TAWAWA1, a regulator of rice inflorescencearchitecture, functions through the suppressionof meristem phase transitionAkiko Yoshidaa,1, Masafumi Sasaoa,1, Naoko Yasunoa,1, Kyoko Takagib, Yasufumi Daimona, Ruihong Chena,Ryo Yamazakia, Hiroki Tokunagaa, Yoshinori Kitaguchia, Yutaka Satoc, Yoshiaki Nagamurac, Tomokazu Ushijimad,Toshihiro Kumamarue, Shigeru Iidab, Masahiko Maekawaf, and Junko Kyozukaa,2

aGraduate School of Agriculture and Life Sciences, The University of Tokyo, Yayoi, Bunkyo, Tokyo 113-8657, Japan; bNational Institute for Basic Biology,National Institutes of Natural Sciences, Okazaki 444-8585, Japan; cNational Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan; dFaculty ofAgriculture, Kyusyu University, Hakozaki, Fukuoka 812-8581, Japan; eInstitute of Genetic Resources, Faculty of Agriculture, Kyusyu University, Hakozaki,Fukuoka 812-8581, Japan; and fResearch Institute for Bioresources, Okayama University, Kurashiki, Okayama 710-0046, Japan

Edited by Sarah Hake, University of California, Berkeley, CA, and approved November 29, 2012 (received for review September 17, 2012)

Inflorescence structures result from the activities of meristems, whichcoordinate both the renewal of stem cells in the center and organformation at the periphery. The fate of a meristem is specified at itsinitiation and changes as the plant develops. During rice inflores-cence development, newly formed meristems acquire a branchmeristem (BM) identity, and can generate further meristems orterminate as spikelets. Thus, the form of rice inflorescence isdetermined by a reiterative pattern of decisions made at themeristems. In the dominant gain-of-function mutant tawawa1-D,the activity of the inflorescence meristem (IM) is extended andspikelet specification is delayed, resulting in prolonged branch for-mation and increased numbers of spikelets. In contrast, reductionsin TAWAWA1 (TAW1) activity cause precocious IM abortion andspikelet formation, resulting in the generation of small inflores-cences. TAW1 encodes a nuclear protein of unknown function andshows high levels of expression in the shoot apical meristem, theIM, and the BMs. TAW1 expression disappears from incipient spike-let meristems (SMs). We also demonstrate that members of theSHORT VEGETATIVE PHASE subfamily of MADS-box genes func-tion downstream of TAW1. We thus propose that TAW1 is a uniqueregulator of meristem activity in rice and regulates inflorescencedevelopment through the promotion of IM activity and suppres-sion of the phase change to SM identity.

ALOG family | meristem identity | grain yield

The timing of each meristem phase change is crucial in thecontrol of inflorescence architecture (1–3). In grass species,

the basic architecture of inflorescence is defined by the spatialarrangement of spikelets, which are small branches containinga variable number of flowers. Among the meristems that aregenerated from the primary inflorescence meristem (IM) duringinflorescence (panicle) development, early ones acquire an in-determinate branch meristem (BM) identity, whereas later onesare specified as determinate spikelet meristems (SMs) (Fig. 1A) (4,5). The BMs themselves are eventually transformed into SMs aftergenerating a certain number of branches and spikelets (Fig. 1B).Thus, the pattern of SM specification is a primary determinant ofgrass inflorescence form. Delays in SM specification lead to iter-ations of branching, resulting in larger panicles that could poten-tially produce more grain. Conversely, the acceleration of SMspecification results in smaller panicles with fewer spikelets. Riceinflorescence development exhibits an additional unique feature inthat the IM loses its activity after producing several BMs, leavinga vestige at the tip of the rachis, the inflorescence main stem (Fig.1A). Therefore, the timing of IM abortion is also a critical factordetermining the form of rice inflorescence.The competence of a meristem to become an SM gradually

increases during inflorescence development; however, the molec-ular basis for the timing of SM specification is largely unknown.

To date, several genes that control inflorescence form throughthe control of meristem phase change have been reported in grassspecies including rice and maize. Three maize genes, RAMOSA1(RA1) and RA2 (encoding transcription factors) and RA3 (en-coding a trehalose-6-phosphate phosphatase), are positive reg-ulators of SM determination and the shift from long-branchinitiation to short-branch initiation (6–9). The mechanisms bywhich these RA genes exert their functions are not well-understood;however, RA1 interacts with a transcriptional corepressor.Orthologs of RA2 and RA3 (but not RA1) are found in therice genome, but their functions are unknown. The rice genesABERRANT PANICLE1 (APO1) and APO2 are orthologs of theArabidopsis genes UNUSUAL FLORAL ORGANS (UFO) andLEAFY (LFY), respectively, and they negatively regulate theshift to SM identity (10–12). In Arabidopsis and other eudicotspecies, UFO and LFY promote determinate floral meristemidentity (10). Thus, APO1 and APO2, which negatively regulatethe shift toward determinacy, play opposing roles to those ofUFO and LFY. The SM is found specifically in grass species andis an intermediate phase between the indeterminate BM andthe floral meristem. This suggests that the genetic frameworkof inflorescence development may have diverged between theeudicots and the grasses. APO1 was also identified as a majorquantitative trait locus (QTL) controlling grain number in the ricepanicle, suggesting a tight link between inflorescence branchingpattern and yield (13). QTL analyses of genes controlling grainnumber also identified DENSE PANICLE1, GRAIN NUMBER1,and OsSPL14 as regulators of inflorescence branching. However,these traits have not been considered from the perspective ofmeristem activity and identity (14–17). To gain a better under-standing of the molecular basis of meristem phase change, weanalyzed mutations of the TAWAWA1 (TAW1) gene, and proposeTAW1 as a unique regulator of SM phase transition.

Results and Discussiontawawa1-D Plants Form Inflorescences Exhibiting an Increased BranchingPhenotype. To understand the molecular links between the controlof meristem phase transition and inflorescence structure, we

Author contributions: J.K. designed research; A.Y., M.S., N.Y., K.T., Y.D., R.C., R.Y., H.T.,Y.K., Y.S., and M.M. performed research; T.U. and T.K. contributed new reagents/analytictools; A.Y., M.S., N.Y., K.T., Y.D., R.C., R.Y., H.T., Y.K., Y.S., Y.N., S.I., and M.M. analyzeddata; and J.K. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1A.Y., M.S., and N.Y. contributed equally to this work.2To whom correspondence should be addressed. E-mail: akyozuka@mail.ecc.u-tokyo.ac.jp.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1216151110/-/DCSupplemental.

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identified mutants with altered inflorescence branching patterns.Two lines with increased branching phenotypes were isolatedfrom a screening population in which nDart1, an endogenous rice

transposon, actively transposes (Fig. 1C) (18). The mutation wasinherited in a semidominant manner in both lines, and sub-sequent analyses revealed mutations in the same gene. The gene

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Fig. 1. Characterization of taw1 dominant mutants.(A) Schematic of a rice inflorescence, called a “pani-cle.” DTP, degenerate tip of the rachis; LS, lateralspikelet; PB, primary branch; SB, secondary branch; TS,terminal spikelet. (B) A conceptualized view of meri-stem identity specification. A newly formed meristem(green) acquires either a spikelet meristem (pink)identity or a branch meristem (blue) identity. The SMproduces a flower and terminates. The BM continuesto produce next-order meristems that repeat the samestep. The BM eventually acquires an SM identity andgrows as a terminal spikelet. Black arrows indicateinitiation of an additional meristem. (C) Inflorescenceof taw1-D mutants. (D) Number of lateral meristemsgenerated on a primary branch. The blue bottombar indicates secondary branches and the pink up-per bar indicates lateral spikelets. Asterisks indicatea significant difference from corresponding control(WT) samples (Student’s t test; **P < 0.01). (E) Fre-quency of tertiary branch formation. In D and E, n =27, 28, and 25 for WT, taw1-D2/+, and taw1-D2, re-spectively. (F) Immature inflorescence of the taw1-D1homozygous plant. (G) Close-up of the taw1-D1 in-florescence shown in F. (H and I) taw1-D2 homozy-gous inflorescence. An aggregation of branchmeristems boxed in H is shown in I. (J–M) Expressionof spikelet meristem marker genes FRIZZY PANICLE (Jand K) and LEAFY HULL STERILE1 (L and M) in wild-type (J and L) and taw1-D1 homozygous plant (K andM) inflorescences at the spikelet meristem initiationstage. (Insets) A spikelet meristem is shown.

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was named TAWAWA1 (TAW1), from a traditional Japaneseword meaning “very fruitful.” taw1-D1 exhibits more severe defectsthan those of taw1-D2. Both mutant lines show normal growthpatterns during the vegetative phase, for example, meristem size,date of leaf initiation, and number of leaves produced. However, intaw1-D1 homozygous plants, stem elongation is suppressed afterthe transition to reproductive growth, and the inflorescence doesnot emerge from the leaves (Fig. S1). In both mutant lines, thenumber of lateral meristems produced on each primary branch iscomparable to that of wild-type plants; however, a higher per-centage of lateral meristems grows as secondary branches (Fig.1D). Reiteration of this pattern results in the production of tertiarybranches, which are not formed on wild-type plants (Fig. 1E).In homozygous taw1-D1 mutants (carrying the more severe al-

lele), the increase in inflorescence branching is so extreme that theinflorescence forms an agglomerate with a massive number of un-differentiated meristems (Fig. 1 F and G). Such aggregated mer-istems are also frequently observed in the inflorescences of taw1-D1heterozygous and taw1-D2 homozygous plants (Fig. 1 H and I).Observations using a scanning electron microscope confirmed thatthese structures are formed from the repeated production of un-differentiated meristems (Fig. S2). The meristems in taw1-D1 ho-mozygous inflorescences were analyzed for expression of two SMmarker genes, FRIZZY PANICLE (FZP) and LEAFY HULLSTERILE1 (LHS1)/OsMADS1 (19–21), by in situ hybridization. Thelack of expression of these genes indicated that the meristems in thehomozygous mutants do not acquire SM identity (Fig. 1 J–M).

TAW1 Encodes a Nuclear Protein Belonging to the ALOG Family. TheTAW1 gene was isolated by transposon tagging. An nDart1

insertion (nDart1-0) showed complete cosegregation with themutant phenotype and was identified by transposon display analysisusing segregating siblings of taw1-D1/+ (Fig. 2A). The transposonwas inserted into chromosome 10, between Os10g0477800 andOs10g0478000. Interestingly, the insertion sites in taw1-D1 andtaw1-D2 were only 16 bp apart (Fig. S3A). Rough mapping alsonarrowed the TAW1 locus to this region. Furthermore, revertantsobtained from taw1-D1 homozygotes (which became taw1-D1/+)and from taw1-D1/+ (which reverted to wild-type) were accom-panied by excisions of nDart1-0 (Fig. S3 B and C). These resultsindicated that the taw1-D defects were caused by the nDart1-0insertions in the region between Os10g0477800 and Os10g0478000.Expression of Os10g0478000 was enhanced in the mutant in-florescences, whereas Os10g0477800 and Os10g0478100 wereexpressed at similar levels in the inflorescences of mutant andwild-type plants (Fig. 2B). Introduction of the genomic regioncontaining Os10g0478000 into wild-type rice plants resulted ina weak taw1-D–like mutant phenotype (Fig. S4). Based on theseresults, we concluded that Os10g0478000 is the TAW1 gene.TAW1 encodes a protein of 205 amino acids belonging to the

ALOG family, which is named after its earliest identified mem-bers (Arabidopsis LSH1 and Oryza G1) (22, 23). The rice genomecontains 10 ALOG genes, whereas Arabidopsis has 11 members

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Fig. 3. Expression pattern of TAW1. (A and B) Localization of TAW1-GFPfluorescence in the rice shoot at the vegetative stage. The TAW1-GFP constructwas expressed under the control of regulatory sequences from the TAW1gene. The construct was introduced into taw1-3 and the rescue of the mutantphenotype was confirmed. A part of the young leaf boxed in A is shown in B.(C–G) In situ hybridization analysis of TAW1 expression in the vegetative shootapex (C), inflorescence meristem (D), very young inflorescence at the earlyprimary branch initiation stage (E), very young inflorescence at the late pri-mary branch initiation stage (F), and an immature inflorescence at the spikeletmeristem initiation stage (G) in wild-type plants. White arrowheads, leaf pri-mordia; red arrowheads, primary branch meristem. (H and I) TAW1 expressionin the taw1-D1 homozygous inflorescence at the SMmeristem initiation stage.The TAW1 signal was more intense in taw1-D1 than in wild-type and the signalcontinued in the immature panicle at the stage when SMs initiate (H) and evenat later stages (I). (J) Quantitative RT-PCR analysis of TAW1 expression in theinflorescence at the spikelet initiation stage. The values represent mean TAW1expression relative to UBIQUITIN ± SD (n = 3).

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(Fig. S5 A and B) (22–26). To obtain a better understanding ofTAW1 function, we obtained two independent loss-of-functionmutants, taw1-3 and taw1-4, from a TILLING population. Thesemutants each carry a missense mutation at the same position(Fig. S5A). taw1-4 mutants have no obvious defects; however,taw1-3 mutants produce small inflorescences containing reducednumbers of primary branches (Fig. 2 C and D). A reduction inTAW1 expression by RNAi resulted in a similar but strongersmall inflorescence phenotype in which both primary and sec-ondary branches were reduced (Fig. 2E). In the developing riceinflorescence, the IM, which is derived from the shoot apicalmeristem (SAM), eventually aborts after producing severalprimary branches, and is left as a vestige at the base of thetopmost primary branch (Fig. 1A). Thus, the timing of IM abortionis a principal determinant for the total number of primary branchesin the rice inflorescence. It is likely that the phenotypes in theseloss-of-function and knockdown mutants result from the earlyonset of IM abortion and the precocious specification of SMidentity. These results again suggest that TAW1 regulates the extentof inflorescence branching through the maintenance of IM activityand suppression of the transition to SM identity. Enhanced TAW1activity results in a prolonged branching phase, whereas reducedTAW1 activity accelerates IM degeneration and the acquisition ofSM identity, giving rise to an abbreviated branching phase.

TAW1 Is Expressed in the Meristem to Suppress the Transition to SMIdentity. TAW1 contains a conserved domain and a nuclear lo-calization signal (Fig. S5A). Indeed, TAW1 localizes to the nucleusand shows slight but significant activity as a transcriptional activator(Fig. 3 A and B and Fig. S6). We examined the spatiotemporalpattern of TAW1 mRNA accumulation by in situ hybridization.During the vegetative phase of development, TAW1 was pre-dominantly expressed in meristems, including the SAM, axillarymeristems, and young leaves (Fig. 3 A and C). After transition tothe reproductive phase, TAW1mRNA continued to accumulate inthe IM (Fig. 3D). The strongest signal was observed in BMs in thegrowing inflorescence (Fig. 3E). After initiation of the primaryBMs, TAW1 expression gradually disappeared from the IM as itdegenerated (Fig. 3F). In wild-type inflorescences, the signal in-tensity in the meristem gradually decreased and became un-detectable at SM initiation (Fig. 3G). On the other hand, in taw1-D inflorescences, stronger TAW1 signals were detected, even atthe stage when the SMs were formed in the wild-type plants (Fig.3 H and I). Quantitative PCR analysis indicated that the levels ofTAW1 expression in the mutant inflorescences roughly coincidedwith the severity of their phenotypes (Fig. 3J). These results implythat TAW1 functions to suppress SM identity and that its activitymust be below a certain threshold level to allow SM specification.Interestingly, TAW1 expression is reduced in the vegetative shootapices, leaves, and roots of taw1-D mutants compared with wild-type plants (Fig. S7). These results suggest that the sequencesurrounding the nDart1 insertion sites is necessary for fine-tuningthe spatial and quantitative expression pattern of TAW1. It may bethat positive regulators of SM identity interact with this sequence,which is located downstream of the transcribed region. It is alsopossible that nDart1 carries a sequence that functions as a tran-scriptional enhancer; however, this is unlikely because not all nDart1insertions activate transcription. Unraveling the molecular mecha-nisms that regulate TAW1 transcription will shed light on the regu-latory networks controlling rice inflorescence architecture.The rice genes G1 and TH1 are ALOG family members that

are expressed in the lemma, palea, and floral organs and controltheir identity and development (23, 26). On the other hand, theArabidopsis ALOG family genes LSH3/OBO1 and LSH4 areexpressed in shoot organ boundary cells (24, 25). The functionsof LSH3/OBO1 and LSH4 are unknown; however, they inducethe overproliferation of shoot meristems and the formation ofextra organs when ectopically expressed (25). A plausible

scenario is that at least some of the ALOG family proteins shareconserved functions, but it may be that their distinctive expres-sion patterns have led to divergent functions. The establishmentof the machinery for meristem-specific expression may be a pri-mary reason for the evolution of TAW1 as a major regulator ofmeristem activity and phase transition.

TAW1 Positively Controls the SVP Family MADS-Box Genes. We nextperformed a transcriptome analysis using a microarray contain-ing genome-wide cDNAs to discover genes that function down-stream of TAW1 (Fig. 4A). Among the 32 type I MADS-boxgenes present in the microarray, three genes (OsMADS22,OsMADS47, and OsMADS55) were extensively up-regulated intaw1-D mutants. These three genes belong to the SHORT VEG-ETATIVE PHASE (SVP) subfamily (27–29). In contrast, genesinvolved in spikelet initiation and spikelet organ development,such as OsMADS7 (SEP3), OsMADS8 (SEP3), OsMADS16 (AP3),OsMADS4 (PI), OsMADS3 (AG), and OsMADS58 (AG), showedsignificant reductions in expression, in keeping with the prolonged

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branching phenotype. Ectopic expression of OsMADS47 in the BMin which TAW1 is expressed was confirmed by in situ hybridizationanalysis (Fig. 4 B and C). These results, together with the knownfunction of SVP genes as suppressors of flowering and in-florescence development (30–33), suggest that the three SVPgenes work downstream of TAW1 to suppress SM identity.Using a two-component induction system, we confirmed thatthe induction of TAW1 transcription does indeed lead to theactivation of the three SVP genes (Fig. 4D). Furthermore, theconstitutive overexpression of OsMADS22 and OsMADS55 bythe cauliflower mosaic virus (CaMV) 35S promoter caused anincrease in inflorescence branching (Fig. 4E). These results supportthe hypothesis that SVP genes function downstream of TAW1.Interestingly, the ectopic expression of SVP genes in barley andmaize also resulted in the severe suppression of SM identity,suggesting that SVP function might be conserved among grasses(34–36). In conclusion, our data indicate that TAW1 suppresses theacquisition of SM identity through the induction of SVP genes,which consequently promote or prolong BM identity.

taw1-D2 Is a Moderate Gain-of-Function Allele That May Be Useful forIncreasing Grain Yields. Grain number per panicle is one of thefour major determinants of rice yield. To test the usefulness ofTAW1 mutations for increasing grain yield, we introgressed themoderate taw1-D2 allele into Koshihikari, which is a leadingcommercial rice cultivar in Japan (Fig. 5 A and B). Field-grownselfed progeny of fifth backcross (BC5F2)-generation plantsshowed ∼45% increases in grain weight per plant (Fig. 5C).These increases were mainly due to extensive increases in grainnumber per panicle (Fig. 5D), resulting from increases in thenumbers of primary, secondary, and tertiary branches (Fig. S8 A–C). Slight decreases in fertility and grain weight were also ob-served (Fig. 5E and Fig. S8D). Despite the increase in paniclesize, the total plant height was slightly reduced in BC5F2 plants

(Fig. S8 E–H). Increases in grain number are often linked todecreases in the numbers of branch shoots, called “tillers,” in rice(16, 17). However, this tradeoff effect was not observed in thetaw1-D2 introgression line (Fig. S8I).

Conclusion and Perspectives.We propose that TAW1 regulates theinflorescence architecture of rice through the promotion of IMactivity and suppression of the phase change to SM identity.The gradual decrease in TAW1 activity during development inwild-type plants may contribute to the increase in competencefor acquiring SM identity. Fine-tuning of the temporal TAW1expression pattern is critical for normal rice inflorescence de-velopment. TAW1 function might have evolved to ensure theproduction of sufficient numbers of spikelets. Unraveling themolecular function of TAW1 will provide an opportunity fora greater understanding of meristem activity and identity, whichis a fundamental issue in plant biology. Furthermore, suchknowledge could be exploited to further increase crop yields. Inaddition, TAW1 is of great interest with respect to the evolutionof inflorescence architecture (37). TAW1 function may be es-sential for the development of compound inflorescences, inwhich the branching pattern is crucial.

Materials and MethodsPlant Materials. taw1-D2 was introduced into Koshihikari through successivebackcrossing five times. Seeds of the resultant BC5F2 plants and Koshihikariwere sown on May 28, 2011. Seedlings were transplanted into a paddy fieldat the Institute of Plant Science and Resources, Okayama University (34.6°N,133.8°E) with a spacing of 15 cm interhill and 40 cm interrow on June 22,2011. Fertilizers were applied at the rate of 5:5:5 kg/10 ha of N:P2O5:K2O asbasal dressing. Two replications were performed.

Transposon Display. Transposon display was performed according to ref. 38.Genomic DNA samples isolated from taw1-D1 and taw1-D2 mutant siblingswere digested with 4-bp restriction enzymes and ligated with adaptorscontaining an MspI site. PCR products from two cycles of amplification, usingthe adaptor primer and Dart5′-3b-first and Dart5′-1,2,3-second/N, were vi-sualized by electrophoresis. Bands cosegregating with the taw1-D pheno-type were isolated from the gel and amplified with a set of primers, theadaptor primer and Dart5′-1,2,3-second/Nt. The DNA sequence of the am-plified band was determined according to ref. 39.Real-time RT-PCR. Total RNAwas extracted using a Plant RNA IsolationMini Kit(Agilent). After DNase I treatment, first-strand cDNA was synthesized usingSuperScript III reverse transcriptase (Invitrogen). PCR was performed withSYBR Green I using a Light Cycler 480 System II (Roche Applied Science). Theprimer sets used to amplify the transcripts are provided in SI Materialsand Methods.Generation of TAW1 knockdown plants. Details of the construction of the binaryvectors are provided in SI Materials and Methods.In situ hybridization. In situ hybridizations were performed as described byKouchi et al. (40). Microtome sections of 10-μm thickness were applied ontoglass slides treated with Vectabond (Vector Laboratories). Details of probesare provided in SI Materials and Methods.Observation of subcellular localization of TAW1-GFP fluorescence. Details of theconstruction of the binary vectors are provided in SI Materials and Methods.Construction of CaMV 35S-SVPs.Details of the construction of the binary vectorsare provided in SI Materials and Methods.

Construction of the GVG-Induction System. Details of the construction of thevectors are provided in SI Materials and Methods.

Tilling. Loss-of-function mutants were obtained from screening populationsmutagenized with N-methyl-N-nitrosourea (41, 42).

Assay of Transcriptional Activity. A modified yeast two-hybrid system wasused to analyze TAW1 activity to activate transcription. Details of thevector construction and activity assays are provided in SI Materialsand Methods.

ACKNOWLEDGMENTS. This work was supported in part by Grants-in-Aid fromthe Ministry of Education, Culture, Sports, Science, and Technology, Japan(22119008, 22247004, and 21027012, to J.K.), Ministry of Agriculture, Forestry

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Fig. 5. Phenotype of taw1-D2–introgressed Koshihikari BC5F2 plants. (A andB) The plant (A) and panicle (B) of Koshihikari (K) (Left) and taw1-D2 BC5F2plants (Right). Tertiary branches are marked with yellow asterisks. (C) Totalgrain weight per plant in grams. (D) Number of grains per panicle. (E) Onethousand grain weight in grams. In C–E, n = 23 plants for Koshihikari and 24plants for BC5F2 homozygous taw1-D2. All data are shown as means ± SD.Asterisks indicate a significant difference from corresponding control samplesof Koshihikari (Student’s t test; *P < 0.05, **P < 0.01).

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