype in 1B-280

Next, we tried to identify the gene affected by thesecond site mutation in 1B-280. We noted several othercharacteristic phenotypes of 1B-280, including acutecurvature of branches and elongation of sterile lemmas,which were not observed in the fon2-3 single mutant(Supplemental Fig. S1). These characteristics are similarto those caused by an asp1mutation (Supplemental Fig.S1, D and H; Yoshida et al., 2012), raising the possibilitythat 1B-280 has a mutation in the ASP1 gene.

To test this possibility, we sequenced ASP1 in 1B-280and found that an essential nucleotide for RNA splicingwas mutated at the donor splice site of intron 14

(Fig. 3A). We therefore examined expression of theASP1 transcript in 1B-280 by RT-PCR analysis. Theexperiment using two primer sets clearly indicated thatmis-spliced ASP1 mRNA containing intron 14 wasexpressed in 1B-280 (Fig. 3B). It is therefore likely thatthis mutation in ASP1, which results in the productionof a truncated protein, is the second mutation respon-sible for enhancement of the fon2 phenotype in 1B-280.We designated this asp1 allele as asp1-fe (fe; fon2enhancer).

Next, we introduced gASP1-GFP, containing a 12.6-kb genomic fragment of theASP1 gene, into fon2-3 asp1-fe (1B-280). The resulting transformants showed rescueof the enhanced flower phenotypes in 1B-280 (Fig. 3C);the frequency of flowers with four or more pistils wasgreatly reduced in the rescued plants (Fig. 3F). Theinflorescence phenotype of 1B-280 was also rescuedby the introduction of gASP1-GFP (Fig. 3E). We thendisrupted the ASP1 gene in fon2-3 using theclustered regularly interspaced short palindromic

Figure 1. Flower phenotypes. A to F, Flower phenotypes of wild typeand the indicated mutants. Arrows indicate the pistils. Bars, 1 mm. G,Frequency of pistil number per flower. n5 50 (wild type), 100 (fon2-3),160 (1B-280), 100 (asp1-10), 100 (asp1-1), 195 (fon2-3 asp1-1); threeplants for each mutant.

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repeats/CRISPR-associated protein 9 (CRISPR/Cas9)system and obtained a biallelic mutant ofASP1 (asp1-ko;Supplemental Fig. S2). The resulting fon2-3 asp1-ko

double mutant showed a marked increase in pistilnumber; that is, it was a phenocopy of 1B-280 (Fig. 3, Dand F). Collectively, these results strongly indicated

Figure 2. Inflorescence phenotypes. A, Schematic representation of the wild-type inflorescence in rice. B to G, Inflorescencephenotypes. Bifurcation of the rachis is observed in 1B-280 (D) and fon2-3 asp1-1 (G). pb, Primary branch; ra, rachis; sb, sec-ondary branch; sp, spikelet. Bars, 2 cm.

Figure 3. Identification of the gene responsible for the enhanced phenotype of fon2 in 1B-280. A, Structure of the ASP1 gene.Boxes and lines between boxes represent exons and introns, respectively. A nucleotide substitution (open arrowhead) is present inthe 59 splice site of intron 14 ofASP1 in 1B-280 (designated asp1-fe). Closed arrowheads labeled a to c indicate the positions of theprimers used for RT-PCR in B. B, RT-PCR analysis of ASP1mRNA. a–b and a–c indicate the primer pairs used for each analysis. C,A fon2-like flower rescued by introduction of gASP1-GFP into 1B-280, showing a decrease in pistil number as comparedwith theoriginal strain 1B-280. Arrows indicate pistils. D, A flower in an ASP1-knockout line produced by CRISPR-Cas9 technology,showing an increase in pistil number as compared with the fon2-3mutant. Arrows indicate pistils. E, An inflorescence rescued byintroduction of gASP1-GFP into 1B-280. The rachis bifurcation phenotype was not observed. F, Frequency of pistil number perflower in each strain. Complementation-1 and Complementation-2 represent independent lines expressing gASP1-GFP in the 1B-280 background. n 5 212 (fon2-3), 104 (1B-280), 26 (Complementation-1), 37 (Complementation-2), 9 (fon2-3 asp1-ko). Bars,1 mm in C and D; 2 cm in E.






that the mutation in ASP1 was responsible for the en-hanced fon2 phenotype of 1B-280.

Genetic Analysis of the Enhancing Effect of asp1

To confirm the phenotype-enhancing effect of asp1, wecombined fon2-3with asp1-1, a previously isolated loss-of-function allele (Yoshida et al., 2012) that produces onepistil in its flower (Fig. 1, E and G). The resulting fon2-3asp1-1 mutant, similar to 1B-280 (fon2-3 asp1-fe), showedan increased number of pistils relative to the fon2-3 singlemutant (Fig. 1, F andG). asp1-1 produced an inflorescencewith short primary branches (Fig. 2F) as described pre-viously (Yoshida et al., 2012). The fon2-3 asp1-1 mutantexhibited a bifurcated rachis (Fig. 2G), similar to 1B-280(fon2-3 asp1-fe), but this rachis bifurcation was not ob-served in either the fon2-3or asp1-1 singlemutant (Fig. 2, Cand F). Therefore, the fon2 and asp1mutations seemed tohave a synergistic effect on inflorescence architecture.

FON1 encodes an LRR receptor-like kinase, a puta-tive receptor of FON2, and the fon1 mutant exhibitsphenotypes similar to those of fon2 (Suzaki et al., 2004,2006). We therefore checked whether the asp1mutationalso enhances the fon1 phenotype. We crossed the fon1-5mutant, harboring a loss-of-function null allele (Suzakiet al., 2008), with the asp1-10 mutant, harboring an allelebearing the same mutation as asp1-fe. The resulting fon1-5asp1-10 double mutant showed an enhanced phenotypeof fon1-5 (Supplemental Fig. S3), suggesting that ASP1functions synergistically with FON signaling.

ASP1 Is Expressed in Various Types ofAbove-Ground Meristem

Weexamined the spatial expression patterns ofASP1 inwild type by in situ hybridization. In the vegetative phase,ASP1 mRNA was expressed in the SAM and leaf pri-mordia (Fig. 4A) and in the premeristem, a transient stateduring axillary meristem formation (Tanaka et al., 2015).In the reproductive phase, ASP1 mRNA was also detec-ted in the inflorescence meristems, primary branch mer-istems, and floral meristems (Fig. 4, B–D), consistent withprevious results (Yoshida et al., 2012). Next, we intro-duced gASP1-GFP into asp1-10 and analyzed the expres-sion pattern of ASP1-GFP in the resulting transformants.ASP1-GFP protein was detected uniformly throughoutthe vegetative SAM and leaf primordia (Fig. 4, E–G).ASP1-GFP was also highly expressed in the premeristem(Fig. 4, H–J). Thus, ASP1 is expressed in various types ofthe above-ground meristems, suggesting that it hasfunctional roles in these meristems.

The Inflorescence Meristem Is Markedly Enlarged infon2 asp1

We examined the early stages of inflorescence de-velopment by using scanning electron microscopy. We

first observed the inflorescence meristem at the stagewhen some primary branch meristems had just startedto initiate. The inflorescence meristem of fon2-3 was alittle larger than that of wild type (Fig. 5, A, B, E, and F),whereas the asp1 meristem was indistinguishable fromthe wild-type meristem (Fig. 5, C and G). In fon2-3 asp1,however, the inflorescence meristem was extremelyenlarged and fasciated (Fig. 5, D and H); furthermore, itwas often split into two or three independentmeristems(Supplemental Fig. S4). A little later, when the primarybranch meristems continued to initiate, no obviousdifferences in the inflorescencemeristemwere observedamong wild type, fon2-3, and asp1 (Fig. 5, I–K andM–O). In fon2-3 asp1, by contrast, two inflorescencemeristems enclosed by a bract were formed, and eachmeristem initiated primary branch meristems (Fig. 5, Dand H). These results indicate that the bifurcated in-florescences observed in fon2-3 asp1 were caused byextreme enlargement of the inflorescence meristem,followed by a subsequent split of this meristem.

fon2 asp1 exhibited phenotypes not observed in eithersingle mutant, such as a fasciated inflorescence meri-stem, indicating that the fon2 and asp1 mutations act

Figure 4. Spatial expression patterns of ASP1 transcripts and ASP1-GFPin the meristem. A, In situ localization of ASP1 transcripts in the vege-tative shoot apex. Arrows indicate the SAM (upper) and the premeristemzone in axillary meristem development (lower). B to D, In situ locali-zation of ASP1 transcripts in the inflorescence meristem (B), primarybranch meristems (C), and floral meristems (D). E to J, Localization ofASP1-GFP in the vegetative SAM (E to G) and premeristem zone inaxillary meristem formation (H to J). The SAM, leaf primordia, andpremeristem are outlined. Transgenic plants expressing gASP1-GFP inthe asp1-10 background, showing complete rescue of the mutantphenotype, were analyzed. br, Bract; fm, floral meristem; im, inflores-cence meristem; lp, leaf primordium; pbm, primary branch meristem;sam, shoot apical meristem. Bars, 100 mm in A to D; 50 mm in E to J.


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synergistically in the control of the size of the inflores-cence meristem.To characterize the extremely enlarged inflorescence

meristems of fon2 asp1, we analyzed the expressionpatterns of ORYZA SATIVA HOMEOBOX1 (OSH1), amolecularmarker of undifferentiatedmeristematic cells(Matsuoka et al., 1993; Sato et al., 1996), and FON2,which probablymarks stem cells similar to ArabidopsisCLV3 (Fletcher et al., 1999; Suzaki et al., 2006). Similarexpression patterns of OSH1 were observed in the in-florescence meristem of wild type, fon2-3, and asp1-1 (Fig. 6, A–C). In fon2-3 asp1-fe (1B-280), by contrast,the expression domain of OSH1 was markedly ex-panded in the enlarged meristem (Fig. 6D). FON2 ex-pression was detected in the apical region of theinflorescence meristem in wild type and asp1-10 (Fig. 6,E andG). FON2was expressed in awider region in fon2-3 than in wild type (Fig. 6F), consistent with our pre-vious observation of increased stem cells in the fon1mutant (Suzaki et al., 2006). The FON2 expression do-main was markedly expanded in the enlarged inflo-rescence meristem of fon2-3 asp1, as compared withfon2-3 (Fig. 6H). Collectively, these results indicatethat the asp1mutation enhances the overproliferation ofstem cells in fon2 and suggest that FON2 and ASP1

together negatively regulate stem cell proliferation inthe inflorescence meristem in wild type.

TAB1 Is Unrelated to Extreme Enlargement of theInflorescence Meristem in fon2 asp1

In Arabidopsis, loss-of-function clv3 mutants exhibitoverproliferation of stem cells due to an expansion ofthe WUS expression domain (Brand et al., 2000;Schoof et al., 2000). We therefore explored whetherthe overproliferation of stem cells in fon2 asp1 is re-lated to WUS-like function in rice. The rice WUSortholog is TAB1, which has been shown to be re-quired for axillary meristem formation but not mer-istem maintenance (Tanaka et al., 2015).Because TAB1 is not expressed in the vegetative

SAM, we first characterized its expression in the inflo-rescence meristem. In wild type, TAB1was expressed ina small group of cells below the FON2 expression do-main (Figs. 6E and 7A). The pattern of TAB1 expressionwas comparable in asp1-10 and wild type (Fig. 7C), butit was markedly expanded in the inflorescence meristemof both fon2-3 and fon2-3 asp-1-1 (Fig. 7, B and D). Theseresults raised two possibilities: either the enhanced

Figure 5. SEM images of developinginflorescences. A to H, Developing in-florescences at an early stage of pri-mary branch meristem initiation. I to P,Developing inflorescences at a laterstage of primary branch meristem ini-tiation. Asterisks indicate primarybranch meristems. Arrowheads indi-cate bracts. im, Inflorescence meri-stem. Bars, 100 mm.





expression of TAB1 caused the overproliferation of stemcells in fon2 and fon2 asp1 or the expanded TAB1 ex-pression domain was a secondary effect due to en-largement of the inflorescence meristems in fon2 andfon2 asp1.

To distinguish between the two possibilities, we ex-amined the genetic interaction of tab1 with fon2 andasp1. If the first possibility is correct, introduction of thetab1 mutation into fon2-3 asp1-fe should lead to a re-duction of the inflorescence meristem size. We found,however, that the inflorescence meristem size of thefon2-3 asp1-fe tab1-1 triple mutant was comparable tothat of the fon2-3 asp1-fe double mutant (Fig. 7L). Sim-ilarly, mutation of tab1 did not affect the size of the in-florescence meristem in wild type, fon2-3, or asp1-fe(Fig. 7, I–K). Quantitative analysis confirmed that thefon2-3 asp1-fe double mutant showed enlargement ofthe inflorescence meristem irrespective of the presenceor absence of tab1mutation (Supplemental Fig. S5). Thephenotype of the mature inflorescence of these mu-tants was consistent with that of the inflorescencemeristem: namely, a fasciated inflorescence wasformed in the fon2-3 asp1-fe tab1-1 triple mutant as inthe fon2-3 asp1-fe double mutant (Supplemental Fig.S6). These results indicated that the tab1mutation didnot suppress the inflorescence phenotypes of fon2 andfon2 asp1. Thus, expansion of the TAB1 expressiondomain in fon2 and fon2 asp1 did not cause the ob-served overproliferation of stem cells but instead wasa secondary effect due to enlargement of the inflo-rescence meristem.

ASP1 Regulates Multiple Genes Controlled by FON2 inthe Meristem

To determine the effects of FON2 and ASP1 on mer-istem maintenance in terms of gene expression, wecarried out transcriptome profiling. Just after primarybranch meristem initiation, the inflorescence meristemswere dissected microsurgically from the shoot apices of

wild type, fon2-3, asp1-10, and fon2-3 asp1-fe, and sub-jected to microarray analysis.

We identified differentially expressed genes (DEGs)between wild type and mutants (false discovery rate,0.05, fold change. 1.5) in three biological replicates. Ascompared with wild type, 852 genes were up-regulatedin fon2, 165 genes in asp1, and 785 genes in fon2 asp1(Fig. 8A); these DEGs were termed “fon2 up,” “asp1up,” and “fon2 asp1 up,” respectively. Downregulatedgenes were similarly identified in each mutant (“fon2down,” “asp1 down,” and “fon2 asp1 down”; Fig. 8B).Notably, about 35% (58/165) of the genes in “asp1 up”were common to both “fon2 up” and “fon2 asp1 up,”indicating that a considerable proportion of the genesrepressed by ASP1 were also negatively regulated byFON signaling. Consistent with the in situ hybridiza-tion analysis, FON2 was upregulated about 2.4-fold infon2 and 4.9-fold in fon2 asp1 (Supplemental Fig. S7A).

Next, we performed Gene Ontology (GO) enrich-ment analysis of the DEGs using agriGO v2.0 (Tianet al., 2017; http://systemsbiology.cau.edu.cn/agri-GOv2/), and found that 21, 39, and 39 GO terms weresignificantly overrepresented in “asp1 up,” “fon2 up,”and “fon2 asp1 up,” respectively (Fig. 8C). Notably, allGO terms enriched in “asp1 up” were shared by “fon2up,” andmost GO terms that were enriched in “fon2 up”were more significantly overrepresented in “fon2 asp1up.” By contrast, this tendency was not observed for theGO terms enriched among the downregulated genes.Taken together, these results suggest that ASP1 andFON signaling negatively regulate a set of genes withsimilar functions.

We visualized the results of the GO analysis on thebasis of semantic similarity (Fig. 8D), which is definedby closeness in meaning among the GO terms (for re-view, see Pesquita et al., 2009). In most cases, the GOterms enriched among the upregulated genes were lo-cated close to each other, but distant from thoseenriched among the downregulated genes. Most of theGO terms that were more significantly overrepresentedin “fon2 asp1 up” than in “fon2 up” were in close

Figure 6. Spatial expression patterns ofmarker genes in the inflorescencemeristem. A to D, In situ localization ofOSH1 transcripts. E to H, In situ local-ization of FON2 transcripts. Bars,100 mm.


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proximity and constituted a cluster, marked “Geneexpression” in Figure 8D. This cluster was rich inGO terms related to transcriptional regulation in-cluding “gene expression” and “transcription, DNA-templated” (Fig. 8, C and D). Thus, the phenotypeenhancement caused by the asp1 mutation in fon2 islikely to be closely associated with transcriptionalregulation.Next, we checked the expression of rice WOX genes

in the microarray data (Nardmann et al., 2007), but nosignificant change in the expression of any WOX genewas detected in wild type or the mutants (SupplementalFig. S7B). Therefore, similar toTAB1, theseWOX genes donot seem to contribute to the fon2 asp1 phenotype. Aphytohormone, cytokinin, is also known to be involved instem cellmaintenance (Leibfried et al., 2005; Gordon et al.,2009; Chickarmane et al., 2012); again, however, the ex-pression of cytokinin-related genes, such as OsAHP,OsRR, and LOGs (Tsai et al., 2012), showed no synergisticchanges in fon2 asp1 (Supplemental Fig. S7C).To confirm the microarray results, we examined the

expression of WOX4 and LOG by in situ hybridization.Although it had very low expression in microarrayanalysis, WOX4 was examined because this WOXgene is known to be involved in meristem mainte-nance in the vegetative SAM (Ohmori et al., 2013).The results showed that both WOX4 and LOG wereexpressed in the inflorescence meristem at a similarlevel in fon2-3 asp1-fe as in wild type and the singlemutants (Supplemental Fig. S8).Finally, we considered whether any of the genes so

far reported to control inflorescence architecture areinvolved in the marked inflorescence phenotypes offon2 asp1 (Komatsu et al., 2003; Ikeda et al., 2007; Rao

et al., 2008; Huang et al., 2009; Jiao et al., 2010; Miuraet al., 2010; Ookawa et al., 2010; Tabuchi et al., 2011;Ikeda-Kawakatsu et al., 2012; Yoshida et al., 2013).Some genes, including APO2, DEP1, and LAX2, wereupregulated in fon2, asp1, or fon2 asp1, indicating thatthey are negatively regulated by FON2 or ASP1(Supplemental Fig. S7A). However, no synergisticeffect of the fon2 and asp1 mutations on these geneswas detected.Collectively, it seems that the marked inflorescence

phenotypes of the fon2 asp1mutant cannot be explainedsimply by the expression of known genes requiredfor stem cell maintenance and proper inflorescencedevelopment.

FON2 and ASP1 Are Involved in Stem Cell MaintenanceAlso in the Vegetative SAM

Lastly, we examined the effect of the fon2 and asp1mutations on vegetative development. At the latevegetative stage, fon2-3 asp1-fe often showed a switchfrom an alternate to an opposite phyllotaxy, which wasrarely observed in wild type, fon2-3, or asp1-10 (Fig. 9,A–D).Because defects in phyllotaxy are sometimes attrib-

uted to a perturbation of meristem size (Jackson andHake, 1999), we measured the size of the vegetativeSAM in each strain. The SAMof fon2-3was a little largerthan that of wild type and asp1-10 (Fig. 9, E–G, M, andN), and FON2 was widely expressed in the apical re-gion of the SAM of fon2 (Fig. 9, I–K). The expressiondomain of FON2 was extremely expanded in the SAMof fon2-3 asp1-fe (Fig. 9, H, L, M, and N). These results

Figure 7. TAB1 expression patternsand the effect of tab1 mutation on theinflorescence meristem. A to D, In situlocalization of TAB1 transcripts in theinflorescence meristem. Arrows indi-cate the signals of TAB1 expression. Eto L, SEM images of the developing in-florescence. Inflorescence meristemsare highlighted in magenta. Bars,100 mm.










Figure 8. Transcriptome analysis. A and B, Venn diagrams of genes that were upregulated (A) and downregulated (B) in eachmutant relative to wild type. C, GO terms overrepresented among the DEGs in each mutant. No GO terms were overrepresentedamong theDEGs in asp1 down. GO terms highlighted in pink or pale pinkwere included in the “Gene expression” category in theREVIGO analysis in D, but those in pale pink were removed by REVIGO from the scatter plot in D. D, Results of the GO analysisvisualized by using the REVIGO web tool (http://revigo.irb.hr/; Supek et al., 2011). Yellow, orange, red, green, and blue circlesrepresent GO terms enriched among the DEGs in asp1 up, fon2 up, fon2 asp1 up, fon2 down, and fon2 asp1 down, respectively.


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suggest that FON2 and ASP1 are involved in stem cellmaintenance, not only in the flower and inflorescencemeristems, but also in the vegetative SAM.To determine whether WOX4 or cytokinin-related

genes are responsible for the enlarged SAM in themutants, we examined the expression of these genesby reverse transcription quantitative PCR (RT-qPCR)analysis (Supplemental Fig. S9). The results showedthat WOX4, LOG, OsRR1, and OsRR5 (abphl1 ortho-log) were expressed at similar levels in wild type,fon2-3, asp1-fe, and fon2-3 asp1-fe, suggesting that theenlargement of the SAM in fon2-3 asp1-fe is unrelatedto eitherWOX4 or these cytokinin-related genes, as inthe case of the inflorescence meristem.

DISCUSSION

asp1 Is a Genetic Enhancer of fon2

We previously reported that maintenance of the flo-ral meristem is negatively regulated by FON signalingin rice, similar to CLV signaling in Arabidopsis (Suzakiet al., 2004, 2006, 2009). To identify additional genesinvolved in stem cell maintenance, we performed agenetic screen and found that the fon2 flower pheno-type was enhanced by a mutation in the ASP1 gene,which encodes a transcriptional corepressor similar toArabidopsis TPL. The characteristic phenotype of fon2,namely an increase in pistil number, was synergisticallyenhanced in the fon2 asp1 double mutant. ASP1 wasfound to be expressed in various types of above-groundmeristems, and its expression domain overlaps withthat of FON2 (Suzaki et al., 2006), suggesting that ASP1functions in stem cells together with FON2. Introduc-tion of an asp1 mutation into the fon1 mutant alsoresulted in an enhanced flower phenotype: that is, in-creased pistil number. These results suggest that ASP1and FON signaling act synergistically in the regulationof meristem maintenance in rice.The fon2 asp1 double mutant exhibited bifurcation of

the rachis, a marked phenotype that, to our knowledge,has not previously been observed in inflorescence mu-tants. This bifurcation seems to result from extremeenlargement of the inflorescence meristem (see below).This result was unexpected, because the single fon2mutation did not lead to obvious defects in the inflo-rescence meristem (Suzaki et al., 2006, and this study).Our previous study showed that FON2 SPARE1(FOS1), a paralog of FON2, functions redundantly withFON2 in maintaining the flower meristem in wild ricespecies (such asOryza rufipogon) andO. sativa indica andthat FOS1 is mutated in all strains of O. sativa japonica(Suzaki et al., 2009). The flower meristem is sensitive tofon2 mutation, because the strains that we used in our

developmental studieswere japonica. Thus, it is possiblethat an unknown gene acts redundantly in the inflo-rescence meristem, similarly to FOS1 in the flowermeristem, and that the inflorescence meristem is regu-lated more robustly than the flower meristem even injaponica strains. The massive enlargement of the inflo-rescence meristem in fon2 asp1 indicates that the asp1mutation might disrupt this robust maintenancemechanism in the inflorescence meristem.In the vegetative phase, leaves initiated in an oppo-

site phyllotaxy in the fon2 asp1 mutant, as comparedwith an alternate phyllotaxy in wild-type rice. In maize,the pattern of leaf initiation changes from an alternate toan opposite phyllotaxy in abphyl mutants owing to en-largement of the SAM (Jackson and Hake, 1999; Yanget al., 2015). Consistent with this, the vegetative SAMwas enlarged in the fon2 asp1 double mutant, relative towild type and the fon2 single mutant. Close examina-tion indicated that the SAM was in fact slightly en-larged in the fon2 single mutant, unlike our previousobservation (Suzaki et al., 2006). Because FCP1 andFCP2 act as major factors in negative regulation of theSAM (Suzaki et al., 2008; Ohmori et al., 2013), the fon2mutation seems to have less effect on this meristem ascompared with the flower meristem.Taken together, our observations indicate that ASP1

is involved in maintaining various types of above-ground meristems; however, the extent of the effect ofasp1 seems to depend on the type of the meristem,probably due to genetic redundancy complementingthe fon2 mutation. Alternatively, the exaggerated in-florescence phenotype of fon2 asp1 might be associatedwith artificial selection to obtain large inflorescencesproducing a large number of grains during domesti-cation and improvement of rice as a crop, similar tomaize ear, which is highly sensitive to td1 or fea2 mu-tation (Taguchi-Shiobara et al., 2001; Bommert et al.,2005, 2013).

ASP1 Negatively Regulates Stem Cell Proliferation inthe Meristem

Scanning electron microscopy analysis showed thatthe inflorescence meristem was enlarged and fasciatedin the fon2 asp1 double mutant and split into two orthree meristems at subsequent stages when the primarybranch meristems were initiated. The expression do-main of FON2was vastly expanded in the inflorescencemeristem of fon2 asp1, relative to fon2, suggesting thatthe stem cells have overproliferated. These findingsindicate that the asp1mutation synergistically enhancesthe enlarged inflorescence meristem in fon2 and suggestthat ASP1 plays an important role in stem cell mainte-nance. This represents clear and direct evidence that

Figure 8. (Continued.)Circle size reflects the2log10 false discovery rate (FDR) value; the larger circle is arranged to be located in the lower layer for thesame GO terms (see upper right). Semantically similar GO terms are presented close together.






TPL-like genes are involved in stem cell maintenance inthe above-ground meristems, although a mutation inREL2, an ortholog of ASP1, was recently shown to beinvolved in the control of the inflorescence meristemsize in maize (Liu et al., 2019). Interestingly, in the rootapical meristem, WOX5 prevents precocious differen-tiation of columella stem cells by recruiting TPL to thetarget gene CYCLING DOF FACTOR4, which promotescell differentiation (Pi et al., 2015).

Microarray analysis of RNAs isolated from micro-dissected inflorescence meristems revealed that a largenumber of genes are up- or down-regulated in fon2 andfon2 asp1, as compared with wild type. By contrast, fewerup- or down-regulated genes were observed in asp1. Thisresult was unexpected because TPL-like corepressors arethought to regulate a large number of genes through in-teractions with various transcription factors (Causieret al., 2012). ASP1-RELATED1, a paralog of ASP1, wasupregulated in asp1 (Supplemental Fig. S7A), raising thepossibility that loss of ASP1 function may be compen-sated by up-regulation of ASP1-RELATED1. It seems,therefore, that a single asp1 mutation would not neces-sarily lead to marked changes in gene expression andmeristem enlargement.

Approximately half (73 of 165) of the genes upregu-lated in asp1were also upregulated in fon2, as comparedwith wild type. This suggests that a substantial pro-portion of the genes repressed by the corepressor actionof ASP1 are also repressed directly or indirectly by FONsignaling. In addition, GO analysis indicated that ASP1and FON2 negatively regulate genes involved in similarbiological processes, such as transcriptional regulation.FON signaling probably acts to fine-tune the expressionof genes responsible for stem cell proliferation in rice,

similar to CLV signaling in Arabidopsis (Schoof et al.,2000; Reddy and Meyerowitz, 2005). It is possible thatmoderate expression levels of those genes are effectivefor this fine-tuning. In this scenario, genes regulated byFON signaling might be partially suppressed by ASP1expression at a moderate level in wild type. In the fon2asp1 double mutant, release from both negative fine-tuning by FON and moderate suppression by ASP1might lead to the observed synergistic effect on the mer-istem.Alternatively, it is possible that the synergistic effectof fon2 and asp1mutationsmight result from the defects inother complex genetic networks, including an ASP1 ac-tion independent of FON signaling.

The expression of WOX genes including WOX4 andthat of the cytokinin-related genes examined so far wasnot altered in these mutants relative to wild type.Similarly, the expression of genes associated with largemeristems and inflorescences, such as APO1/ STRONGCULM2 andDEP1 (Ikeda et al., 2007; Huang et al., 2009;Ookawa et al., 2010), was not affected by fon2 and fon2asp1 mutations. It is possible that, by physically inter-acting with various transcription factors, ASP1 mayrepress multiple genes at a relatively low level; in thiscase, the effect of asp1 mutation on individual genesmight be difficult to observe, but the cumulative effectwould result in a marked enlargement of the inflores-cence meristem in fon2 asp1.

Putative Roles of TAB1 and ASP1 in StemCell Maintenance

In Arabidopsis, WUS promotes stem cell prolifera-tion in all types of above-ground meristem, such as the

Figure 9. Phenotypes of the vegetativeSAM. A to D, Phyllotaxy phenotypes. Ar-rowheads indicate the nodes of the flagleaves. E to H, The SAM after treatmentwith a clearing agent. I to L, In situ lo-calization of FON2 transcripts. M and N,Quantification of SAM width (M) andheight (N). n5 20 (wild type), 18 (fon2-3),16 (asp1-10), 20 (fon2-3 asp1-fe). Differ-ent letters (a, b, and c) indicate significantdifferences between samples (P , 0.01,Tukey’s test). fl, flag leaf. Bars, 1 cm in A toD; 50 mm in E to H; 100 mm in I to L.


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vegetative SAM and inflorescence/flower meristems,and its expression domain is expanded in the enlargedmeristems of the clv1 and clv3 mutants (Mayer et al.,1998; Brand et al., 2000; Schoof et al., 2000). By contrast,TAB1, the rice ortholog of WUS, is not expressed in thevegetative SAM, and loss of function of TAB1 does notaffect primary shoot growth, suggesting that TAB1 isnot involved in SAMmaintenance (Tanaka et al., 2015).Instead, TAB1 has a critical role in the initial stages ofaxillary meristem formation.In contrast to the vegetative SAM, however, TAB1

was expressed in the inflorescence meristem, and itsexpression domain was expanded in fon2 asp1. Thus,we hypothesized that overexpression of TAB1might bea major factor in the extreme enlargement of the inflo-rescence meristem in fon2 asp1. Surprisingly, however,genetic analysis revealed that this meristem abnor-mality was not suppressed in the fon2 asp1 tab1 triplemutant, indicating that our hypothesis should be dis-missed. The fon2 asp1 phenotype seems to result fromcumulative changes in multiple genes due to the lack ofASP1 function as a transcriptional corepressor, asdiscussed above.In general, WUS-like gene promotes stem cell activ-

ity, and its loss-of-functionmutation leads to prematuretermination of the meristem (Laux et al., 1996; Mayeret al., 1998; Kieffer et al., 2006). However, genetic in-troduction of the tab1 mutation did not lead to an ob-vious significant effect on inflorescencemeristem size inwild type, fon2, asp1, or fon2 asp1. This result clearlyindicated that TAB1 is not involved in promoting stemcell proliferation in the inflorescence meristem, eventhough it is expressed in this meristem. Thus, weshowed that the function of TAB1 in rice differs fromthat of WUS in Arabidopsis, as described in our previ-ous study (Tanaka et al., 2015). In Arabidopsis, agenome-wide study revealed that WUS regulates theexpression of several genes, including CLV1, TPL, andTPRs, suggesting that SAM maintenance is modulatedby a complex regulatory network (Busch et al., 2010). Inaddition, WUS physically interacts with TPL-like co-repressors (Kieffer et al., 2006). In rice, the physical in-teraction of TAB1 and TPL-like proteins has beenreported (Lu et al., 2015). Therefore, we cannot excludethe possibility that TAB1 has some role in meristemmaintenance in rice, although our genetic analysis in-dicated that it is not a key gene in this regulation.Similar to rice, REL2 is associated with inflorescence

architecture in maize. In addition, the recent reportsuggested the possibility that REL2 is involved in thecontrol of meristem maintenance (Liu et al., 2019), asdescribed above. It will be of great interest to deter-mine how REL2 interacts genetically with regulatorsof stem cell maintenance such as FEA2 and TD1 inmaize. Genetic redundancy among TPL-like genesseems to be relatively low in rice and maize, becausesingle mutation and/or combination with anothergene results in phenotypic alterations (Gallavottiet al., 2010; Yoshida et al., 2012; Liu et al., 2019). Itis therefore expected that our understanding of the

function of individual TPL-like genes in stem cellmaintenance and other developmental processes willprogress in rice and maize, in analogy to the discov-ery of a new signaling pathway that regulates stemcell maintenance in addition to CLV signaling (Suzakiet al., 2008; Je et al., 2016).

MATERIALS AND METHODS

Plant Materials

Rice (Oryza sativa. ssp. japonica) variety Taichung 65 (T65) was used as thewild type. The fon2-3, asp1-1, and fon1-5mutants have been reported previously(Yamaki et al., 2005; Suzaki et al., 2006, 2008; Yoshida et al., 2012). fon2-3 has apoint mutation at a conserved amino acid in the CLE domain and exhibitsflower phenotypes very similar to those of a deletion allele. 1B-280 (fon2-3 asp1-fe) was identified in the genetic screen described by Yasui et al. (2017). asp1-10(TCM697) was identified as a mutant showing acute curvature of branches in acollection of mutants (Institute of Genetic Resources, Faculty of Agriculture,Kyushu University) obtained by N-methyl-N-nitrosourea treatment of T65 andsubsequently confirmed to contain a mutation in ASP1 by direct sequencing.tab1-1was isolated by the TILLING (targeting induced local lesions in genomes)approach and had a nucleotide substitution at the splice site of the first intron,leading to premature termination of the protein (Tanaka et al., 2015). All strainsused in this study, except for asp1-1 (Nipponbare background), had the samegenetic background as T65.

RT-PCR and RT-qPCR Analyses

For RT-PCR analysis of ASP1 mRNA, total RNA was extracted from T65,fon2-3, and fon2-3 asp1-fe using TRIsure (BIOLINE). After DNase I treatment,first-strand cDNA was synthesized from 150 ng of total RNA using the Su-perScript III First-Strand Synthesis System (Thermo Fisher Scientific) and theoligo(dT)15 primer. RT-PCR was then performed with primers listed inSupplemental Table S1.

For RT-qPCR, shoot apices of 8-week-old plants including SAMs and P1–P3leaf primordia were pooled (10 apices per pool) and used for RNA isolation.Total RNA extraction and first-strand cDNA synthesis were performed as de-scribed above. RT-qPCR was carried out on three biological replicates using anApplied Biosystems 7300 Real-Time PCR System with Power SYBR GreenMaster Mix (Thermo Fisher Scientific). The primers used for RT-qPCR analysisare listed in Supplemental Table S1. ACTIN1 was used as an internal control.

Plasmid Construction and Transformation

To construct gASP1-GFP, a 5-kb genomic fragment of the ASP1 upstreamregion was amplified with the primers 59-CACCCTCACACGGCCGATGGTACG-39 and 59-GGCTCCGCCGATCCCAGCCT-39, and the PCR product wascloned into a pENTR/D-TOPO vector (Thermo Fisher Scientific). The resultingplasmid, designated pENTR-pASP1, was digested with SacI (in the 59 UTRregion of ASP1), blunted, and then digested with AscI (just upstream of theattL2 site in the original vector) for subsequent ligation. Next, an approximate7.8-kb genomic fragment containing the entire ASP1 gene (25 exons and 24introns) except the stop codon was amplified with the primers 59-AGCTCTGGGTTTATTAATTTTTTTTGG-39 and 59-AAAAGGCGCGCCCGACTTCTGGTTTGTTAGCTG-39 (the AscI site is underlined). The blunt-end PCR product,digested withAscI, was ligatedwith pENTR-pASP1. The resulting plasmid wasconfirmed to carry an approximate 12.6-kb genomic sequence comprising the 5-kb upstream region and the entireASP1 gene except for the stop codon by directsequencing. The 12.6-kb fragment was inserted into pGWB4, a binary vectorcarrying an sGFP gene (Nakagawa et al., 2007), by LR recombination (ThermoFisher Scientific) to produce gASP1-GFP.

To prepare a construct for knockout of theASP1 gene using theCRISPR-Cas9system, a 20-bp sequence in the second exon was selected as the guide RNA(gRNA) target site (Supplemental Fig. S2). The target sequence was theninserted into an all-in-one vector carrying the Cas9/gRNA expression cassette,derived from pU6gRNA-oligo and pZH_OsU3gYSA_MMCas9, according tothe method of Mikami et al. (2015).








The constructs were introduced into Agrobacterium tumefaciens EHA101strain and transformed into scutellum-derived calli according to the method ofHiei et al. (1994). fon2-3 asp1-fe, asp1-10, and fon2-3 were used as hosts forcomplementation, observation of ASP1-GFP localization, and knockout ofASP1, respectively.

In Situ Hybridization

The two probes for detection of ASP1 transcripts were prepared as follows.Partial cDNA fragments were amplified with the primer pair 59-AATCGGGTCCAAAAAAACCAAAAGC-39 and 59-TCCAGCTCTCAAAGCCGAACT-39 forprobe1 (located in the 59 UTR) and 59-CATACTTACAACAACAGATTGTGACGG-39 and 59-GCTGGTTGCCACATTTGAGG-39 for probe2 (located in theregion between the two b-propeller domains). The PCR products were thencloned into a pCRII vector (Thermo Fisher Scientific). The resulting plasmidswere linearized and transcribed with Sp6 and T7 RNA polymerase for probe1and probe2, respectively, by using a DIG RNA Labeling Kit (Roche).

The FON2 probe was transcribed as described previously (Suzaki et al.,2006) and then treated by alkaline hydrolysis. The OSH1, TAB1, WOX4, andLOG probes were prepared as described by Yasui et al. (2017), Tanaka et al.(2015), Ohmori et al. (2013), and Kurakawa et al. (2007), respectively.

Plant tissues were fixed and dehydrated as described by Toriba and Hirano(2018). Next, the samples were embedded in Paraplast Plus (McCormick) andsectioned at a thickness of 10 mm using a microtome. In situ hybridization andimmunological detection were carried out as described in Toriba and Hirano(2018).

Imaging and Measurement of Meristems

To observe ASP1-GFP fluorescence, shoot apices of transgenic plants (T0generation) were embedded in 5% (w/v) agar and sliced into 30-mm sectionsusing a vibratome. GFP fluorescence was observed with a confocal microscope(LSM510, Zeiss).

Inflorescence meristems were observed by scanning electron microscopy asdescribed by Tanaka et al. (2015). A top view of the oval-shaped inflorescencemeristem was used for measurement of the major and minor axes by ImageJ(https://imagej.nih.gov/ij/). Tomeasure SAM size, shoot apices of 8-week-oldplants were dissected and fixed in acetic alcohol (1:3). After treatment with aclearing agent (16 g of chloral hydrate dissolved in 8 mL of 25% [v/v] glycerol),the samples were observed under DIC optics. The SAM size was measured justabove the P1 primordium by using ImageJ.

Microarray Experiments

Inflorescencemeristems at the In1 – In2 stage (Itoh et al., 2005) including 1 – 2bracts of each strain were pooled (nine per pool) and used for RNA isolation.Total RNA was extracted with TRIsure (BIOLINE) and treated with DNaseI.Microarray analysis was carried out using the Rice (US) gene 1.0 ST array(Thermo Fisher Scientific) as described by Yasui et al. (2018) with three bio-logical replicates per assay. The resulting data were analyzed by using R soft-ware and the Bioconductor package limma (Ritchie et al., 2015). GO enrichmentanalysis was performed with agriGO v2.0 (Tian et al., 2017; http://systemsbiology.cau.edu.cn/agriGOv2/), and the results were visualized byREVIGO (Supek et al., 2011; http://revigo.irb.hr/) and the R package ggplot2(Wickham, 2016).

Statistical Analyses

Statistical analysis was performed using R (version 3.5.0; http://www.r-project.org/). Tukey’s test was performedwith the “TukeyHSD” function of thepackage “stats.” P values, 0.05 were considered to be significant. Sample sizeis indicated in the figure legends.

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBL/DDBJdatabases under the following accession numbers: AB245090 (FON2),AB638269 (ASP1), D16507 (OSH1), AB218894 (TAB1), JF836159 (WOX4), andAK071695 (LOG). All microarray data were deposited in the Gene ExpressionOmnibus (https://www.ncbi.nlm.nih.gov/geo/) under accession numberGSE120489.

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. asp1-like phenotypes in rice strain 1B-280.

Supplemental Figure S2. Generation of a knockout line of ASP1 in thefon2-3 mutant by the CRISPR-Cas9 system.

Supplemental Figure S3. Phenotypes of fon1-5 asp1-10.

Supplemental Figure S4. Split inflorescence meristems in fon2-3 asp1-fe.

Supplemental Figure S5. Effects of the tab1 mutation on inflorescencemeristem size.

Supplemental Figure S6. Effects of the tab1 mutation on inflorescencephenotype.

Supplemental Figure S7. Relative expression levels of genes frommicroarray data.

Supplemental Figure S8. Expression patterns of WOX4 and LOG.

Supplemental Figure S9. Expression levels of WOX4, LOG, and OsRRs inthe vegetative SAM.

Supplemental Table S1. Primers used in this study.

ACKNOWLEDGMENTS

The authors thank Toshihiro Kumamaru, Dai-Suke Sato, Taiyo Toriba,Takuya Suzaki, and Akiko Yoshida for preparing plant materials; KyokoOhashi-Ito and Yukiko Sugisawa for microarray analysis; Masaki Endo andMasafumi Mikami for kindly providing the pU6gRNA-oligo and pZH_OsU3-gYSA_MMCas9 vectors; Mitsutomo Abe for technical support; Eiko Oki andAkiko Takahashi for technical assistance; and technicians at the Institute forSustainable Agro-ecosystem Services of the University of Tokyo forcultivating rice.

Received April 10, 2019; accepted April 29, 2019; published May 11, 2019.

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