Transcription Factor OsTGA10 Is a Target of the MADSProtein OsMADS8 and Is Required forTapetum Development1[OPEN]

Zhi-Shan Chen,a Xiao-Feng Liu,b Dong-Hui Wang,a Rui Chen,c,d Xiao-Lan Zhang,b Zhi-Hong Xu,a andShu-Nong Baia,2

aState Key Laboratory of Protein and Plant Gene Research, College of Life Sciences, Peking University, Beijing100871, ChinabDepartment of Vegetable Sciences, Beijing Key Laboratory of Growth and Developmental Regulation forProtected Vegetable Crops, China Agricultural University, Beijing 100193, ChinacMolecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee 37212dVanderbilt Genetics Institute, Vanderbilt University, Nashville, Tennessee 37212

ORCID IDs: 0000-0002-5893-8048 (Z.-S.C.); 0000-0003-3478-1581 (D.-H.W.); 0000-0002-4341-2908 (R.C.); 0000-0002-9521-4073 (S.-N.B.).

This study aimed at elucidating regulatory components behind floral organ identity determination and tissue development. Itremains unclear how organ identity proteins facilitate development of organ primordia into tissues with a determined identity,even though it has long been accepted that floral organ identity is genetically determined by interaction of identity genesaccording to the ABC model. Using the chromatin immunoprecipitation sequencing technique, we identified OsTGA10,encoding a bZIP transcription factor, as a target of the MADS box protein OsMADS8, which is annotated as an E-class organidentity protein. We characterized the function of OsTGA10 using genetic and molecular analyses. OsTGA10 was preferentiallyexpressed during stamen development, and mutation of OsTGA10 resulted in male sterility. OsTGA10 was required for tapetumdevelopment and functioned by interacting with known tapetum genes. In addition, in ostga10 stamens, the hallmark cell wallthickening of the endothecium was defective. Our findings suggest that OsTGA10 plays a mediator role between organ identitydetermination and tapetum development in rice stamen development, between tapetum development and microsporedevelopment, and between various regulatory components required for tapetum development. Furthermore, the defectiveendothecium in ostga10 implies that cell wall thickening of endothecium is dependent on tapetum development.

The ABCmodel for the genetic control of floral organidentity determination is the most influential theory inplant developmental biology in the last three decades(Coen and Meyerowitz, 1991). This model proposesthat transcription factors encoded by three classes ofgenes, namely A, B, and C, determine organ identities,acting either alone or in conjunction with one another,for sepals, petals, stamens, and carpels, which consti-tute the four whorls of floral organs. Following initial

description of the ABC model, it was demonstrated tofacilitate flower development in a wide range of plantspecies, albeit with a number of species-specific modi-fications (Soltis and Soltis, 2014; Chanderbali et al.,2016; Theißen et al., 2016).

In addition to the ABC genes, D- and E-class geneshave been subsequently characterized; these genesare considered responsible for ovule development(Colombo et al., 1995; Favaro et al., 2003) and deter-mination of organ identity in all four whorls (Pelazet al., 2000; Honma and Goto, 2001; Theißen and Sae-dler, 2001; Ditta et al., 2004), respectively. D- and E-classgenes in turn were also described in other plant species,where they generally perform conserved roles. In rice(Oryza sativa), five MADS-box genes, i.e. OsMADS1,OsMADS5, OsMADS7, OsMADS8, and OsMADS34,have been classified as E-class genes (Pelucchi et al.,2002; Malcomber and Kellogg, 2004; Zahn et al., 2005;Cui et al., 2010).

However, regardless of the widespread acceptance ofthe ABC model, closer inspection reveals that what isreferred to as “organ identity” is defined by two typesof criteria: the gene expression patterns present in theprimordial tissue or earlier meristematic cells and theorgan’s morphological characteristics once it is formed.

1 This project was supported by the Ministry of Science and Tech-nology of China through grants 2013CB126901 and 2009CB941502.

2 Address correspondence to shunongb@pku.edu.cn.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:Shu-Nong Bai (shunongb@pku.edu.cn).

Z.-S.C., Z.-H.X. and S.-N.B. conceived and designed the research;Z.-S.C. performed most of the experiments; X.-F.L., D.-H.W., andX.-L.Z. provided technical assistance and performed in situ hybridi-zation; R.C. provided technical assistance and Perl scripts for ChIP-seqanalysis; Z.-S.C. and S.-N.B. analyzed the data and wrote the manu-script; all authors read and approved the final manuscript.

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These criteria cover the initial events in the primordiumand the end result of the determination of identity butleave out what happens between those stages. We werethus intrigued to explore the events that occur duringthe development of primordial tissue into a definedorgan, specifically how a primordium develops via cellproliferation and differentiation into an organ with adefined identity and particular morphological charac-teristics.

Diverse research efforts have focused on character-izing the roles of ABCDE genes in the development ofprimordia into determined organs through geneticanalyses (Larkin et al., 1994; Bowman and Smyth, 1999;Sawa et al., 1999; Schiefthaler et al., 1999; Yang et al.,1999; Byrne et al., 2000; Semiarti et al., 2001; Schellmannet al., 2002; Ito et al., 2004; Gómez-Mena et al., 2005; Shiet al., 2011; Ó Maoiléidigh et al., 2013; Pajoro et al.,2014). In recent years, systematic analyses of targets ofthe ABCDE proteins have been performed using chro-matin immunoprecipitation sequencing (ChIP-seq), re-vealing numerous ABCE targets, including 3,475 forSEP3, 2,298 for AP1, 1,958 for AG, and 1,500 for AP3and PI (Kaufmann et al., 2009; Kaufmann et al., 2010a;Wuest et al., 2012; Ó’Maoiléidigh DS et al., 2013). In ourprevious work, we found that OsMADS58, annotatedas a C-class protein, can bind to DNA at numerousphotosynthetic genes, thereby inhibiting their expres-sion and subsequently affecting chloroplast differenti-ation in rice stamens (Chen et al., 2015). These findingscan potentially be used to characterize how primordiawith particular identities develop to have shapes,structures, and functions that correspond to specificorgan types.

Among the four floral organ types, stamens functionto produce microspores, thereby facilitating the differ-entiation of microgametophytes (pollen) and assistingwith their dispersal upon maturation. The regulation ofstamen development has been intensively investigatedbecause, in addition the key role that stamens play incompletion of the plant life cycle, male sterility causedby defects in stamen development can be exploited forthe generation of crop plants that exhibit heterosis(Goldberg et al., 1993; Ma, 2005; Chang et al., 2011;Kelliher et al., 2014; Walbot and Egger, 2016).

The tapetum is a hallmark tissue in the stamen, andmany mutants that are defective in stamen develop-ment display defects in tapetum development. Anumber of genes altered in such tapetum-defectivemutants encode transcription factors such as DYT1,TDF1, AMS, and MS1 in Arabidopsis (Arabidopsisthaliana; Wilson et al., 2001; Sorensen et al., 2003; Zhanget al., 2006; Zhu et al., 2008) and UDT1, GAMYB, TIP2,TDR, and EAT1 in rice (Jung et al., 2005; Li et al., 2006;Liu et al., 2010; Niu et al., 2013; Fu et al., 2014; Ko et al.,2014). Some of the genes involved in tapetum devel-opment encode enzymes and other proteins, such as thefasciclin glycoprotein MTR1, the Cys protease CP1, theaspartic proteases AP25 and AP37, and the anti-apoptosis protein API5 and its interacting partnersAIP1,AIP2, andHUB1/2 in rice (Lee et al., 2004; Li et al.,

2011; Tan et al., 2012; Niu et al., 2013; Cao et al., 2015).TIP2 interacts with TDR to modulate EAT1 expression,which in turn regulates AP25 and AP37 expression andtriggers programmed cell death in yeast and plants(Niu et al., 2013; Ko et al., 2014). In addition, TDR isinvolved in tapetum degradation by regulating CP1expression (Li et al., 2006). However, no regulation oftapetum genes by organ identity proteins has beendescribed.

In plants, the basic Leu zipper (bZIP) proteins form atranscription factor superfamily that regulates pro-cesses including pathogen defense, light and stresssignaling, seed maturation, and flower development(Jakoby et al., 2002; Nijhawan et al., 2008). There is adistinct subclade of bZIP transcription factors, namelythe TGA transcription factors, which bind to the con-served sequence TGACG (Jakoby et al., 2002). In Ara-bidopsis, there are 10 TGA transcription factors: TGA1to TGA7, PERIANTHIA, TGA9, and TGA10, whichhave been reported to function in floral patterning andanther development (Chuang et al., 1999; Murmu et al.,2010). To date, 89 genes are annotated as encoding bZIPtranscription factors in the rice genome (Nijhawanet al., 2008); however, functions of rice TGA transcrip-tion factors in flower development have not yet beendescribed.

Building on a report that the potential C-class proteinOsMADS58 can regulate the expression of chloroplastgenes and affect chloroplast differentiation (Chen et al.,2015), we employed ChIP-seq to investigate the targetsof other B- and E-class proteins in order to decipher theregulatorymechanisms of stamen development. For theE-class protein OsMADS8, we identified a bZIP geneLOC_Os09g31390 as a target and designated it OsTGA10based on its homology to Arabidopsis TGA10. OsTGA10was found to be specifically expressed during stamendevelopment, and OsTGA10 mutant plants were malesterile. Based on these observations, we systematicallyanalyzed OsTGA10 function. Our results revealed thatOsTGA10 functions in tapetum development via inter-action with TIP2 and TDR and also affects AP25 andMTR1 expression induction during tapetum and micro-spore development. We propose that OsTGA10 serves asa mediator between organ identity determination andtapetum development in rice stamen development, aswell as between tapetum development and microsporedevelopment and between various regulatory compo-nents required for tapetum development. Furthermore,the lack of the hallmark cell wall thickening of the endo-thecium in ostga10 implies that endothecium develop-ment is dependent on tapetum development.

RESULTS

LOC_Os09g31390 Is a Target of OsMADS8

To explore whether B- and E-class proteins, like theC-class OsMADS58, function in stamen developmentfollowing organ identity determination, we attempted

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to generate antibodies recognizing known rice B- andE-class proteins for use in ChIP-seq analyses. An antibodyagainst OsMADS8 was produced and found suitable forChIP-seq analysis (Supplemental Fig. S1). Using this an-tibody, ChIP-seq analysis revealed that 828 genes werebound by OsMADS8 in the rice genome (SupplementalFig. S2). By querying the previously established dynamictranscriptome during early rice stamen development(Chen et al., 2015), it was revealed that 12 transcriptionfactor genes were among those genes that were bothexpressed in the stamen and targets of OsMADS8. Ofthese, LOC_Os09g31390, annotated as encoding a TGA-like transcription factor, was preferentially expressed inthe stamen (Supplemental Fig. S3), prompting us to in-vestigate it further.To confirm DNA binding, we analyzed the DNA se-

quence of the LOC_Os09g31390 enriched region targetedby OsMADS8 (Supplemental Fig. S4). One CC[A/T]6GGmotif (CArG-box), the MADS protein binding element(Schwarz-Sommer et al., 1992; Treisman and Ammerer,1992; Riechmann et al., 1996), was identified in the P3region (Fig. 1A). An electrophoretic mobility shift assay(EMSA) demonstrated that the P3 region sequence wasindeed bound by recombinant OsMADS8 (Fig. 1B).ChIP-quantitative PCR (qPCR) assay further demon-strated that OsMADS8 binding enriched the P3 regionbut not the P1 and P2 regions, which do not contain aCArG-box, as revealed by analysis of rice paniclesamples (Fig. 1C). Combined, these results suggest thatOsMADS8 targets LOC_Os09g31390 through directprotein-DNA binding.

LOC_Os09g31390 Is Homologous to Arabidopsis TGA10

LOC_Os09g31390was annotated as a bZIP gene.Amongthe 89 bZIP genes in the rice genome, nine were clusteredwithArabidopsis TGA-like genes in aphylogenetic analysis(Supplemental Fig. S5A). LOC_Os09g31390 was placedwithin the same clade as Arabidopsis TGA10, and a de-tailed comparison revealed that the LOC_Os09g31390amino acid sequence shares 54% identity with that ofAtTGA10 (Supplemental Fig. S5B). Therefore, we namedLOC_Os09g31390 as OsTGA10. There was another gene(LOC_Os09g10840) observed in the same clade asAtTGA10and OsTGA10; as this gene was neither bound byOsMASD8 nor expressed in rice stamens, we did notstudy it further in this work.

Arabidopsis TGA10 is a transcription factor. To exam-ine if OsTGA10 performs a similar function, we investi-gated OsTGA10 protein localization. In rice protoplasts,an eGFP fusion protein containing the full-length OsTGA10amino acid sequence driven by the 35S promoter waslocalized in the nucleus (Fig. 2A). Furthermore, a tran-scriptional activity assay using yeast and rice protoplastsrevealed that OsTGA10 could activate the transcriptionof target genes (Fig. 2, B–D). These data suggest thatOsTGA10 does function as a transcription factor.

OsTGA10 Is Preferentially Expressed duringStamen Development

Database transcriptomeanalyses indicate thatOsTGA10is preferentially expressed during stamen development

Figure 1. Os09g31390 is a target of OsMADS8. A, Schematic diagram of Os09g31390 gene structure. Exons and introns arerepresented by black boxes and lines, respectively. The red line indicates the binding region of OsMADS8 revealed through ChIP-seq. P1 to P3 are the regions amplified in the ChIP-qPCR validation assay. B, EMSA assay of recombinant OsMADS8 interactingwith biotin-labeled probe in the P3 region. The 50-, 100-, and 200-fold excess unlabeled probes were used for competition.Arrows indicate the shifted bands and free probes. C, ChIP-qPCR assay showed binding of OsMADS8 to the P3 region containingCArG-box. The promoter of UBIQUITIN was used as an internal control, and N1 represents a DNA fragment away from thebinding region used as a negative control. Error bars indicate the SD of three biological replicates. Student’s paired t test: *P, 0.05.

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(Supplemental Fig. S3). To explore the detailed spatio-temporal OsTGA10 expression pattern in the stamen, weexamined OsTGA10 expression using in situ hybridiza-tion. The expression pattern revealed that during stamendevelopment,OsTGA10 expression was initially apparentat stage 3 (Fig. 3C; Supplemental Fig. S6) and increasedthrough stages 4 to 6 primarily in tapetum and meioticcells (Fig. 3, D–F and I–L). No signals were observed in thenegative control using sense probe, showing the specificityof this experiment (Fig. 3, G andH). Yang et al. (2016) havereported in situ results similar to ours and interpreted thatOsTGA10 is specifically expressed in tapetum, not inmeiotic cells. To clarify it, we examined the gene expres-sion profile data of stamen meiotic cells generated inTang’s and Matsuoka’s labs. Their data clearly revealedthat OsTGA10 is detected in meiotic cells in rice stamen(Hobo et al., 2008; Tang et al., 2010).

In addition, we also examined expression pattern ofOsMADS8 by in situ hybridization and found thatOsMADS8 expression was detected from stage 2 to6 stamens (Fig. 3, M–R). This result indicates that ex-pression of OsMADS8 is overlapped with that ofOsTGA10 during anther development. Taken together,these analyses suggest that OsTGA10 is a downstreamtarget of OsMADS8 and functions in the tapetum andmeiotic cells.

A Male-Sterile Phenotype Is Associated with ostga10

To study whether OsTGA10 plays a role in rice stamendevelopment,weobtained anOsTGA10 insertionalmutant

from the POSTECH T-DNA collection (Yi and An, 2013).The mutant line designated ostga10 contained T-DNA in-serts in the fifth intron of the genomic OsTGA10 sequence(Supplemental Fig. S7A). Three primer pairs weredesigned to examine OsTGA10 transcripts in ostga10plants. The first of these primer pairs annealed to the se-quence upstream of the T-DNA insertion, whereas theremaining primer pairs delimited the sequence that con-tained the T-DNA inserts (primer pair 2) and targeted thesequence downstreamof the T-DNA insertion (primer pair3; Supplemental Fig. S7A). In ostga10, PCR ampliconswereobtained only using primer pair 1, whereas amplificationwas observed in the wild type using all three primer pairs(Supplemental Fig. S7B). These results suggest thatOsTGA10 expression was indeed altered by the T-DNAinsertion. In addition, we examined expression levels oftwo OsTGA10 flanking genes and observed no change intheir transcript levels in ostga10 (Supplemental Fig. S7C),suggesting that T-DNA insertional mutation in ostga10 af-fected the expression of OsTGA10 alone.

Characterization of the ostga10 mutant phenotyperevealed no obvious difference in plant morphologyand growth rate before heading; however, obviouslylower seed set was apparent inmutant plants comparedto that in the wild type (Fig. 4, A, B, and I). Close in-spection of ostga10 flowers revealed anther abnormali-ties regarding overall shape (Fig. 4, C–F) and pollendevelopment, with the latter indicated by significantlyreduced pollen grain number (Fig. 4J) and severe de-fects in starch accumulation (Fig. 4, G and H). To ex-amine whether mutation of OsTGA10 affected femalefloral organs, we pollinated ostga10 pistils with wild-

Figure 2. Characterization of OsTGA10 as a transcription factor. A, Subcellular localization of OsTGA10 in rice protoplasts. Top,localization of OsTGA10 fused with GFP. Bottom, localization of signal from empty vector containingGFP alone. B, Transcriptionalactivity assay in yeast. Full-length OsTGA10 amino acid sequence was fused with the DNA binding domain in pGBKT7. Yeast cellscoexpressing pGBKT7-OsTGA10 and pGADT7 grew normally on selective medium lacking Leu, Trp, His, and Ade. Yeastcotransformedwith pGBKT7-53 andpGADT7 served as a positive control, andyeast cotransformedwith pGBKT7-LamandpGADT7served as a negative control. C, Schematic diagram showing the constructs used in the transient expression assays in D. D, Tran-scriptional activity assay in rice protoplasts. Compared to the negative control (GAL4BD), OsTGA10-GAL4BD significantlyup-regulated the LUC/REN ratio. Error bars indicate the SD of four biological replicates. Student’s paired t test: *P , 0.05.

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type pollen grains, which rescued the sterility of ostga10flowers and resulted in a seed set rate of approximately58% (Fig. 4I; Supplemental Fig. S8). To further verify themale-sterile phenotype caused by OsTGA10 mutation,we generated two differentOsTGA10-targetedCRISPR-Cas9 lines (Supplemental Fig. S9, A and B). Both ofthese OsTGA10-mutated lines exhibited a male-sterilephenotype that was comparable to that in the T-DNAinsertion line ostga10 (Supplemental Figs. S9C and S10),which further supported the conclusion that a male-sterile phenotype arises when OsTGA10 gene functionis compromised.

Tapetum Development Is Impaired in ostga10

To clarify the effects of OsTGA10 mutation on ricestamen development, we used stamen cross sections toobserve the developmental process. While there wereno observable differences in anther transverse patternsbefore stage 4 (Supplemental Fig. S6; Sanders et al.,1999; Lu et al., 2006; Chen et al., 2015), following thispoint, the two adaxial locules displayed slow differen-tiation in ostga10 (Fig. 5, A–H). However, the moststriking ostga10 phenotypic abnormality was observedafter stage 7, where, in addition to obvious alterationof tapetum development, there was an observable col-lapse in anther locules after stage 9 (Fig. 5, I–P).

Meanwhile, there were severe defects in microsporedevelopment, which is consistent with the impairedpollen grains described above (Fig. 4).

To further observe the developmental status oftapetal cells, we performed transmission electron mi-croscopy. Wild-type stamens contained tapetal cellsthat exhibited a condensed cytoplasm with clear or-ganelles such as endoplasmic reticulum (ER) and nucleiat stage 7 (Fig. 6, A and B). These cells were thinner atstage 8 compared to how they appeared at stage 7, andwhile a condensed cytoplasm was maintained, the or-ganelles were no longer readily observable and thenuclear membrane had become indistinct (Fig. 6, C andD). By contrast, the tapetal cells in ostga10 stamens atstage 7 did not exhibit cytoplasm condensation, and, incomparison to that in the wild type, organelles werenormally distributed and nuclei were larger (Fig. 6, Eand F). Unlike in the wild type, tapetal cells in ostga10did not become thinner at stage 8, and the organelles inthese cells such as mitochondria remained clearly visi-ble, with a greater abundance of ER observable sur-rounding a distinct nucleus (Fig. 6, G and H). Theseobservations were consistent with those describedabove using semithin cross sections (Fig. 5, I–P). Atstage 9, stamen tapetal cells in the wild type were fur-ther reduced in size, with no nuclei or other organellesclearly visible and the deposition of ubisch bodies

Figure 3. Expression pattern of OsTGA10. A to H, Detection of OsTGA10 via in situ hybridization in wild-type anthers beforemeiosis stage. The anther developmental process is described in detail in Supplemental Figure S6. The anther sections werehybridized with antisense probe (A–F) or sense probe (G and H). A, Anther at stage 1; B, anther at stage 2; C, anther at stage 3;D and G, anther at stage 4; E and H, anther at stage 5; F, anther at stage 6-1. Bar = 50 mm. I to L, Magnified view of boxes inC to F. M to R, Detection of OsMADS8 via in situ hybridization in wild-type anthers before meiosis stage. M, anther at stage 1;N anther at stage 2; O anther at stage 3; P, anther at stage 4; Q, anther at stage 5; R, anther at stage 6-1. Bar = 50 mm. MMC,Microspore mother cell; PPC, primary parietal cell; SPC, secondary parietal cell; T, tapetum.

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observable on the internal side of the cell (Fig. 6, I and J).The tapetal cells of ostga10 stamens were seen to sud-denly collapse at stage 9 (Fig. 6K). Although ubischbodies were still observable in these cells, they exhibi-ted an altered deposition pattern (Fig. 6L).

During tapetum development, one of the hallmarkevents is programmed cell death, which is generallydetectable using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay fromapproximately stage 7 to 10 (Li et al., 2006) (corre-sponding to stages 6-2 to 9 in our study, because thewhole process of meiosis was defined as stage 6,whereas it was defined as stage 6 and stage 7 in Liet al. [2006]). As expected, we found dynamic TUNELsignals in wild-type tapetal cells from stages 6-2 to 9(Fig. 7, A–D). By contrast, no such signals weredetected in ostga10 tapetal cells during the same de-velopmental period (Fig. 7, E–H).

Based on these observations, we conclude that theprocess of tapetum development is severely impaired

during ostga10 stamen development. Since OsTGA10 ispreferentially expressed during stamen development, thisostga10 mutant phenotype suggests that OsTGA10 mayplay a role in the regulation of tapetum development.

OsTGA10 Affects Tapetum Development throughInteractions with Known Tapetum Genes

Thus far, 15 genes have been demonstrated to be in-volved in tapetum development in rice (herein referredto as “tapetum genes”; Jung et al., 2005; Li et al., 2006; Liet al., 2011; Liu et al., 2010; Tan et al., 2012; Niu et al.,2013; Fu et al., 2014; Ko et al., 2014; Cao et al., 2015; Yiet al., 2016; Yu et al., 2016; Cui et al., 2017). To investi-gate if OsTGA10 plays a role in regulating tapetumdevelopment, we first examined the expression levels ofthe known tapetum genes. Among the 12 tapetumgenes examined, which encompassed all characterizedtapetum genes at the time of performing these analyses,nine genes exhibited expression patterns during the

Figure 4. Comparison between wild-type and ostga10 phenotypes. A, Comparison of wild-type (WT) and ostga10mutant plantsafter seedmaturation. Bar = 10 cm. B, Panicles in wild-type and ostga10mutant plants. Bar = 10 cm. C andD, Florets in wild-type(C) and ostga10 mutant (D) plants. Half of the lemma and palea were removed. Bar = 500 mm. E and F, Scanning electron mi-croscopy observation of wild-type (E) and ostga10 (F) anthers. Bar = 200 mm. G and H, Wild-type (G) and ostga10 (H) pollengrains were stained by I2-KI solution. Bar = 100 mm. I to L, Statistical analysis of seed setting (I), pollen grains (J and K), andabnormal anthers (L) in 24 panicles from eight independent ostga10 mutant lines and seven panicles from four independentwild-type lines.

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developmental process from stages 5 to 10 that werealtered in ostga10 stamens compared to those in wild-type stamens (Fig. 8). For the remaining three genes,namely AP37, AIP1, and AIP2, no altered expressionwas observed in ostga10. Aside from TDR (Fig. 8D), the

expression levels of the affected tapetum genes weredecreased to various extents in ostga10 stamens com-pared to those in wild-type stamens (Fig. 8). These re-sults serve as molecular evidence for the impairment oftapetum development in the ostga10.

Figure 5. Observation of sections representing wild-type and ostga10 anther development. The anther developmental processwas described in detail in Supplemental Figure S6. The transverse sections from the middle of anthers were observed. A to H,Paraffin sections of wild-type (A–D) and ostga10 (E–H) anthers beforemeiosis stage. A and E, Anthers at stage 3; B and F, anthers atstage 4; C and G, anthers at stage 5; D and H, anthers at stage 6-1. Bar = 50 mm. I to P, Semithin sections of wild-type (I–L) andostga10 (M–P) anthers after meiosis stage. I andM, Anthers at stage 7; J andN, anthers at stage 8; K andO, anthers at stage 9; L andP, anthers at stage 11. Bar = 20 mm; E, Epidermis; En, endothecium; M, middle layer; MC, meiotic cell; MMC, microspore mothercell; MP, mature pollen; Msp, microspore; PPC, primary parietal cell; Sp, sporogenous cells; SPC, second parietal cells;T, tapetum; Td, tetrads.

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To explore the role of OsTGA10 in tapetum devel-opment, the most straightforward approach was toperform ChIP-seq to identify target genes; however,no suitable OsTGA10 antibody was obtained for thispurpose. Therefore, two alternative approaches wereadopted to investigate the potential function of OsTGA10in the known regulatory network of tapetum develop-ment (Ko et al., 2014).

The first approach to characterize the role ofOsTGA10 was to explore potential protein interactionsbetween OsTGA10 and other known tapetum devel-opment regulatory players. Through a yeast two-hybrid screen using OsTGA10 as the bait protein toproducts of the 12 known tapetum genes describedabove, four proteins were observed to interact withOsTGA10 (Fig. 9A; Supplemental Fig. S11A). These

protein-protein interactions were verified via two fur-ther assays: in vitro pull-down and in vivo split lucif-erase complementation based on gene coexpression inN. benthamianamesophyll cells. Both in vitro pull-downand in vivo split luciferase assays showed that TIP2 andTDR interacted with OsTGA10 (Fig. 9, B and C).However, neither of the additional assays demon-strated an interaction between OsTGA10 and eitherUDT1 or EAT1 (Supplemental Figure S11, B and C;Supplemental Table S2).

We also explored whether OsTGA10 binds to DNA attapetumgenes andactivates their expression. By analyzingthe promoter sequences of the 12 tapetum genes charac-terized at the time of these analyses, we revealed TGACGelements associated with five genes, namely AP25,MTR1,AIP1, AIP2, and CP1 (Fig. 10A; Supplemental Fig. S12).

Figure 6. TEM analysis of wild-type and ostga10 anther development. A, C, and I, Cross sections of wild-type anthers atstage 7 (A), stage 8 (C), and stage 9 (I). Bar = 5 mm. B, D, and J, Magnifications of tapetal cells highlighted by boxes in A, C, and I.Bar = 1 mm. E, G, and K, Cross sections of ostga10 anthers at stage 7 (E), stage 8 (G), and stage 9 (K). Bar = 5 mm. F, H, and L,Magnifications of tapetal cells highlighted by boxes in E, G, and K. Bar = 1 mm. Dashed boxes in J and L indicate ubisch bodes intapetal cells. E, Epidermis; En, endothecium; ER, endoplasmic reticulum; M, middle layer; Msp, microspore; Mt, mitochondria;Nu, nucleus; T, tapetum; Ub, ubisch bodies.

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These elements are a hallmark for the binding of TGAtranscription factors, which includeOsTGA10. Among thefive genes containing a TGACG element in their promotersequence, three genes (AP25, MTR1, and CP1) exhibitedaltered expression levels in ostga10 (Fig. 8). We thereforecarried out an EMSA to examine whether OsTGA10physically binds the AP25, MTR1, or CP1 promoters. TheTGACG elements in the AP25 andMTR1 promoters wereindeed bound by OsTGA10 (Fig. 10, A–C); however, nobinding effectwas observed forCP1. In addition,we testedfor an effect of OsTGA10 on the promoter activities ofAP25 and MTR1 using a transient reporter assay. Sur-prisingly, OsTGA10 suppressed the promoter activities ofboth genes rather than activating the bound promoters aswas expected (Fig. 10, E and F). Furthermore,we identifiedanE-box, the hallmarkbinding site of basic helix-loop-helixtranscription factors, in both AP25 and MTR1 promoters(Fig. 10A). Thus,we also examinedwhether TIP2 andTDRaffected AP25 and MTR1 promoter activity. We observedthatAP25 expression in the presence of OsTGA10was notaffected further by either TIP2 or TDR (Fig. 10E). ForMTR1promoter activity, the presence of TIP2 enhanced the ex-pression suppression effect of OsTGA10 (Fig. 10F). No ef-fects onMTR1 promoter activity were observedwith TDRalone nor did TDR presence alter the extent of OsTGA10-mediated promoter activity suppression (Fig. 10F).The above results revealed that OsTGA10 affects tape-

tum development through interacting with known tape-tum genes, via both protein-protein and protein-promoter

interactions. Since multiple transcription factors are in-volved in tapetum development and the interaction be-tween these factors is complicated, the precise manner ofOsTGA10 function in this network is intriguing.

Cell Wall Thickening of Endothecium Is Affectedin ostga10

In addition to tapetum development impairment inostga10, locule collapse after stage 9 was also a prom-inent phenotype (Fig. 5, O and P). To explore the un-derlying cause of locule collapse, we observed cell wallthickening of wild-type and ostga10 stamens under ul-traviolet (UV) illumination. This revealed that prom-inent secondary cell wall thickening occurred in theendothecium cells of wild-type stamens at late stages9 and 11 (Fig. 11, A–D). However, although structuralcompoundswere synthesized, based on autofluorescencebeing observed, no such cell wall thickening was presentin ostga10 stamens during comparable developmentalstages (Fig. 11, E–H).

While little is known concerning the genes involvedin endothecium development, rice genes involved insecondary cell wall thickening have been reported.These genes include cellulose synthase catalytic subunitgenes (Tanaka et al., 2003; Zhang et al., 2009; Wanget al., 2012, 2016), BRITTLE CULM1 (Liu et al., 2013), aNAC domain transcription factor, and a MYB familytranscription factor (Zhong et al., 2011). The expression

Figure 7. Comparison of DNA fragments in the tapetum of wild-type and ostga10 anthers. A to D, DNA fragments in the tapetumof wild-type anthers. Bars = 50 mm. E to H, DNA fragments in the tapetum of ostga10 anthers. Bars = 50 mm. A and E, Anthers atstage 6-2. B and F, Anthers at stage 7. C and G, Anthers at stage 8. D and H, Anthers at stage 9. The yellow fluorescence indicatesTUNEL-positive signal, which is merged with red fluorescence from background staining and green fluorescence from TUNELpositive nuclei staining. Arrowheads indicate tapetal cells.

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of 16 genes involved in secondary cell wall thickeningwas investigated in stage 11 stamens, which revealedthat 11 of the genes exhibited decreased expression inostga10 stamens (Fig. 11I). Although this result does notexplain the large changes observed in the ostga10 anthertransverse pattern, the decreased expression of genesinvolved in secondary cell wall thickening suggests thatcell wall strength is weakened in ostga10, which mayexplain the collapse of the locules.

DISCUSSION

To further characterize the regulatory network un-derlying stamen organ formation following identitydetermination, we characterized OsTGA10, which en-codes a bZIP transcription factor identified as a target ofthe E-class MADS box protein OsMADS8. AlthoughOsTGA10 is designated based on the sequence simi-larity to Arabidopsis TGA10 (Supplemental Fig. S5),the two genes obviously have different functions. Wedemonstrate that OsTGA10 is preferentially expressedduring stamen development, is required for the regu-lation of tapetum development, and functions throughinteraction with known tapetum genes. In addition, wefound that the impairment of tapetum development

alters endothecium development. Thus, in addition toidentifying a novel component of the regulatory net-work mediating organ identity determination and ta-petum development, this study reveals a complicatedrelationship among the regulatory components requiredfor the tapetum development, which suggests thatOsTGA10 plays a mediator role at three levels.

The first-level mediator role of OsTGA10 is linkingorgan identity determination and tapetum develop-ment. It is known that E-class MADS box proteins playindispensable roles in the determination of floral organidentities in all angiosperms examined to date, includ-ing in rice (Pelaz et al., 2000; Honma and Goto, 2001;Ditta et al., 2004; Zahn et al., 2005; Cui et al., 2010). Withthe exception of ChIP-seq analysis of SEP3 targets inArabidopsis (Kaufmann et al., 2009; Pajoro et al., 2014),little is known regarding downstream events duringorgan formation. Here, we demonstrate that OsTGA10is a target of the rice E-class MADS box proteinOsMADS8 (Fig. 1). Furthermore, we demonstrate thatOsTGA10 is required for development of the tapetum(Figs. 4–10). This is the first description of a role forOsMADS8, via its effect on the downstream transcrip-tion factor OsTGA10, in tapetum development, whichis one of the critical events during stamen development.On the other hand, OsTGA10 plays a mediator role to

Figure 8. RT-qPCR analysis of expression levels of tapetum genes in ostga10. S5 to S10 represent stages 5 to 10 of anther de-velopment (see Supplemental Fig. S6). Error bars indicate the SD of three biological replicates. Student’s paired t test: *P, 0.05,**P , 0.01.

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link the involvement of OsMADS8 in organ identitydetermination with tapetum development.A question arising from the above conclusion is

whether tapetum development is affected in osmads8mutant plants. Unfortunately, such mutants do notexist and the further E-class MADS protein OsMADS7shares high sequence similarity with OsMADS8and has similar expression pattern with OsMADS8(Supplemental Fig. S13). Cui et al. (2010) reported noobvious aberrant phenotype in RNAi lines where eitherOsMADS8 or OsMADS7 gene expression was sup-pressed; however, severe morphological floral organalterations were observed in RNAi lines where the ex-pression of both genes was suppressed. We found anincrease inOsTGA10 expression in theOsMADS8RNAiline (Supplemental Fig. S14A). In conjunction, the ex-pression of OsMADS7 was also increased in theOsMADS8 RNAi line (Supplemental Figure S14A; Cuiet al., 2010). We therefore examined the binding ofOsMADS7 to the second intron of OsTGA10, asOsMADS7 possesses the same DNA binding domain asOsMADS8. As anticipated, OsMADS7 does bind thesecond intron of OsTGA10 (Supplemental Fig. S14B).These findings suggest that OsMADS7 has a compen-satory effect following decreasedOsMADS8 expressionin the OsMADS8 RNAi line, which prevents the mani-festation of a mutant tapetum phenotype.The second-level mediator role of OsTGA10 is based

on its interaction both with regulatory elements

required for tapetum development, such as TDR andTIP2 via protein-protein interaction and AP25 viaprotein-promoter interaction (Figs. 9 and 10), and withregulatory elements required for microspore develop-ment, such as MTR1 via protein-promoter interaction(Fig. 10). It is well known that microspore developmentis dependent on tapetum development for the supply ofenergy and metabolites. The interaction of OsTGA10with both tapetum genes and microspore genes sug-gests that OsTGA10 may play a mediator role betweenthe developments of both cell types.

The third-level mediator role of OsTGA10 is based onits interaction with both up- and down-stream com-ponents in the regulatory pathways of tapetum devel-opment. It is known that the AP25 is a key player intapetum development and supposed to be preciselyregulated. EAT1 transcription factor, which interactswith TDR at the protein level, directly binds AP25promoter (Niu et al., 2013); both TDR and TIP2 regulateEAT1 transcription by directly binding to the EAT1promoter (Ko et al., 2014), and TIP2 may functiondownstream of UDT1, as inferred by altered TIP2 ex-pression in udt1 mutant plants (Ko et al., 2014). Basedon these data, it seems clear that TDR and TIP2 areupstream regulators of AP25 expression. Our resultsrevealed that while OsTGA10 directly interacts withTDR and TIP2, it also directly binds AP25 pro-moter itself, interacting with both upstream regulatorsand their downstream target AP25. This phenomenon

Figure 9. Interaction between OsTGA10and TIP2 and between OsTGA10 andTDR. A, Yeast two-hybrid assay test for in-teraction. Yeast cells cotransformed withdifferent constructs were grown on selec-tive medium lacking Leu, Trp, His, andAde. 2.5 mM and 5 mM 3-amino-1,2,4-triazolewere added to inhibit self-transcriptional activation of OsTGA10.B, In vitro pull-down assay test for in-teraction. OsTGA10 fused with TriggerFactor (TF), TIP2 fused with GST, andTDR fused with GST were expressed inE. coli. Interactions were determined byimmnoblot analysis using anti-TF anti-body. Lanes without interactions betweenOsTGA10 and other proteins were notincluded in this immunoblot image.C, Split-luciferase complementation as-say test for interaction inN. benthamianaleaf tissue. Luciferase signals were detectedin leaf cells 48 h following coinfiltrationwith OsTGA10-nLuc and TIP2-cLuc andwith OsTGA10-nLuc and TDR-cLuc. nLucrepresents the N-terminal fragment ofluciferase, whereas cLuc represents theC-terminal fragment of luciferase.

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Figure 10. Analysis of genes bound by OsTGA10. A, Distribution of E-box and TGACG motifs in AP25 and MTR1 promotersequences. B and C, EMSA analysis showed interaction between recombinant OsTGA10 and biotin-labeled probes containingthe TGACG motif in the promoter of AP25 (B) and MTR1 (C). Unlabeled probes were used for competition. Arrows indicate theshifted bands and free probes. D, Schematic diagram depicting the constructs used in transient expression assays in E and F. E, Thetranscriptional repression of the AP25 promoter by OsTGA10 in N. benthamiana leaves. F, OsTGA10 further enhancedthe transcriptional repression of theMTR1 promoter by TIP2 inN. benthamiana leaves. Error bars in E and F indicate the SD of sixbiological replicates. Student’s paired t test: *P , 0.05, **P , 0.01, ***P , 0.001.

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Figure 11. Defective endothecium secondary cellwall thickening inostga10 anthers. A, C, E, andG, Sections ofwild-type (A andC)and ostga10 (E and G) anthers. A and E, Anthers at stage 9, and C and G, anthers at stage 11. Bar = 20 mm. B, D, F, and H, Themagnified views of anthers in A, C, E, and G illuminated by UV light. Arrowheads in B and D indicate signals of secondary cell wallthickening in endothecium cells. I, RT-qPCR analysis of expression levels of genes involved in rice secondary cell wall thickening inostga10. Error bars indicate SD of three biological repeats. Student’s paired t test: *P , 0.05, **P , 0.01.

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suggests that regulation of AP25 expression is not lin-ear, but rather multilayered. From this perspective, amultilayered regulatory network is required for theproper tapetum development, and OsTGA10 is an im-portant role in such a complicated network.

In addition to detailing the role of OsTGA10 in ta-petum development, this work revealed the intriguingphenomenon of defective secondary cell wall thicken-ing in the endothecium of ostga10 anthers. While theregulatory mechanism for endothecium secondary cellwall thickening is poorly understood, the results of thisstudy suggest that either OsTGA10 is directly involvedin regulating the genes required for this process or en-dothecium development is dependent on correct tape-tum development. The relationship between tapetumand endothecium development is not currently welldescribed (Lee et al., 2004; Mizuno et al., 2007; Li et al.,2011; Ko et al., 2014; Yi et al., 2016). Our observationssuggest that the relationship between tapetum andendothecium development could be a fruitful topic forfuture investigation.

MATERIALS AND METHODS

Plant Materials and Growth Conditions

Seeds of the T-DNA insertion mutant ostga10 (2A-10206L) and its corre-sponding wild type (Dong Jing) were obtained from the Postech Rice mutantdatabase (Yi andAn, 2013). The seeds were sown in soil, and plants were grownin a paddy field during the normal growing season in Beijing, China, and in agrowth chamber (30°C 6 2°C, 11-h light/13-h dark regime) during the winter.

To create the OsTGA10 CRISPR lines, 23-bp targeting sequences (includingprotospacer adjacent motif (PAM)) were chosen and confirmed using a BLASTsearch against the rice (Oryza sativa) genome. Target sequences were synthe-sized and ligated into the pBGK032 vector, which was used to transformEscherichia coil competent cells for plasmid propagation. An Agrobacteriumtumefaciens strain EHA105 carrying the CRISPR plasmid was used to transformwild-type rice plants as described previously (Nishimura et al., 2006). Trans-genic plants were grown in a growth chamber (30°C 6 2°C, 11-h light/13-hdark light regime) during the winter.

For mutant genotyping, genomic DNA was extracted from wild-type andmutant leaves. For ostag10, the genotype was determined by PCR using specificprimer pair 2A-10206L-LP and 2A-10206L-RP, and a T-DNA primer 2715-LB-BP.The genotypes of CRISPR lines were determined by PCR using specific primerpairs GP4434-F/R and GP4435F/R flanking the designed target sites, and PCRproducts (300–500 bp) were sequenced directly and identified using the De-generate Sequence Decoding method (Ma et al., 2015). Primers used for geno-typing are listed in Supplemental Table S1.

Characterizations of Mutant Phenotypes

Wild-type and ostga10 plants and panicles following seed maturation werephotographed using a Nikon digital camera. Floret images were generatedusing a ZEISS Lumar V12 stereomicroscope (Carl Zeiss). Mature pollen grainswere stained by I2-KI solution in darkness for a fewminutes and then visualizedusing an Imager D2microscope and a ZEISS AxioCam ICc5 digital camera (CarlZeiss).

For cross section images, panicles or spikeletswerefixed immediately in FAA(50% Ethanol, 10% Formaldehyde (37%), 5% Acetic Acid) solution overnight at4°C. Paraffin was applied to samples according to procedures described in Haoet al. (2003). For semithin sectioning, dehydrated samples were infiltrated witha 1:1 ethanol:resin (v/v) solution for 4 h and then with 100% resin overnight.Following this, samples were embedded in 100% resin (LR white resin, Sigma)and polymerized for 24 h at 65°C. The embedded samples were sectioned (3mmthick) before staining with toluidine blue O (Urchem) and imaging using anImager.D2 microscope with a ZEISS AxioCam ICc5 digital camera (Carl Zeiss).

The preparation of spikelets for scanning electron microscopy and trans-mission electron microscopy (TEM) was conducted according to proceduresdescribed in Bai et al. (2004) with some modifications. For TEM, spikelets werefixed in 2.8% glutaraldehyde with 0.02% Triton X-100 overnight at 4°C andwere postfixed in 1% OsO4 prepared in phosphate-buffered saline for 24 h at4°C. Ultrathin sections were observed using a Tecnai G2 20 Twin (FEI) trans-mission electron microscope.

TUNEL Assay

Spikelets were prepared for the TUNEL assay as described above for thegeneration of paraffin sections. The paraffin sections were dewaxed in xyleneand rehydrated in an ethanol series. TUNEL assay was performed using an InSitu Cell Death Detection Kit, Fluorescein (Roche, #11684795910), according tothe manufacturer’s instructions with some modifications. Signals were ob-served and imaged using an Imager.D2 microscope (Carl Zeiss).

Secondary Cell-Thickening Observation

Semithin anther sections representing wild-type and ostga10 anthers weregenerated according to the sectioning procedure described above. Sectionswere illuminated using UV light, and secondary cell-thickening signal wasimaged with an Imager.D2 fluorescence microscope (Carl Zeiss).

ChIP-qPCR Assay

OsMADS8polyclonal antibody71987wasproducedby injecting rabbitswitha synthesized peptide corresponding to the OsMADS8 C terminus and purifiedthrough an antigen-specific affinity chromatography column (Beijing ProteinInnovation, China). The specificity of the resulting antibody was tested viaimmunoblot analysis using OsMADS8 recombinant protein and plant proteinextracts as described by Ng et al. (2009) with somemodifications. Proteins weretransferred onto a nitrocellulose membrane, incubated with secondary anti-rabbit immunoglobulin G (IgG) coupled to horseradish peroxidase.

Panicles between 0.5 and 2 cm in length (covering S1–S6 stamens) werecollected and tissue was fixed. ChIP was performed using OsMADS8 antibodyand IgG antibody according to previously described protocols (Kaufmannet al., 2010b). Immunoprecipitated DNA fragments were used as templates forreal-time qPCR (RT-qPCR) performed using specific primers (see SupplementalTable S1). UBIQUITIN was used as the internal reference gene. IgG and ge-nomic fragments from nonbinding sites were used as negative controls. Threebiological repeats, each with three technical repeats, were included, allowingsubsequent statistical analysis.

RT-qPCR

Total RNA was isolated with a Plant RNA Kit (OMEGA, #R6827), digestedwithDNase I (Promega, #M6101), andused to synthesize cDNAwith a qPCRRTKit (TOYOBO, #FSQ-101) according to the manufacturer’s instructions.RT-qPCR analyses were performed in an ABI7500 machine using SYBR PremixEx Taq Mix (Takara #RR420A). ACTINwas used as the internal reference gene.Specific primers for each target gene are listed in Supplemental Table S1. Eachsample was included in three biological repeats, each with three technique re-peats, allowing subsequent statistical analysis.

In Situ Hybridization

Panicle and spikelets were fixed in FAA (50% Ethanol, 10% Formaldehyde(37%), 5% Acetic Acid) overnight at 4°C followed by dehydration in an ethanolseries, after which tissue was embedded in Paraplast High Melt (Lecia) forsectioning. In situ hybridization was performed as previously described (Sunet al., 2016). OsTGA10-specific regions were amplified with correspondingprimer pairs (see Supplemental Table S1) and transcribed in vitro to createprobes using the Digoxigenin RNA labeling kit (Roche #11175025910).

Phylogenetic Analyses

The OsTGA10 sequence was used to search for homologous rice proteins. Amultiple sequence alignment was performed with Clustal W (http://www.clustal.org/clustal2/) using rice and Arabidopsis (Arabidopsis thaliana) TGA

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transcription factor sequences. A phylogenetic tree of aligned sequences wasconstructed using the neighbor-joining method with the complete deletionoption, the Jones-Taylor-Thornton model, a Gamma Distribution (G) of 1, and1,000 bootstrap replicates in MEGA version 6 (Tamura et al., 2013).

EMSA Assay

The full-length coding sequences of OsMADS8, OsMADS7, and OsTGA10were cloned into the pCold-TF vector (Takara). The verified constructs weretransformed into Transetta (DE3) competent cells (TransGen Biotech, #CD801),and transformants were cultured at 37°C until 0.4 to 0.6 OD600 and then inducedwith 0.1 mM isopropylthio-b-galactoside (IPTG) for 24 h at 16°C. The recom-binant proteins were purified with Ni-NTA agarose (QIAGEN #30210). Probescontaining CArG or TGACG (TGA) motif were amplified with specific primerslabeledwith biotin, while unlabeled primers were used to produce competitors.EMSA assays were performed according to the manufacturer’s instructions of aLightShift R Chemiluminescent EMSA Kit (Thermo #20148). The probe se-quences for EMSA are listed in Supplemental Table S1.

OsTGA10 Subcellular Localization

The full-length OsTGA10 coding sequence without the terminal codon wasamplified with specific primer pairs (see Supplemental Table S1) and cloned intothe pM999 vector to produce anOsTGA10-GFP fusionprotein. Verified constructswere transformed into rice protoplasts via polyethylene glycol (PEG)/calcium-mediated transformation alongside pM999-GFP, which was included as a nega-tive control. Fluorescence in the transformed protoplasts was observed andphotographed under a confocal laser-scanning microscope (TCS SP2, Lecia).

Yeast Two-Hybrid Assay

Yeast two-hybrid assays were performed according to the manufacturer’sinstructions using the Matchmaker GAL4 two-hybrid system (Clontech). Thefull-lengthOsTGA10 coding sequence was cloned into pGBKT7, and full-lengthcoding sequences of genes of interest were cloned into pGADT7. Plasmids werecotransformed into AH109 yeast stains, which were initially grown onmediumlacking Leu and Trp for 3 d at 30°C. Six of the resulting independent colonieswere transferred onto medium without Leu, Trp, His, and Ade and containing3-amino-1,2,4 triazole for 3 d at 30°C to confirm protein interactions.

Pull-Down Assay

The full-length OsTGA10 coding sequencing was cloned into pCold-TF, gen-erating OsTGA10-TF. The full-length coding sequences of those genes of interestwere cloned into pGEX-4T-2, generating proteins of interest fusedwith GST. Pull-down assays were performed as described previously (Zhao et al., 2017)

Dual-Luciferase Transient Expression Assay

For transcription activity assay using the GAL4/UAS-based system in riceprotoplasts, the full-length OsTGA10 coding sequence was cloned into 62-SK-GAL4BD to create the effector, whereas 35S-UAS-LUCwas used as the reporter.The plasmids containing OsTGA10-GAL4BD and 35S-UAS-LUC were used totransform rice leaf protoplasts as previously described (Bart et al., 2006).Transformation with plasmids containing GAL4BD and 35S-UAS-LUC wasused as a negative control.

To measure the effect of OsTGA10 on transcriptional activity of target genes inNicotiana benthamiana leaves, the full-length coding sequences of OsTGA10, TIP2,and TDR were cloned into pGreen II 62-SK as effectors. The 1,500-bp sequencesupstream ofAP25 andMTR1 coding regions were cloned into pGreen II 0800-LUCas reporters. Agrobacterium GV3101 strain carrying the verified constructs werecultured toOD600 = 0.5 before cells were collected by centrifugation, resuspended ininfiltration buffer (10mMMES, 10mMMgCl2, 150mMacetosyringone), andplaced inthe darkness for 3 h. A 9:1 effector:reporter construct cell culture mixture wasinfiltrated into N. benthamiana leaves. As negative controls, leaves were infiltratedwith strains carrying reporter and empty effector constructs.

The dual-luciferase reporter system (Promega #E1910) was used to analyzetransient expression in transfected riceprotoplasts followingovernight incubation indarkness and in infiltrated N. benthamiana levels 48 h following infiltration. Theactivities of firefly (Photinus pyralis) and Renilla reniformis luciferases were mea-sured sequentially within a single sample on a GLO-MAX 20/20 Luminometer

(Promega). The ratio of LUC to REN was calculated as a measure of final tran-scriptional activity. Four biological repeats were included for each rice protoplastexperimental sample, whereas six biological repeats were included for each N.benthamiana experimental sample.

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBL data librariesunder accession numbers OsTGA10 (Os09g31390), OsMADS8(Os09g32948),OsMADS7 (Os08g41950), UDT1 (Os07g36460), TDR (Os02g02820), TIP2(Os01g18870), GAMYB(Os01g59660), EAT1(Os04g51070), MTR1 (Os02g28970),API5 (Os02g20930), AP25 (Os03g08790); AP37 (Os04g37570), CP1 (Os04g57490),CESA4 (Os01g54620), CESA7 (Os10g32980), CESA9 (Os09g25490), BC1(Os03g30250), BC3 (Os02g50550), BC10 (Os05g07790), BC12 (Os09g02650), BC14(Os02g40030), BC15 (Os09g32080), SWN1 (Os06g04090), SWN2 (Os08g02300),MYB46 (Os12g33070), MYB61 (Os01g18240).

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Antibody test for OsMADS8.

Supplemental Figure S2. ChIP-seq for OsMADS8.

Supplemental Figure S3. Expression pattern of Os09g31390 in transcrip-tome database.

Supplemental Figure S4. Distribution in Os09g31390 of sequenced DNAfragments from OsMADS8 ChIP-seq.

Supplemental Figure S5. Os09g31390 is homologous to ArabidopsisTGA10.

Supplemental Figure S6. Morphological criteria for rice stamen develop-ment before release of mature pollen grains used in this study.

Supplemental Figure S7. Characterization of ostga10.

Supplemental Figure S8. The ostga10 panicle pollinated with wild-typepollen grains.

Supplemental Figure S9. Phenotype of ostga10mutants created by CRISPRtechnology.

Supplemental Figure S10. Analysis of pollen grains in ostga10 mutants.

Supplemental Figure S11. No interaction between OsTGA10 and UDT1,nor between OsTGA10 and EAT1.

Supplemental Figure S12. Distribution of TGACG motif in the promotersof AIP1, AIP2, and CP1.

Supplemental Figure S13. The expression pattern of OsMADS7 duringanther development.

Supplemental Figure S14. Os0931390 was also bound by OsMADS7.

Supplemental Table S1. Primers used in this study.

Supplemental Table S2. Summary of demonstrated protein-proteininteractions.

ACKNOWLEDGMENTS

The authors thank Prof. Ming-Hua Deng, School of Mathematics, PekingUniversity and his student Zeng-Miao Wang for their assistance with ChIP-seqdata analysis; Prof. Da-Bing Zhang, Shanghai Jiaotong University, and hisassistant Li Yang for providing us with the tapetum gene mutant plants; Prof.Zheng Meng for providing us with the OsMADS8 RNAi line; Prof. Xiao-HuaFang, The Institute of Genetics and Developmental Biology, Chinese Academyof Sciences (CAS), for his contributions toward improving the protocol ofTUNEL assay for tapetum development; Prof. Chun-Ming Liu, The Instituteof Botany, CAS, and Prof. Cheng-Cai Chu, The Institute of Genetics and Devel-opmental Biology, CAS, for their assistance in providing rice growth facilities.

Received October 3, 2017; accepted November 16, 2017; published November20, 2017.

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