mir156-targeted sbp-box transcription factors interact ... · mir156-targeted sbp-box transcription...

miR156-Targeted SBP-Box TranscriptionFactors Interact with DWARF53 to RegulateTEOSINTE BRANCHED1 and BARREN STALK1Expression in Bread Wheat1

Jie Liu,a,2 Xiliu Cheng,b,2 Pan Liu,a and Jiaqiang Suna,3

aNational Key Facility for Crop Gene Resources and Genetic Improvement, Institute of Crop Science, ChineseAcademy of Agricultural Sciences, Beijing 100081, ChinabBiotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China

ORCID IDs: 0000-0002-2031-8783 (J.L.); 0000-0003-3908-6633 (P.L.); 0000-0002-3448-6956 (J.S.).

Genetic and environmental factors affect bread wheat (Triticum aestivum) plant architecture, which determines grain yield. In thisstudy, we demonstrate that miR156 controls bread wheat plant architecture. We show that overexpression of tae-miR156 inbread wheat cultivar Kenong199 leads to increased tiller number and severe defects in spikelet formation, probably due to thetae-miR156-mediated repression of a group of SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes. Furthermore,we found that the expression of two genes TEOSINTE BRANCHED1 (TaTB1) and BARREN STALK1 (TaBA1), whose orthologousgenes in diverse plant species play conserved roles in regulating plant architecture, is markedly reduced in the tae-miR156-OEbread wheat plants. Significantly, we demonstrate that the strigolactone (SL) signaling repressor DWARF53 (TaD53), whichphysically associates with the transcriptional corepressor TOPLESS, can directly interact with the N-terminal domains ofmiR156-controlled TaSPL3/17. Most importantly, TaSPL3/17-mediated transcriptional activation of TaBA1 and TaTB1 can belargely repressed by TaD53 in the transient expression system. Our results reveal potential association between miR156-TaSPLsand SL signaling pathways during bread wheat tillering and spikelet development.

Breadwheat (Triticum aestivum) is a major staple cropworldwide. Global demand for bread wheat is in-creasing with world population growth. To guaranteeglobal food security, people have been seeking eliteagronomic traits of bread wheat to improve its yield.Genetic and environmental factors affect plant archi-tecture, which strongly influences crop productivity.The identification and characterization of the regula-tory genes associated with wheat plant architecture isindispensable for both understanding the genetic basis

of phenotypic variation and facilitating the breedingof elite varieties with ideal wheat plant architecture.However, in addition to the wheat “green revolution”gene Reduced height 1 (Peng et al., 1999), the main genesthat determine wheat plant architecture remain to beidentified.

MicroRNA156 (miR156) targets members of theSQUAMOSA PROMOTER BINDING PROTEIN-LIKE(SPL) gene family for cleavage and/or translationalrepression, which encodes transcription factors that, inturn, regulate a large network concerning plant growthand development (Cardon et al., 1999; Wu and Poethig,2006; Schwarz et al., 2008; Wang et al., 2009; Wu et al.,2009; Yamaguchi et al., 2009; Yu et al., 2010; Gou et al.,2011). SPL transcription factors share a highly con-served DNA binding domain named the SQUAMOSAPROMOTER BINDING PROTEIN (SBP)-box and havebeen shown to associate with motifs with the consensussequence TNCGTACAA (N represents any base) topromote the downstream target genes transcription(Cardon et al., 1999; Yamasaki et al., 2004). In the pastfew years, a number of key genes controlling importantagronomic traits have been cloned from rice (Oryzasativa), among which are several transcription factorsfrom the SPL gene family (Chuck et al., 2007; Jiao et al.,2010; Miura et al., 2010; Wang et al., 2012; Xie et al., 2012;Si et al., 2016). The rice IDEAL PLANTARCHITECTURE1(IPA1) gene, isolated by the map-based approach,

1 This research was supported by the National Key Research andDevelopment Program of China (grant no. 2016YFD0100302), theMinistry of Agriculture of China (grant no. 2016ZX08009003-003),the Institute of Crop Science, Chinese Academy of Agricultural Sci-ences (CAAS), the Agricultural Science and Technology InnovationProgram of CAAS, and Youth Talent Plan of CAAS.

2 These authors contributed equally to the article.3 Address correspondence to [email protected] 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:Jiaqiang Sun ([email protected]).

The authors have declared that no competing interests exist.J.S. conceived the original screening and research plans; J.S. and

J.L. designed research and analyzed the data; J.L., X.C., and P.L.performed the experiments; J.S., J.L., and X.C. wrote the article; J.S.supervised and complemented the writing.

www.plantphysiol.org/cgi/doi/10.1104/pp.17.00445

Plant Physiology�, July 2017, Vol. 174, pp. 1931–1948, www.plantphysiol.org � 2017 American Society of Plant Biologists. All Rights Reserved. 1931 www.plantphysiol.orgon November 1, 2020 - Published by Downloaded from

Copyright © 2017 American Society of Plant Biologists. All rights reserved.

http://orcid.org/0000-0002-2031-8783

http://orcid.org/0000-0002-2031-8783

http://orcid.org/0000-0002-2031-8783

http://orcid.org/0000-0002-2031-8783

http://orcid.org/0000-0002-2031-8783

http://orcid.org/0000-0002-2031-8783

http://orcid.org/0000-0002-2031-8783

http://orcid.org/0000-0003-3908-6633

http://orcid.org/0000-0003-3908-6633

http://orcid.org/0000-0003-3908-6633

http://orcid.org/0000-0003-3908-6633

http://orcid.org/0000-0003-3908-6633

http://orcid.org/0000-0002-3448-6956

http://orcid.org/0000-0002-3448-6956

http://orcid.org/0000-0002-3448-6956

http://orcid.org/0000-0002-3448-6956

http://orcid.org/0000-0002-3448-6956

http://orcid.org/0000-0002-2031-8783

http://orcid.org/0000-0003-3908-6633

http://orcid.org/0000-0002-3448-6956

http://crossmark.crossref.org/dialog/?doi=10.1104/pp.17.00445&domain=pdf&date_stamp=2017-06-20

mailto:[email protected]

http://www.plantphysiol.org

mailto:[email protected]

http://www.plantphysiol.org/cgi/doi/10.1104/pp.17.00445


encodes OsSPL14, and the ipa1 mutant shows idealplant architecture with decreased tiller number andincreased plant height and panicle branches (Jiao et al.,2010). WEALTHY FARMER’S PANICLE, another over-expression allele ofOsSPL14, resulted froman epigeneticchange in the OsSPL14 promoter and shows a similarphenotype (Miura et al., 2010). The quantitative traitlocus GW8, encoding OsSPL16, regulates rice grain size,shape, and quality (Wang et al., 2012). The major quan-titative trait locus GLW7, encoding OsSPL13, positivelyregulates cell size in the grain hull, resulting in enhancedrice grain length and yield (Si et al., 2016). Therefore, ithas been suggested that elite SPLs alleles have great po-tential for improving crop agronomic traits and enhancinggrain yield.

In addition to SPLs, several key genes associatedwithcrop architecture have been identified from maize (Zeamays) and rice. For example, the maize TEOSINTEBRANCHED1 (TB1) and its orthologous genes riceOsTB1 (FINE CULM1) and Arabidopsis (Arabidopsisthaliana) BRANCHED1, belonging to members of theTEOSINTE BRANCHED1, CYCLOIDEA AND PCFTRANSCRIPTION FACTOR (TCP) gene family, functionas conserved negative regulators of lateral branching(Doebley et al., 1997; Takeda et al., 2003;Aguilar-Martínezet al., 2007). Also, themaize BARRENSTALK1 (BA1) geneencodes a noncanonical bHLH protein, and the tassel ofba1 mutants is unbranched, shortened, and predomi-nantly sterile owing to the often complete lack of spikelets(Gallavotti et al., 2004).

Strigolactones (SLs) are a group of newly identifiedplant hormones that suppress plant shoot branching(Gomez-Roldan et al., 2008; Umehara et al., 2008). Inrecent years, great advances have been made in SLsignaling in the model plants Arabidopsis and rice. Inrice, SL signaling requires degradation of the SL sig-naling repressor DWARF53 (D53), which is mediatedby a receptor complex including D14 and D3 (Jianget al., 2013; Zhou et al., 2013). In Arabidopsis, theSL receptor complex including AtD14 and the D3ortholog MORE AXILLARY GROWTH2 mediatesdegradation of the D53-like proteins, SUPPRESSOROF MORE AXILLARY GROWTH 2-LIKE6 (SMXL6),SMXL7, and SMXL8 (Soundappan et al., 2015; Wanget al., 2015; Yao et al., 2016). However, the down-stream transcription factors repressed by the D53transcriptional repressors in the SL signaling remain tobe identified.

Here, we report the characterization of tae-miR156 asa critical regulator of bread wheat plant architecture.We first showed that overexpression of miR156 inbread wheat plants triggers a bushy phenotype andsevere defects in spikelet formation. Meanwhile, agroup of putative SPL genes were identified to be tar-geted and repressed by tae-miR156. Next, we showedthat the expression of two genes, TaBA1 and TaTB1, wasmarkedly reduced in the miR156-overexpression(miR156-OE) wheat plants. Furthermore, our assaysrevealed that the SL signaling repressor TaD53 func-tions as a transcriptional repressor through interaction

with the transcriptional corepressor TOPLESS (TaTPL).Significantly, we found that TaD53 could physicallyinteract with the miR156-controlled SPL proteinsTaSPL3/17 and largely repress TaSPL3/17-mediatedtranscriptional activation of TaBA1 and TaTB1 ex-pression. Our findings provide new insight into themiR156-TaSPLs module in regulating bread wheatplant architecture.

RESULTS

Identification of a Tandem tae-microRNA156 Gene inBread Wheat

Previous analysis on the whole-genome shotgundraft sequence of the bread wheat A-genome progeni-tor Triticum urartu uncovered a large group of scaffoldsthat contain putative microRNA precursors in T. urartugenome (Ling et al., 2013), which makes it easier toidentify miRNA genes in bread wheat plants underly-ing important agronomic traits. Among these scaffolds,we noticed that the scaffold14333 potentially contains atandem tae-microRNA156 (tae-miR156) gene, in whichthreeMIR156 precursors (annotated asMIR156a, b, andc in this study) were aligned in tandem (SupplementalFig. S1). To identify the homologous sequences of thistandem tae-miR156 gene in hexaploid bread wheat, weused scaffold14333 as query to search the wheat surveysequences, which include the chromosome-based draftsequence of the hexaploid bread wheat (https://urgi.versailles.inra.fr/blast/; Deng et al., 2007; InternationalWheat Genome Sequencing Consortium, 2014). Finally,three highly conserved sequences separately located onchromosomes 3A, 3B, and 3D were obtained (Fig. 1A).Similar to that in T. urartu genome, the three sequencesinclude potential MIR156a/b/c precursors in tandem(separately marked by red, blue, and orange boxes inFig. 1A), indicating a high-level conservation of thistandem miR156 gene in bread wheat during the evo-lutionary process from its progenitor species. We fur-ther validated the tandem miR156 gene by predictingthe RNA secondary structures of these precursors.Certainly, each of these sequences can give rise tothree tandem stable stem-loop structures with ex-tremely low free energies (described as DG in Fig. 1Band Supplemental Fig. S2), which are reminiscent ofmiRNA precursors. Importantly, all of the mature tae-miR156 sequences are located on the stem regions of thestem-loop structures (highlighted by red in Fig. 1B andSupplemental Fig. S2) and are completely identical tothe known miR156 sequences in barley (Hordeum vul-gare), purple false brome (Brachypodium distachyon),maize, rice, and Arabidopsis annotated by miRBase(http://www.mirbase.org/; Fig. 1C). To further testwhether the identified tandem MIR156 precursorscould generate the mature tae-miR156 in vivo, wegenerated transgenic bread wheat lines using the breadwheat cultivar Kenong199 (KN199) as the background,in which a 1-kb nucleotide sequence from chromosome

1932 Plant Physiol. Vol. 174, 2017

Liu et al.

www.plantphysiol.orgon November 1, 2020 - Published by Downloaded from Copyright © 2017 American Society of Plant Biologists. All rights reserved.

http://www.plantphysiol.org/cgi/content/full/pp.17.00445/DC1


https://urgi.versailles.inra.fr/blast/




http://www.mirbase.org/


Figure 1. Identification of a tandemmicroRNA156 gene in breadwheat. A, The nucleotide sequences from chromosomes 3A, 3B,and 3D of the bread wheat cultivar KN199 encoding tandem miR156 genes. The three MIR156 precursors (MIR156a, b, and c)aligned in tandem are separately marked by red, blue, and orange boxes. The black shade boxes represent the positions of maturemiR156. B, Predicted secondary structure of tandem tae-MIR156a/b/c precursors from chromosome 3A. DG (kcal mol21) cal-culated byMfold represents the minimum free energy of the RNA secondary structure. nt, Nucleotides. C, Nucleotide sequencesof miR156 from different plant species. tae, Triticum aestivum; hvu,Hordeum vulgare; bdi, Brachypodium distachyon; zma, Zeamays; osa,Oryza sativa; ath, Arabidopsis thaliana. D, Stem-loop qRT-PCR quantification of tae-miR156 and tea-miR319 in wild-type bread wheat KN199 as well as in transgenic bread wheat plants overexpressing the tandem tae-MIR156-3A cluster driven bythe ubiquitin promoter. Three independent transgenic lines (1#, 2#, and 3#) were analyzed. The tae-miR156 and tae-miR319levels were all normalized against TaU6, and the mean values in wild-type KN199 were set to 1. Error bar represents SD amongthree independent biological replicates; asterisks above the bars denote significant differences compared with wild-type KN199plants at P , 0.01 (Student’s t test).

Plant Physiol. Vol. 174, 2017 1933

Repression of TaSPLs Activity by TaD53



A of KN199 containing the tandem MIR156 clusterwas overexpressed by using the ubiquitin promoter(Supplemental Fig. S3). We next checked the accumu-lation of mature tae-miR156 in transgenic plants usingthe stem-loop quantitative real-time PCR (qRT-PCR)and observed 5- to 12-fold increase of tae-miR156compared with the wild-type KN199 plants (Fig. 1D).Meanwhile, as a negative control, we also determinedthe level of another bread wheat endogenous miRNA,miR319, by the stem-loop qRT-PCR. Our results con-firmed that miR319 was similarly expressed in wild-typeand transgenic bread wheat plants (Fig. 1D), furtherconfirming the specific up-regulation of tae-miR156. Insummary, these data strongly support the conclusion thatthe identified tandem MIR156 cluster encodes functionaltae-miR156 gene in bread wheat.

tae-miR156 Controls Bread Wheat Plant Architecture

To our knowledge, the roles of miR156 in controllingbreadwheat plant development have not been reportedup to now. By sowing the tae-miR156-OE transgenicbread wheat lines under field conditions (Beijing) andinvestigating their vegetative and reproductive devel-opment phenotypes, we characterized the biologicalroles of tae-miR156 in regulation of bread wheat plantarchitecture in detail in this study. The most conspicu-ous aspect of the phenotypes of tae-miR156-OE plantsis the enhanced tillering. When the bread wheat plantswere at the vernalization stage, we already observedmore visible tiller buds in tae-miR156-OE seedlingscompared with wild-type KN199 (Fig. 2A), suggestingthat overexpression of tae-miR156 led to more easierand earlier production of lateral branching. Afterturning green in spring, the tae-miR156-OE transgeniclines generated more tillers than those in wild-typeKN199 and showed bushy phenotype at all their de-velopmental stages (the jointing stage as shown in Fig.2B and the heading and mature stages as shown in Fig.2C). Statistical analyses showed that overexpression oftae-miR156 caused a dramatic increase of tillers from;10 in the wild type to more than 40 in transgenic lines(Fig. 2E). Moreover, we observed that a certain numberof axillary buds in leaf axil in the tae-miR156-OEtransgenic plants could outgrow and thus producehigh-node tillers that rarely occur in wild-type plants(Supplemental Fig. S4). These observations indicatethat tae-miR156 plays a positive role in promoting theoutgrowth of axillary buds. In addition, the stem of tae-miR156-OE plants became thinner than that in wild-type KN199 (Supplemental Fig. S4).

In addition to the enhanced tillering phenotype,overexpression of tae-miR156 also led to severe defectsin spike morphology. A typical spike from wild-typeKN199 includes a central long rachis (8–10 cm in length)on which several tightly packed rows of spikelets (in-cluding 20–30 spikelets) are located (Fig. 2, D and E).However, in the tae-miR156-OE lines, an abnormallysmall spike was observed containing the extremely

shortened rachis (1–2 cm in length) and a single spikeletlocated on the top (Fig. 2, D and E; SupplementalFig. S4). By comparing the spikelets from the tae-miR156-OE plants and wild-type KN199, we noticedthat both spikelets equally contained at least threeflorets, and each floret had a lemma and a palea(Supplemental Fig. S4). However, the main difference isthat unlike a pair of glumes in wild-type spikelets,only one glume was observed in the tae-miR156-OEspikelets (Supplemental Fig. S4). The reduced spike-let number and defective spikelet morphology of tae-miR156-OE plants suggest that tae-miR156 is a keyregulator of bread wheat domestication-related traitson spike architecture.

tae-miR156 Targets a Group of Putative TaSPL Genes

The plant microRNAs regulate plant growth anddevelopment mainly through the down-regulation oftheir corresponding target genes. To further elucidatethe molecular bases of the defects in plant architecturecaused by overexpression of tae-miR156, we focused onthe analyses of tae-miR156 targeting genes. We pre-dicted the potential tae-miR156 target genes based onthe whole wheat cDNA library (T. aestivum, cDNA,Ensemblplants, release-31) using the Web-based plantsmall RNA target analysis tool psRNATarget (Dai andZhao, 2011). Meanwhile, 10 previously cloned and an-notated bread wheat SPL genes were also included forthe prediction (Zhang et al., 2014). Finally, 16 potentialmiR156-targeting genes were obtained with the max-imum expectation as 2.0 (Supplemental Table S1).Consistent with previous studies (Jones-Rhoades andBartel, 2004; Wang et al., 2009; Wu et al., 2009), all thepredicted tae-miR156 target genes encode putativeplant specific SPL transcription factors (SupplementalTable S1). Among these targets, T8 and T9 had alreadybeen annotated as TaSPL3 and TaSPL17, respectively, inbread wheat in a previous study (Zhang et al., 2014).

If these TaSPL genes are indeed the tae-miR156 targetgenes, whose transcripts can be cleaved by tae-miR156,their transcripts should have lower levels when tae-miR156 is overexpressed. In support of our hypothesis,qRT-PCR assays showed that all the tested miR156-targeting genes (T1 to T9 in Fig. 3A) were significantlydownregulated in three independent tae-miR156-OEtransgenic bread wheat lines, compared with those inwild-type KN199 (Fig. 3A), suggesting the repression ofthese putative TaSPL genes by the overexpression of tae-miR156. To further confirm the specificity of tae-miR156in regulation of these putative TaSPL genes, we simulta-neously examined the expression of two other TaSPLgenes, TaSPL20 and TaSPL21, which were predicted to benot regulated by tae-miR156 due to their low-level com-plementarity with tae-miR156 (Supplemental Fig. S5).Indeed, our results showed that the transcription levels ofTaSPL20 and TaSPL21 were similarly expressed in bothwild-type KN199 and tae-miR156-OE transgenic breadwheat lines (Supplemental Fig. S5), also supporting our


Liu et al.















Figure 2. Morphological characters of tae-miR156-OE bread wheat plants. A, Appearance of the visible tiller buds in wild-typeKN199 and tae-miR156 overexpression (tae-miR156-OE) bread wheat seedlings at the vernalization stage (in the middle ofNovember, Beijing). The red arrows point to the positions of tiller buds. Column diagram shows the statistical analyses of thenumbers of visible tiller buds of wild-type KN199 and three independent transgenic lines (n$ 5). B, Vegetative phenotypes of tae-miR156-OE transgenic bread wheat plants at jointing stage grown in the field. The whole seedling (top) and the stem bases

Plant Physiol. Vol. 174, 2017 1935 www.plantphysiol.orgon November 1, 2020 - Published by Downloaded from

Copyright © 2017 American Society of Plant Biologists. All rights reserved.


prediction that TaSPL20 and TaSPL21 are not the targetsof tae-miR156.

Next, we validated the tae-miR156-mediated cleavageon its targeting TaSPL genes in vivo through a previouslydescribed RACE procedure (Liu et al., 2014). Here weemployed T8_6DS/TaSPL3 and T9_7DS/ TaSPL17 for theRACE assays. Our results showed that tae-miR156 couldpredominantly cleave the TaSPL3 and TaSPL17 mRNAsat the position 10 to 11 of tae-miR156 from its 59 end (Fig.3B), which has been considered as a canonical miRNAcleavage site (Peters andMeister, 2007; Höck andMeister,2008); as negative controls, no cleavageproduct ofTaSPL20and TaSPL21 mRNAs was detected.

It has been proved in Arabidopsis that miR156 levelsare higher in young seedlings and decrease in an age-dependent manner; meanwhile, the expression patternsof miR156-targeting SPLs are inverse to that of miR156(Wang et al., 2009; Wu et al., 2009). Therefore, wewondered whether tae-miR156 and its targets alsodisplay similar temporal expression patterns in breadwheat plants. As expected, our results confirmed thattae-miR156 was highly accumulated in bread wheatjuvenile leaves that were collected from KN199 seed-lings at trefoil stage, but significantly decreased in flagleaves at heading stage (Supplemental Fig. S6), indi-cating a decrease tendency of tae-miR156 accompany-ing with the transition from juvenile to adult phases.The lowest level of tae-miR156 was detected in KN199young spikes at heading stage when comparedwith theleaf tissues (Supplemental Fig. S6). On the contrary, thetranscription levels of tae-miR156-regulated TaSPL3 andTaSPL17 displayed an increase tendency. qRT-PCRassays showed that the expression levels of TaSPL3 andTaSPL17 were much lower in juvenile leaves and in-creased in flag leaves (Supplemental Fig. S6). Strikingly,their maximum transcript levels were observed in youngspikes at heading stage (Supplemental Fig. S6). The in-verse expression patterns of tae-miR156 and TaSPL3/17further support our conclusion that TaSPL3/17 are tar-geted and cleaved by tae-miR156 in vivo.

In summary, we define a group of TaSPL genes astargets of tae-miR156 in breadwheat, whose expressioncan be repressed by tae-miR156.

The tae-miR156-Targeted TaSPLs Positively RegulateTaTB1 Expression

To further explore the underling mechanism of theenhanced tillering in tae-miR156-OE transgenic plants,we focused on the TB1 gene. Previous studies have

revealed that TB1 genes in maize and rice act as nega-tive regulators of lateral branching (tillering) and re-press the outgrowth of axillary buds (Doebley et al.,1997; Wang et al., 1999; Takeda et al., 2003). Theenhanced tillering phenotype of the tae-miR156-OEtransgenic bread wheat lines prompted us to determinewhether the TB1 ortholog in bread wheat is transcrip-tionally affected. First, we used the coding sequence ofZmTB1 (GenBank accession no. JQ900502) as query tosearch the wheat survey sequences using BLAST andobtained three nucleotide sequences with high simi-larity. Annotation and BLAST analyses revealed thatthe three homologous coding sequences are separatelylocated on chromosomes 4A, 4B, and 4D (SupplementalFig. S7) and encode proteins sharing ;56% identitieswith OsTB1 (LOC_Os03g49880) and;54%with ZmTB1(Supplemental Fig. S8), suggesting that they are mostlikely the candidates of TaTB1-4A, -4B, and -4D inbread wheat. Then, we designed specific primers todetermine the transcription of TaTB1 by qRT-PCR.Considering that TB1 gene is involved in lateralbranching control, we used the stem bases from breadwheat seedlings grown at the vernalization stage forour analyses. Our results showed that in all detectedtae-miR156-OE lines, TaTB1 was dramatically down-regulated (Fig. 4A).

The correlation between the compromised tran-scription of TaTB1 and the down-regulation of TaSPL3and TaSPL17 followed by the overexpression of tae-miR156 in transgenic bread wheat plants led us to askwhether TaTB1 is a potential downstream gene regu-lated by TaSPLs. To confirm this hypothesis, we firstcloned the 2-kb promoter sequences of TaTB1-4A,-4B, and -4D from the KN199 genome using specificprimers. As expected, we identified a number of SBP-box binding motifs (including CGTACAA, GTACAA,CGTACA, GTACA, and GTAC) on each of the threeidentified TaTB1 promoters (Fig. 4B; Supplemental Fig.S9), in support of our hypothesis that TaSPLs mightregulate TaTB1 genes through binding to their pro-moter regions. To experimentally confirm the regula-tory effect of the miR156-regulated TaSPLs on TaTB1genes, we carried out transient transcriptional activityassays by Agrobacterium tumefaciens-mediated transientinfiltration in Nicotiana benthamiana leaves. First, wegenerated the reporters by fusing the 2-kb identifiedTaTB1 promoters (TaTB1-4Apro, -4Bpro, and -4Dpro) withfirefly luciferase (LUC) gene to produce constructsTaTB1-4Apro:LUC, -4Bpro:LUC, and -4Dpro:LUC. To checkthe basal activities of these reporters, the constructsof TaTB1-4Apro:LUC, -4Bpro:LUC, and -4Dpro:LUC were

Figure 2. (Continued.)(bottom) from three independent transgenic bread wheat lines as well as wild-type KN199 were observed in early April. Bars =10 cm. C, The bushy phenotype of tae-miR156-OE bread wheat plants at heading (top, in late April) and mature (bottom, in lateJune) stages. Bars = 20 cm. D, Spike phenotypes of wild-type KN199 and tae-miR156-OE bread wheat plants at the heading (topleft), grain filling (top right, in early May), or mature (middle and bottom) stages. Bars = 5 cm. E, Statistical analyses of the plantarchitecture characters of wild-type KN199 and tae-miR156-OE bread wheat plants. Three independent transgenic lines wereanalyzed at the mature stage, and error bar represents SD (n$ 5). Asterisks above the bars in A and E denote significant differencescompared with wild-type KN199 plants at P , 0.01 (Student’s t test).


Liu et al.












separately infiltrated into N. benthamiana leaves, fol-lowed by the determination of LUC activity. Interest-ingly, LUC activity was exclusively observed in theTaTB1-4Apro:LUC infiltrated samples (with lumines-cence intensity ;6,000 counts per second [CPS], asshown in Fig. 4C, infiltration 2), whereas no obviousLUC signal was detected either in TaTB1-4Bpro:LUC or-4Dpro:LUC expressed samples (the luminescence in-tensitieswere,2,000 CPS; Fig. 4C, infiltrations 3 and 4),

compared with the empty vector (EV) control. We at-tributed these variations to divergent driving activi-ties among the three types of TaTB1 promoters.Subsequently, we selected TaTB1-4Apro:LUC (with highdriving activity) and TaTB1-4Dpro:LUC (with low drivingactivity) constructs for further activation analyses.Our results confidently confirmed that coexpressionof 35S:TaSPL3 with TaTB1-4Apro:LUC and TaTB1-4Dpro:LUC all led to almost 30- to 40-fold increases of the LUC

Figure 3. tae-miR156 targets a group of putative TaSPL genes in breadwheat. A, Determination of the transcript levels of putativetae-miR156 targets in tae-miR156-OE bread wheat lines. The transcript levels of indicated genes were first normalized againstTaGAPDH, and the mean values in wild-type KN199 were set to 1. Error bars represent SD among three independent biologicalreplicates; asterisks above the bars denote significant differences compared with wild-type KN199 plants at **P, 0.01 or *P, 0.05(Student’s t test). B, Mapping of cleavage sites of tae-miR156 by 59RACE. Nucleotide sequences of potential tae-miR156 target sites onTaSPL3 and TaSPL17, togetherwith theWatson-Crick base pairings to the tae-miR156 are shown. The free energies of duplex structuresrepresented by DG (kcal mol21) are calculated by Mfold. The red arrows indicate the cleavage sites, and numbers below the arrowsshow the frequency of clones with matching 59RACE product from this site out of total clones confirmed by sequencing.





reporter activity compared with that in the EV controls(with luminescence intensities higher than 30,000 CPS;Fig. 4D), suggesting that TaSPL3 could dramatically ele-vate the driving activities of TaTB1-4Apro and TaTB1-4Dpro.

Parallel experiments were performed using TaSPL17, andsimilar results were observed (Supplemental Fig. S10), in-dicating that both TaSPL3 and TaSPL17 are activators ofTaTB1 expression.

Figure 4. TaSPL3 positively regulates the expression of TaTB1 in bread wheat. A, Quantification of the TaTB1 transcript levels instem base tissues of KN199 and tae-miR156-OE bread wheat seedlings by qRT-PCR. The stem base of the bread wheat seedlingwere collected at the vernalization stage (in the middle of November, Beijing), and the transcript levels of TaTB1were quantifiedby normalizing against TaGAPDH. Mean values of TaTB1 in wild-type KN199 were set to 1. **P , 0.01 (Student’s t test). B,Schemes illustrating the 2-kb promoters of TaTB1-4A, 4B, and 4D. The black boxes display the positions of SBP-box binding coremotifs. C, LUC activity assays showing the promoter-driving activities of TaTB1-4Apro/4Bpro/4Dpro. The 2-kb promoter sequencesof TaTB1-4Apro, -4Bpro, and -4Dpro were used to drive the LUC gene expression, and the LUC activities were determined by A.tumefaciens-mediated infiltration in N. benthamiana 48 h postinfiltration (hpi). Left, A representative leaf image; right, quanti-fication of the relative luminescence intensities (n = 10); the colored scale bar indicates the luminescence intensity (CPS). Themean values in EV were set to 1, and asterisks above the bars represent significant differences against EV control at P , 0.01(Student’s t test). D, Transient expression assays illustrating the activation of TaTB1 transcription by TaSPL3. TaTB1-4Apro and-4Dpro were employed for the analyses. Left panel shows representative leaf images, and the right column diagram represents thequantification of the relative luminescence intensities (n = 15). The mean values in combinations 1 and 3 were all set to 1.Asterisks above the bars represent significant differences against combinations 1 or 3 at P, 0.01 (Student’s t test). Error bars in A,C, and D represent SD among three independent biological replicates.


Liu et al.




In summary, the above data imply that TaTB1 is prob-ably involved in the miR156-TaSPLs module-mediatedsignaling through transcriptional activation by TaSPL3and TaSPL17 transcription factors.

The tae-miR156-Targeted TaSPLs Are Essential for theTranscription of TaBA1

Numerous genes have been reported to be essentiallyinvolved in the inflorescence/panicle/spike develop-ment in diverse plant species (Pelaz et al., 2000; Honmaand Goto, 2001; Chuck et al., 2002; Ritter et al., 2002;Gallavotti et al., 2004, 2010; Bortiri et al., 2006;Shitsukawa et al., 2006; Paolacci et al., 2007; Derbyshireand Byrne, 2013; Liu et al., 2013; Ren et al., 2013; Linet al., 2014; Dobrovolskaya et al., 2015; Guedira et al.,2016). To dissect the molecular basis of defective spikearchitecture in the tae-miR156-OE lines, we made effortsto determine whether the expressions of these genesare altered in our transgenic lines, especially in youngspikes before the heading stage. According to our results,the expression of some inflorescence/panicle/spikedevelopment-related genes, such asTaVERNALIZATION1(GenBank accession no. AY747604), TaWFL (GenBankaccession no. AB231888), TaSEPALLATA1 (GenBankaccession no. AM502866), and TaSEP2 (GenBank ac-cession no. AM502867), were not affected by the over-expression of tae-miR156, indicating that these genesmight not participate in the miR156-TaSPL-regulatedsignaling pathway in bread wheat (SupplementalFig. S11). Interestingly, TaBA1 was markedly down-regulated by tae-miR156 overexpression in breadwheat (Fig. 5A). The ZmBA1 gene, an ortholog ofTaBA1, was initially isolated from the maize recessivemutant ba1, in which the tassel (apical male inflores-cence in maize) is seriously defective in branchingand spikelet formation (Ritter et al., 2002; Gallavottiet al., 2004), suggesting that ZmBA1 is a positiveregulator of spikelet formation in maize. Three copiesof intronless TaBA1 genes (Supplemental Fig. S12),which are separately located on 3A, 3B, and 3Dchromosomes in hexaploid bread wheat, all encodetranscription factors with a conserved bHLH do-main and share ;58% identity with ZmBA1 protein(GenBank accession no. NP_001105271; Fig. 5B).Significantly, the bHLH domain in TaBA1, which isinvolved in DNA binding activity, is 100% identicalto that in ZmBA1 (Fig. 5B), implying that BA1 pro-teins in maize and bread wheat might be involvedin similar signaling pathways in regulating spikearchitecture.Based on the finding that the transcription of TaBA1

was drastically compromised, we tested whetherTaBA1 is a potential target gene controlled by thetae-miR156-regulated TaSPLs. First, 2-kb promoter se-quences corresponding to the TaBA1-3A, -3B, and -3Dgenes were identified from bread wheat KN199 basedon the information of wheat survey sequences. Analy-ses further revealed that several SBP-binding motifs

(such as GTACA and GTAC) were included in TaBA1-3Apro and TaBA1-3Dpro, and one (GTACAA) in TaBA1-3Bpro (Fig. 5C; Supplemental Fig. S13). The transientexpression assays inN. benthamianawere carried out byusing the promoters of TaBA1-3A, -3B, and -3D fusedwith the LUC gene as reporters (TaBA1-3Apro:LUC,-3Bpro:LUC, and -3Dpro:LUC) to identify the drivingactivities of TaBA1 promoters. Results showed thatonly TaBA1-3Dpro could constitutively drive the ex-pression of LUC gene (with luminescence intensity;5,000 CPS), compared with the EV negative con-trol, while no obvious LUC signal was observed inTaBA1-3Apro:LUC or -3Bpro:LUC expression samples(with luminescence intensity ,1,000 CPS; Fig. 5D).These results suggest that TaBA1-3Dpro has the highestdriving activity among the three types of TaBA1promoters. We further carried out transient tran-scriptional activation assays in N. benthamiana usingTaBA1-3Bpro:LUC (with low driving activity) and TaBA1-3Dpro:LUC (with high driving activity) as the reportersto determine the effects of TaSPL3 on the transcrip-tion of TaBA1 genes. In contrast to the low levels ofLUC activity in the TaBA1-3Bpro:LUC or TaBA1-3Dpro:LUC expressed samples (Fig. 5E, coinfiltrations 1 and3), dramatically enhanced luminescence intensitieswere observed in all samples coexpressing 35S:TaSPL3(with luminescence intensities higher than 20,000CPS; Fig. 5E, coinfiltrations 2 and 4), indicating thatTaSPL3 could efficiently activate the transcription ofTaBA1 genes. This conclusion was further confirmedby using TaSPL17 as the activator (Supplemental Fig.S14).

Taken together, the above data lead to the conclu-sion that the tae-miR156-targeted TaSPLs, includingTaSPL3 and TaSPL17, are potential activators of TaBA1expression.

TaD53 Directly Interacts with TaSPL3 and TaSPL17

The enhanced tillering of tae-miR156-OE breadwheat lines is reminiscent of the phenotypes of somerice tillering dwarf mutants (d mutants) defective in SLbiosynthesis and signaling (Ishikawa et al., 2005; Zouet al., 2006; Arite et al., 2007, 2009; Lin et al., 2009; Jianget al., 2013; Nakamura et al., 2013). Among them, thed53 mutant, which shows increased tillering and lessbranched panicle, was well characterized (Jiang et al.,2013; Zhou et al., 2013). Previous studies revealed thatD53 shares predicted features with the class I ClpATPase proteins and functions as a repressor of SLsignaling in rice (Jiang et al., 2013; Zhou et al., 2013).Based on the plant architecture phenotypes of rice d53mutant, we supposed a hypothesis that the breadwheatD53 homologs TaD53 might be associated with themiR156-TaSPLs signaling module in controlling breadwheat plant architecture. To test this hypothesis, we firstidentified the bread wheat TaD53 genes (SupplementalFig. S15) based on the coding sequence of OsD53(LOC_Os11g01330). BLAST analyses revealed that three













Figure 5. TaSPL3 positively regulates the expression of TaBA1 in breadwheat. A, Determination of the TaBA1 transcript levels byqRT-PCR in KN199 and tae-miR156-OE breadwheat plants. The expression levels of TaBA1were normalized against TaGAPDH,and the mean values of TaBA1 in wild-type KN199were set to 1. **P, 0.01 (Student’s t test). B, Sequence comparison of TaBA1-3A/3B/3D and ZmBA1 proteins. The bHLH domains are indicated by red lines. The black shade represents the identity, and graymeans the similarity. C, Schemes represent the promoters of TaBA1-3A, -3B, and -3D. The black boxes display the positions ofSBP-box binding core motifs. D, LUC activity assays illustrating the promoter-driving activities of TaBA1-3Apro/3Bpro/3Dpro. The2-kb promoter sequences of TaBA1-3Apro, -3Bpro, and -3Dprowere constructed to drive the LUC gene, and the LUC activities weredetermined by A. tumefaciens-mediated infiltration in N. benthamiana. Left panel, a representative leaf image; right panel, thequantification of the relative luminescence intensities by using n = 10 independent leaves; the scale bar illustrates the lumi-nescence intensity of CPS by color. The mean values in EV were set to 1, and asterisks above the bars represent significant dif-ferences against EV control at P , 0.01 (Student’s t test). E, Transient expression assays illustrating the activation of TaBA1transcription by TaSPL3. TaBA1-3Bpro and -3Dpro were employed for the assays. Left panel shows representative leaf images, and


Liu et al.



copies of TaD53 encode proteins sharing 70% iden-tity with OsD53 and 42% with the D53-like proteinAtSMXL7 in Arabidopsis (Supplemental Fig. S16). Wefurther asked whether TaD53 physically interacts withthe tae-miR156-regulated TaSPL transcription factors.To this end, we fused TaD53 to the N-terminal part ofLUC (nLUC) to generate nLUC-TaD53, and TaSPL3and TaSPL17 to the C-terminal part of LUC (cLUC) toproduce cLUC-TaSPL3 and cLUC-TaSPL17. We thanperformed LUC complementation imaging (LCI)assays inN. benthamiana leaves. Interestingly, obviousLUC activities were observed both in nLUC-TaD53/cLUC-TaSPL3 and nLUC-TaD53/cLUC-TaSPL17 co-expression samples (Fig. 6A, coinfiltrations 4 and 6),demonstrating the physical association of TaD53 withTaSPL3 and TaSPL17. The direct interaction betweenTaD53 and TaSPL3/TaSPL17 was further confirmedby bimolecular fluorescence complementation (BiFC)assays in N. benthamiana, by which the fluorescencesignal of yellow fluorescent protein (YFP) could beobserved exclusively in the nuclei of nYFP-TaSPL3/cYFP-TaD53 and nYFP-TaSPL17/cYFP-TaD53 coex-pression samples, but not in the negative controls (Fig.6B). Therefore, we proposed that the SL signaling re-pressor TaD53 might be functionally associated with thetae-miR156-regulated TaSPLs transcription factors, suchas TaSPL3 and TaSPL17.

The N Terminus of TaSPL3 Mediates the Interaction withTaD53, While Its C Terminus Has the TranscriptionalActivation Activity

To further define the interaction domains of TaSPL3/17 with TaD53, the full-length TaSPL3 and TaSPL17proteins were divided into three truncated partsaccording to the positions of the highly conserved SBPdomains, i.e. NT (N terminus), MD (middle domain,containing the intact SBP domain), and CT (C terminus;Fig. 7A; Supplemental Fig. S17). The LCI results showedthat the strongest interaction signals were observed be-tween TaD53 and NT parts of TaSPL3 and TaSPL17(Fig. 7B; Supplemental Fig. S17), suggesting that theN-terminal domains of TaSPL3/17 mainly mediate thephysical interaction with TaD53.

As is well known, the SBP domains of SPL tran-scription factors are responsible for the DNA binding totheir target genes. We here performed transcriptionalactivity assays in AH109 yeast (Saccharomyces cerevisiae)cells for the full length as well as truncated forms ofTaSPL3 protein to define its transcriptional regulationdomain. We separately fused the full-length as well asthe NT, MD, and CT parts of TaSPL3 with pGBKT7(BD) vector and transformed into yeast strain AH109.The transformants were selected on synthetic dextrosemedium lacking Trp and His (SD-W/H) to check their

Figure 5. (Continued.)the right column diagram represents the quantification of the relative luminescence intensities using n = 15 independent leaves.The mean values in combinations 1 and 3 were all set to 1. Asterisks above the bars represent significant differences againstcombinations 1 or 3 at P , 0.01 (Student’s t test). Error bars in A, D, and E represent SD among three independent biologicalreplicates.

Figure 6. ThemiR156-regulated TaSPL3and TaSPL17 physically interact withTaD53. A, Firefly LCI assay detecting theinteraction between TaSPL3/17 andTaD53. The signals were collected48 hpi. B, BiFC assays confirming theinteraction between TaSPL3/17 andTaD53. The YFP fluorescence signalswere collected 48 hpi. BF, Bright field.Bars = 50 mm. In A and B, five inde-pendent N. benthamiana leaves wereused for the assays and three biologicalreplications were performed with simi-lar results.








growth status. As expected, the yeast cells harboringthe full-length TaSPL3 grew well on the selective me-dium, suggesting that TaSPL3 could efficiently activatethe transcription of the report gene (Fig. 7C); moreimportantly, the yeast cells containing TaSPL3-CTshowed similar growth status with those containing thefull-length TaSPL3 on the selective medium (Fig. 7C),indicating that the C-terminal part of TaSPL3 is re-sponsible for its transcriptional activation activity.

In summary, our analyses suggest that the three dif-ferent regions of TaSPL3 protein have distinct functions,

i.e. MD (the SBP domain) for the DNA binding ability,CT for the transcriptional activation activity, andNT forthe physical interaction with the transcriptional re-pressor TaD53.

Interaction of TaD53 with TaTPL

A previous study has shown that OsD53 couldphysically associate with the TPL/TOPLESS-RELATEDPROTEIN (TPR) class of transcriptional corepressorsthrough ethylene-responsive element binding factor-associated amphiphilic repression (EAR) motifs (Jianget al., 2013). Like OsD53, we found that TaD53 alsocontains three putative EARmotifs: 571Leu-Val-Leu-Asn-Leu-575 (EAR1), 792Leu-Asp-Leu-Ser-Leu-796 (EAR2),and 970Phe-Asp-Leu-Asn-Leu-974 (EAR3; SupplementalFig. S16). Thus, we speculated that TaD53 might also beassociatedwith the TPL orthologs in breadwheat. In thiscontext, we identified a TaTPL gene (Supplemental Fig.S18) from breadwheat based on the sequence ofOsTPR1(LOC_Os01g15020). Analysis revealed that the TaTPLprotein candidate shares high level identities withOsTPR1 (62% identity), OsTPR2 (LOC_Os08g06480; 67%identity), OsTPR3 (LOC_Os03g14980; 94% identity),and AtTPL (At1g15750; 77% identity) (SupplementalFig. S19). Next, we conducted LCI assays in N. ben-thamiana and confirmed that TaTPL indeed could in-teract with TaD53 (Fig. 8A). The interaction was furthersupported by the BiFC assays, in which an obviousinteraction signal was observed exclusively in the cellnucleus of nYFP-TaTPL/cYFP-TaD53 coexpressionsample (Fig. 8B). These results confirm the conservationof D53-TPL regulatory module in both rice and breadwheat.

TaD53 Represses the Transcriptional Activation Activity ofTaSPL3 on TaBA1 and TaTB1 Transcription

To evaluate the effects of TaD53 on the transcrip-tional activities of TaSPLs, we coexpressed 35S:TaD53and 35S:TaSPL3 together with the reporter TaTB1-4Apro:LUC in a 1:1:1 ratio in N. benthamiana (Fig. 9A, coinfil-tration 4). Meanwhile, the coexpression of 35S:TaSPL3/35S:GFP, 35S:TaD53/35S:GFPwith the LUC reporter in1:1:1 ratio (Fig. 9A, coinfiltrations 2 and 3) and the ex-pression of 35S:GFPwith the reporter in a 2:1 ratio (Fig.9A, coinfiltration 1) were simultaneously carried out ascontrols. Similar to the above results, coexpressionof 35S:TaSPL3 dramatically elevated the luminescenceintensity (Fig. 9A, coinfiltration 2), compared with the35S:GFP expressed negative control (Fig. 9A, coinfil-tration 1). However, after the coexpression of 35S:TaD53 with 35S:TaSPL3, the induction of LUC activityby TaSPL3 was dramatically compromised by morethan 70% (Fig. 9A, coinfiltration 4), suggesting that thetranscriptional activation activity of TaSPL3 was largelysuppressed by TaD53 in regard to the activationof TaTB1 transcription. Parallel experiments using

Figure 7. The N-terminal domain of TaSPL3 is sufficient for the inter-action with TaD53 and its C-terminal domain has transcriptional acti-vation activity. A, Schemes display full length as well as truncatedversions of TaSPL3 protein. NT, N-terminal domain; MD, middle do-main; CT, C-terminal domain; aa, amino acids. B, LCI assay showing theinteraction between the truncated TaSPL3 versions and the full-lengthTaD53. The LUC signals were observed at 48 hpi (n $ 5). Three bio-logical replications were performed, and similar results were observed.C, Transcriptional activation activity determination of TaSPL3 in yeastAH109 (S. cerevisiae). SD-W, Synthetic dextrose medium lacking Trp;SD-W/H, synthetic dextrose medium lacking Trp and His; 1021, 1022,1023, and 1024 denote the different dilution series.


Liu et al.









TaBA1-3Dpro:LUC as the reporter were carried out. Asexpected, the transcriptional activation activity ofTaSPL3 in enhancing TaBA1 transcription was largelyblocked by TaD53 (Fig. 9B, coinfiltrations 5–8). Theseresults suggest a repressive effect of TaD53 on the ac-tivation activities of tae-miR156-targeted TaSPLs inregulating the transcription of their downstream targetgenes.

DISCUSSION

miR156 Controls Bread Wheat Plant Architecture

Previous study on themaizeCorngrass1 (Cg1) mutantproved that overexpression of miR156 in maize led torepeated initiation of tillers and transformedmaize intobush (Chuck et al., 2007). Overexpression of miR156 inrice also led to enhanced tillering phenotype (Luo et al.,2012; Xie et al., 2012). Similarly, in the model plantArabidopsis, a bushy phenotype was observed uponthe overaccumulation of miR156 (Schwab et al., 2005).These studies, coupled with the bushy phenotype inour tae-miR156-OE transgenic bread wheat plants (Fig.2), confidently illustrate that miR156 is functionallyconserved in promoting tillering/branching in differentplant species.However, the potential molecular function of miR156

in inflorescence/panicle/spike development still needsto be elucidated. In this study, we identified a potentialrelationship between tae-miR156 and bread wheatspike morphology. Interestingly, unlike its positive ef-fect on tillering initiation, tae-miR156 was shown to actas a repressor of bread wheat spikelet development,according to the observation that overdose of tae-miR156 led to defective spikes with single or fewspikelet(s) (Fig. 2; Supplemental Fig. S4). In contrast toless affected panicle morphology in rice miR156-OEtransgenic plants (Xie et al., 2012), miR156-OE in breadwheat appears to trigger an extremely severe defect inspike shape, including dramatically shortened rachis(1–2 cm) and single spikelet (Fig. 2; Supplemental Fig.S4). Previous studies showed that miR156 is highly

accumulated in young seedlings but subsequently de-creases along with the vegetative-reproductive phasetransitions (Xie et al., 2006; Chuck et al., 2007; Wanget al., 2008, 2009; Xie et al., 2012). A similar expressionpattern was observed in this study that tae-miR156 ishighly accumulated in juvenile but decreases in flagleaves and young spikes (Supplemental Fig. S6).However, how plants precisely control miR156 levels indifferent developmental stages remains unclear. Futureworks on this issue should be of benefit to answer whyand how tae-miR156 is simultaneously involved in theregulation of bread wheat plant architecture throughmodulation of two distinct aspects, including the tillernumber per plant and the spikelet number per spike.

Based on bioinformatics prediction and experimentalconfirmation, we further identified a group of breadwheat putative TaSPLs as potential tae-miR156 targetinggenes (Fig. 3; Supplemental Table S1). Upon the over-expression of tae-miR156, the expression of these TaSPLgenes was significantly repressed (Fig. 3), while the otherTaSPLs, such as TaSPL20 and TaSPL21, that are not tar-geted by tae-miR156were not affected (Supplemental Fig.S5). Previous studies in rice have revealed that IPA1, en-coding the miR156-controlled SPL14 transcription factor,could profoundly determine rice ideal plant architecture,including decreased tillers but increased panicle branch-ing (Jiao et al., 2010; Miura et al., 2010; Luo et al., 2012;Wang et al., 2017). More importantly, among our identi-fied tae-miR156 targets, TaSPL17 is the orthologous geneof rice IPA1, which shares high identity with IPA1 at theprotein level. These results promote us to assume that thephenotypes of tae-miR156-OE plants, including enhancedtillering and decreased spikelet number, might be par-tially due to the compromise of these tae-miR156-targetedTaSPL genes. Although our current data could not sup-port the conclusion that all these miR156 targets arefunctional in regulation of breadwheat plant architecture,further detailed investigations, such as generation of thedominant andmiR156-resistantmutations for specific tae-miR156 targeted TaSPL genes, may provide clues forunderstanding the potential roles of theseTaSPLs in breadwheat plant architecture modulation.

Figure 8. Physical interaction of TaD53with TaTPL. A, LCI assay showing the in-teraction of TaD53 with TaTPL. B, BiFCconfirming the physical interaction be-tween TaD53 and TaTPL. The LUC and YFPfluorescence signals were all collected48 hpi. BF, Bright field. Bars = 50 mm. In Aand B, five independent N. benthamianaleaves were used for the assays and threebiological replicationswere performedwithsimilar results.












The miR156-Controlled TaSPLs Regulate the Expression ofTaTB1 and TaBA1 Genes

The TB1 homologous genes from diverse plant spe-cies were reported to play a conserved role in sup-pressing lateral branching, especially tillering (Doebleyet al., 1997; Takeda et al., 2003; Aguilar-Martínez et al.,2007), while ZmBA1 acts as a pivotal regulator inmodulating maize plant architecture, especially thespike morphology (Ritter et al., 2002; Gallavotti et al.,2004). In this study, we noticed that the expression ofTaTB1 was significantly reduced in the stem bases oftae-miR156 overexpression bread wheat plants (Fig.4A); similarly, TaBA1 gene expression was also largelycompromised in young spikes of tae-miR156-OE breadwheat lines (Fig. 5A). These observations correlatedwith the extremely enhanced tillering and defectivespike phenotype in tae-miR156-OE transgenic breadwheat plants, which led us to ask whether these genesare potentially involved in the miR156-TaSPL-mediated

signaling pathway. Analyses of TaTB1 and TaBA1 pro-moter sequences identified a certain number of SBP-binding motifs (Supplemental Figs. S9 and S13), raisingthe possibility that TaSPL transcription factors mightbind to the promoters of these genes to regulate theirexpression. In vitro assays indeed showed that bothTaSPL3 and TaSPL17 could significantly activate thetranscription of TaTB1 and TaBA1 (Figs. 4 and 5;Supplemental Figs S10 and S14). According to theseanalyses, we assume that TaTB1 and TaBA1 might bepotentially located downstream of miR156-TaSPLssignaling, and their transcriptional expression shouldpartially require the activation activities of miR156-controlled TaSPLs. In agreement with our findings, afunctional link between the miR156-SPLs module andTB1 has been established by a previous study in riceshowing that IPA1/OsSPL14 directly regulates the ex-pression of OsTB1 to suppress rice tillering, thus plac-ing the miR156-SPLs module upstream of OsTB1 (Luet al., 2013). Together, we propose that the miR156-SPLs-TB1 signaling cascade might be highly conservedin diverse crop species. In the future, it would be in-triguing to confirm the functions of TaTB1 and TaBA1 insuppressing tillering and promoting spike/panicle de-velopment in bread wheat and thus provide new po-tential genetic resources for crop molecular breeding.

Repression of TaSPL Activities by the SL SignalingRepressor TaD53

In recent years, several studies greatly advanced ourunderstanding on SL signaling, especial the signalperception coupled with the degradation of SL signal-ing repressors D53 proteins. As a relatively new classof plant hormone, more components involved in SLsignaling remain to be identified. For example, thedownstream transcription factors regulated by the re-pressor D53 in SL signaling remain largely unknown upto now. In this study, we showed that TaD53 interacts

Figure 9. TaD53 represses the transcriptional activation activity ofTaSPL3 for TaBA1 and TaTB1 transcription. Transient expression assaysseparately illustrating the repressive effect of TaD53 on TaSPL3-mediatedactivation of TaBA1 (A) and TaTB1 (B) expression. The coinfiltrations of35S:GFP, 35S:TaSPL3-GFP/35S:GFP, 35S:TaD53-GFP/35S:GFP, and35S:TaD53-GFP/35S:TaSPL3-GFPwith the reportersTaTB1-4Apro:LUC orTaBA1-3Dpro:LUC in 2:1 or 1:1:1 ratios were carried out as indicated. Leftpanels show representative leaf images, and the right column diagramsrepresent quantification of the relative luminescence intensities (n = 15).The values in combinations 3 and 7 were all set to 1. Asterisks above thebars represent significant differences against combinations 3 or 7 at P ,0.01 (Student’s t test). Error bar represents SD among three independentbiological replicates.

Figure 10. A proposed working model of the miR156-TaSPLs moduleand TaD53 in controlling bread wheat plant architecture.


Liu et al.






with the transcription corepressor TaTPL (Fig. 8), whichsupports the conclusion that TaD53 functions as atranscriptional repressor. Significantly, we found thatTaD53 directly interacts with tae-miR156-controlledTaSPL transcription factors, such as TaSPL3 andTaSPL17 (Fig. 7; Supplemental Fig. S17). Furthermore,our results showed that the transcriptional activity ofTaSPL3 could be largely suppressed by TaD53 in regardto the activation of its downstream target genes TaTB1and TaBA1 (Fig. 9). These data support the hypothesisthat the tae-miR156-controlled TaSPLs might also bedirect targets of TaD53 at the protein activity layer,which provides new insight into the SL signalingpathway in plants. However, it should be more com-plex in real situations. A previous study in rice showedthat overexpression of the OsSPL14 gene could largelysuppress the tillering phenotype of SL biosynthesis/signaling mutants d10-2 and d3-2 (Luo et al., 2012).Meanwhile, Luo et al. (2012) also showed that thetreatment with GR24 (a synthetic SL analog) still couldsuppress the tiller outgrowth in miR156-OE transgenicrice plants. These results support a hypothesis that SPLsmight act independently or downstream of SL signal inthe control of tillering. In this study, we showed that theSL signaling repressor TaD53 could physically interactwith the miR156-regulated TaSPLs and repress theiractivities (Figs. 6, 7, and 9), indicating that SLmight alsoact, at least in part, through D53-mediated regulation ofSPLs transcription factors to control tillering. However,we could not rule out the possibility that SL might alsoparticipate in the control of bread wheat plant archi-tecture through other transcription factors besides themiR156-regulated SPLs, which may to some extent ex-plain the observation that GR24 is still effective inmiR156-OE transgenic rice lines in suppressing ricetillering (Luo et al., 2012). The findings in rice and breadwheat together suggest that SL might act through theSPL-dependent pathway via direct repression of theSPLs activities by D53 and the SPL-independent path-way via other types of transcription factors.Based on our findings in this study, we propose a

working model on the miR156-TaSPL regulatorymodule and TaD53 in controlling bread wheat plantarchitecture (Fig. 10). In this model, the miR156-controlled TaSPLs, such as TaSPL3 and TaSPL17, actas transcriptional activators to enhance the expressionof some critical downstream genes including TaTB1and TaBA1, which might potentially contribute to thebread wheat plant architecture with limited tillers andnormal spike morphology; meanwhile, several types ofrepressors are involved in this signaling pathway, liketae-miR156 that may down-regulate TaSPL mRNAs atthe posttranscriptional level, and the TaD53-TaTPLmodule that may directly interact with and suppressthe transcriptional activation activities of TaSPLs.However, besides the tae-miR156/TaD53-controlledTaSPL signaling pathway, other TaSPL-independentpathways might also exist, in which the SL-controlledTaD53 might functionally antagonize the activities ofsome other unidentified transcription factors to regulate

bread wheat tillering (Fig. 10). Finally, we might be in-clined to assume that instead of complete antagonism,the complex mutual regulation is more likely the themeamong these positive and negative regulators in vivo,aiming to fine tune the establishment of appropriatebread wheat plant architecture.

MATERIALS AND METHODS

Plant Materials and Growth Conditions

For gene transformation, bread wheat (Triticum aestivum) cultivar wild-typeKenong199 was selected as the receptor plant. The indicated tae-miR156 genewas ligated to the BamHI/KpnI double-digested pUbi:cas vector and transformedinto the 1-month-old embryogenic calli of KN199 using a PDS1000/He particlebombardment system (Bio-Rad) with a target distance of 6.0 cm from the stoppingplate at helium pressure 1100 p.s.i., as described previously (Shan et al., 2013).

The wild-type KN199 as well as tae-miR156-OE transgenic lines (T2 gen-eration) were planted in an experimental field (39°96’N, 116°33’E) of the Insti-tute of Crop Science, the Chinese Academy of Agricultural Sciences, Beijing.Briefly, the seeds were sown at the beginning of October before the winter andharvested in mid-June next year. Nicotiana benthamiana was grown in a green-house at 22°C with a 16-h-light/8-h-dark cycle.

RNA Extraction and Gene Expression Analyses

For gene expression assays, leaves, stem bases, or young panicles werecollected form wild-type KN199 or tae-miR156-OE lines, and total RNA wasextracted using Trizol (Invitrogen) reagent. For detection of miRNAs by stem-loop qRT-PCR, specific reverse transcription primer for mature miRNAs withstem-loop structure was designed as previously described (Chen et al., 2005).About 2 mg of total RNA was used for reverse transcription. For quantificationof coding genes, about 2 mg of total RNA and M-MLV reverse transcriptase(Promega) were further used for reverse transcription. SYBR Premix Ex Taq(Perfect Real Time; TaKaRa) was used for qRT-PCR assay, and expression levelsof target genes were normalized to TaU6 or TaGAPDH. The statistical signifi-cance was evaluated by using data from three independent biological replica-tions (Student’s t test).

The miRNA Gene Identification, Cloning, and SecondaryStructure Prediction

The scaffold14333 from wheat A-genome progenitor Triticum urartu wasused as the query sequence to search the wheat survey sequences using BLAST(https://urgi.versailles.inra.fr/blast/), and three scaffolds, i.e. scaffold211901,225387, and 272705 from chromosomes 3A, 3B, and 3D, were obtained based onwheat TGAC (The Genome Analysis Centre) whole-genome shotgun assemblydatabase. The cloning primers then were designed according to the flankingsequences of the tandem hairpin structures, and PCR were performed by usingwild-type KN199 genomeDNA as the template. The obtained PCR product wasligated to cloning vector pEASY-Blunt (Transgen Biotech; CB101). The se-quences containing the tandem miR156 derived from chromosomes 3A, 3B,and 3D were identified by sequencing. The primer sequences are shown inSupplemental Table S2.

The secondary structures of the tandem miR156 gene containing threeMIR156 precursors (MIR156a/b/c) from chromosomes 3A, 3B and 3D wereseparately predicted using the RNA-folding program Mfold (Zuker, 2003).

Validation of miRNA Cleavage Site by 59RACE Assay

The RLM-RACE kit (TaKaRa; code D315) was used for 59RACE assayaccording to the manufacturer’s instruction. About 2 mg of wild-type KN199total RNAswere used for the ligation ofRNAOligo adaptorwithout calf intestinalphosphatase treatment. For the first round PCR, the 59RACEouter primer togetherwith gene-specific outer primers were used; a nested PCR amplification was thencarried out using the 59RACE inner primer together with gene-specific innerprimers. The obtained PCR products were then were ligated to cloning vector forsequencing. The primer sequences are shown in Supplemental Table S2.









Gene and Promoter Cloning and Sequence Analyses

For coding gene cloning, a BLASTn search against the database of wheatsurvey sequences (https://urgi.versailles.inra.fr/blast/) was initially conductedby using the coding sequence of orthologs from other plant species as the query.According to the obtained sequences from the hexaploid bread wheat, specificprimers were designed for the cloning inwild-type KN199 cDNAs. The obtainedcloning products were further verified by sequencing. Encoded protein se-quences were aligned with orthologous proteins from other plant species byMegAlign using the ClustalW method, and the identities among the protein se-quences were calculated.

For promoter cloning, the corresponding scaffolds from the hexaploid breadwheat containing the target geneswerefirst identified by screening the databaseofwheat survey sequences. Specific cloning primerswere designed based on the2-kb sequences upstream of the genes, and the promoters were cloned in KN199by using its genome DNA as the template. Sequences were verified by se-quencing. The primer sequences are shown in Supplemental Table S2.

Generation of DNA Constructs

The constructs used in this study are based on several expression vectors.The constructs used for LCI assays were based on the vectors p1300-35S-

nLUC and p1300-35S-cLUC (Chen et al., 2008). Briefly, the target genes werePCR amplified and separately ligated into the KpnI/SalI-digested p1300-35S-nLUC and KpnI/BamHI digested p1300-35S-cLUC using ligation free cloningmastermix (Applied Biological Materials; E011-5-A) according to the manu-facturer’s instruction.

The vectors for BiFC assays were generated based on the Gateway vectorspEarleygate201-YN (nYFP) and pEarleygate202-YC (cYFP) (Lu et al., 2010). Theconstructs for transcriptional activation assays were based on the Gatewayvector pGWBs (Nakagawa et al., 2007). All the gene sequences were cloned intothe entry vector pQBV3 and subsequently introduced into the destinationvectors pEarleygate201-YN (nYFP), pEarleygate202-YC (cYFP), or pGWBsfollowing Gateway technology (Invitrogen).

All the primers used for generation of constructs are shown in SupplementalTable S2.

Transcriptional Activity Assays in N. benthamiana

The transcriptional activity assays were performed in N. benthamiana leavesas previously described (Sun et al., 2012). The 2-kb promoter sequences werefused with the luciferase reporter gene LUC through Gateway reactions(Invitrogen) into the plant binary vector pGWB35 (Nakagawa et al., 2007) togenerate the reporter constructs. For effector construction, the coding se-quences of indicated genes were cloned into the plant binary vector pGWB5(Nakagawa et al., 2007) using pQBV3 as the entry. The reporter and effectorconstructs were separately introduced into Agrobacterium tumefaciens strainGV3101 (pMP90), to carry out the coinfiltration in N. benthamiana leaves. LUCactivities were observed and quantified 48 h after infiltration using Night-SHADE LB 985 (Berthold). In each experiment, 10 independentN. benthamianaleaves were infiltrated and analyzed, and three biological replications wereperformed with quantification.

LCI Assays

The LCI assays for the protein interaction detection was performed in N.benthamiana leaves as described previously (Sun et al., 2013). In brief, the in-dicated genes were separately fused with the N- and C-terminal parts of theluciferase reporter gene LUC and separately introduced into A. tumefaciensstrain GV3101. A. tumefaciens cells harboring the nLUC and cLUC derivativeconstructs were coinfiltrated into N. benthamiana leaves, and the LUC activitieswere analyzed 48 h after infiltration using NightSHADE LB 985 (Berthold). Ineach analysis, five independent N. benthamiana leaves were infiltrated and an-alyzed, and three biological replications were performed with similar results.

BiFC Assays

For infiltration, A. tumefaciens cells harboring the nYFP and cYFP derivativeconstructs were coinfiltrated into N. benthamiana leaves, and YFP signal wasimaged 48 h after infiltration using the confocal microscope (Carl Zeiss;

LSM880). Five independent N. benthamiana leaves were observed for analyses,and totally three biological replications were performed with similar results.

Yeast Experiments

For transcriptional activation activity assays in yeast (Saccharomyces cer-evisiae) cells, AH109 strain was used. In brief, the full-length TaSPL3 as well asits truncated versions (including the NT, MD, and CT parts) were fused withGAL4-BD vector pGBKT7 and separately transformed into yeast AH109 strain.The transformants were first selected on synthetic dextrose growth mediumlacking Trp (SD-W). Then the yeast strains were dropped on synthetic dextroseselection medium lacking Trp and His (SD-W/H) for transcriptional activationactivity evaluation according to their growth status.

Accession Numbers

Sequence data from this study can be found in the NCBI data library underthe following accession numbers: TaSPL3, KF447885; TaSPL17, KF447877;TaSPL20, KF447878; TaSPL21, KF447882; TaTB1-4Apro, KY363317; TaTB1-4Bpro,KY363318; TaTB1-4Dpro, KY363319; TaBA1-3Apro, KY363320; TaBA1-3Bpro,KY363321; TaBA1-3Dpro, KY363322; TaD53, KY363316; and TaTPL, KY363324.

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Nucleotide sequence of partial scaffold14333from Triticum urartu.

Supplemental Figure S2. Predicted secondary structure of tandem tae-MIR156a/b/c precursors from chromosomes 3B and 3D.

Supplemental Figure S3. Nucleotide sequence of tae-MIR156-3A clusterused for gene transformation in bread wheat plants.

Supplemental Figure S4. Morphological characterization of wild-typeKN199 and tae-miR156-OE transgenic bread wheat plants (3#).

Supplemental Figure S5. Quantification of the expression levels ofTaSPL20 and TaSPL21 in tae-miR156-OE bread wheat lines.

Supplemental Figure S6. Expression of tae-miR156, TaSPL3 and TaSPL17in wild-type KN199 in different development phases/tissues.

Supplemental Figure S7. The coding sequences of TaTB1 genes.

Supplemental Figure S8. Sequence comparison of TaTB1-4A/4B/4D,ZmTB1, and OsTB1 proteins.

Supplemental Figure S9. Nucleotide sequences of TaTB1-4A/4B/4Dpromoters.

Supplemental Figure S10. Transient expression assays illustrating the ac-tivation of TaTB1 transcription by TaSPL17.

Supplemental Figure S11. Quantification of the expression levels of someinflorescence/panicle/spike development relevant bread wheat genes intae-miR156-OE transgenic lines.

Supplemental Figure S12. The coding sequences of TaBA1 genes.

Supplemental Figure S13. Nucleotide sequences of TaBA1-3A/3B/3Dpromoters.

Supplemental Figure S14. Transient expression assays illustrating the ac-tivation of TaBA1 expression by TaSPL17.

Supplemental Figure S15. The coding sequences of TaD53 genes.

Supplemental Figure S16. Sequence alignment of TaD53, OsD53, andAtSMXL7 proteins.

Supplemental Figure S17. The N-terminal domain of TaSPL17 physicallyinteracts with TaD53.

Supplemental Figure S18. The coding sequence of TaTPL gene.

Supplemental Figure S19. Alignment of TaTPL with TPL-related proteinsin rice and Arabidopsis.

Supplemental Table S1. Predicted target genes of tae-miR156.

Supplemental Table S2. Primers used in this study.


Liu et al.




























ACKNOWLEDGMENTS

We thank the Biotechnology Facility of the Institute of Genetics and Devel-opmental Biology, Chinese Academy of Sciences for assistance in bread wheatgene transformation.

Received March 31, 2017; accepted May 16, 2017; published May 19, 2017.

LITERATURE CITED

Aguilar-Martínez JA, Poza-Carrión C, Cubas P (2007) ArabidopsisBRANCHED1 acts as an integrator of branching signals within axillarybuds. Plant Cell 19: 458–472

Arite T, Iwata H, Ohshima K, Maekawa M, Nakajima M, Kojima M,Sakakibara H, Kyozuka J (2007) DWARF10, an RMS1/MAX4/DAD1ortholog, controls lateral bud outgrowth in rice. Plant J 51: 1019–1029

Arite T, Umehara M, Ishikawa S, Hanada A, Maekawa M, Yamaguchi S,Kyozuka J (2009) d14, a strigolactone-insensitive mutant of rice, showsan accelerated outgrowth of tillers. Plant Cell Physiol 50: 1416–1424

Bortiri E, Chuck G, Vollbrecht E, Rocheford T, Martienssen R, Hake S(2006) ramosa2 encodes a LATERAL ORGAN BOUNDARY domainprotein that determines the fate of stem cells in branch meristems ofmaize. Plant Cell 18: 574–585

Cardon G, Höhmann S, Klein J, Nettesheim K, Saedler H, Huijser P(1999) Molecular characterisation of the Arabidopsis SBP-box genes.Gene 237: 91–104

Chen C, Ridzon DA, Broomer AJ, Zhou Z, Lee DH, Nguyen JT, Barbisin M,Xu NL, Mahuvakar VR, Andersen MR, et al (2005) Real-time quantificationof microRNAs by stem-loop RT-PCR. Nucleic Acids Res 33: e179

Chen H, Zou Y, Shang Y, Lin H, Wang Y, Cai R, Tang X, Zhou JM (2008)Firefly luciferase complementation imaging assay for protein-proteininteractions in plants. Plant Physiol 146: 368–376

Chuck G, Cigan AM, Saeteurn K, Hake S (2007) The heterochronic maizemutant Corngrass1 results from overexpression of a tandem microRNA.Nat Genet 39: 544–549

Chuck G, Muszynski M, Kellogg E, Hake S, Schmidt RJ (2002) The controlof spikelet meristem identity by the branched silkless1 gene in maize.Science 298: 1238–1241

Dai X, Zhao PX (2011) psRNATarget: a plant small RNA target analysisserver. Nucleic Acids Res 39: W155–W159

Deng W, Nickle DC, Learn GH, Maust B, Mullins JI (2007) ViroBLAST: astand-alone BLAST web server for flexible queries of multiple databasesand user’s datasets. Bioinformatics 23: 2334–2336

Derbyshire P, Byrne ME (2013) MORE SPIKELETS1 is required for spikeletfate in the inflorescence of Brachypodium. Plant Physiol 161: 1291–1302

Dobrovolskaya O, Pont C, Sibout R, Martinek P, Badaeva E, Murat F,Chosson A, Watanabe N, Prat E, Gautier N, et al (2015) FRIZZYPANICLE drives supernumerary spikelets in bread wheat. Plant Physiol167: 189–199

Doebley J, Stec A, Hubbard L (1997) The evolution of apical dominance inmaize. Nature 386: 485–488

Gallavotti A, Long JA, Stanfield S, Yang X, Jackson D, Vollbrecht E,Schmidt RJ (2010) The control of axillary meristem fate in the maizeramosa pathway. Development 137: 2849–2856

Gallavotti A, Zhao Q, Kyozuka J, Meeley RB, Ritter MK, Doebley JF, PèME, Schmidt RJ (2004) The role of barren stalk1 in the architecture ofmaize. Nature 432: 630–635

Gomez-Roldan V, Fermas S, Brewer PB, Puech-Pagès V, Dun EA, PillotJP, Letisse F, Matusova R, Danoun S, Portais JC, et al (2008) Strigo-lactone inhibition of shoot branching. Nature 455: 189–194

Gou JY, Felippes FF, Liu CJ, Weigel D, Wang JW (2011) Negative regu-lation of anthocyanin biosynthesis in Arabidopsis by a miR156-targetedSPL transcription factor. Plant Cell 23: 1512–1522

Guedira M, Xiong M, Hao YF, Johnson J, Harrison S, Marshall D, Brown-Guedira G (2016) Heading date QTL in winter wheat (Triticum aestivumL.) coincide with major developmental genes VERNALIZATION1 andPHOTOPERIOD1. PLoS One 11: e0154242

Höck J, Meister G (2008) The Argonaute protein family. Genome Biol 9: 210Honma T, Goto K (2001) Complexes of MADS-box proteins are sufficient to

convert leaves into floral organs. Nature 409: 525–529International Wheat Genome Sequencing Consortium (IWGSC) (2014) A

chromosome-based draft sequence of the hexaploid bread wheat (Triti-cum aestivum) genome. Science 345: 1251788

Ishikawa S, Maekawa M, Arite T, Onishi K, Takamure I, Kyozuka J(2005) Suppression of tiller bud activity in tillering dwarf mutants ofrice. Plant Cell Physiol 46: 79–86

Jiang L, Liu X, Xiong G, Liu H, Chen F, Wang L, Meng X, Liu G, Yu H,Yuan Y, et al (2013) DWARF 53 acts as a repressor of strigolactonesignalling in rice. Nature 504: 401–405

Jiao Y, Wang Y, Xue D, Wang J, Yan M, Liu G, Dong G, Zeng D, Lu Z, ZhuX, Qian Q, Li J (2010) Regulation of OsSPL14 by OsmiR156 defines idealplant architecture in rice. Nat Genet 42: 541–544

Jones-Rhoades MW, Bartel DP (2004) Computational identification ofplant microRNAs and their targets, including a stress-induced miRNA.Mol Cell 14: 787–799

Lin H, Wang R, Qian Q, Yan M, Meng X, Fu Z, Yan C, Jiang B, Su Z, Li J,Wang Y (2009) DWARF27, an iron-containing protein required for thebiosynthesis of strigolactones, regulates rice tiller bud outgrowth. PlantCell 21: 1512–1525

Lin X, Wu F, Du X, Shi X, Liu Y, Liu S, Hu Y, Theißen G, Meng Z (2014)The pleiotropic SEPALLATA-like gene OsMADS34 reveals that the‘empty glumes’ of rice (Oryza sativa) spikelets are in fact rudimentarylemmas. New Phytol 202: 689–702

Ling HQ, Zhao S, Liu D, Wang J, Sun H, Zhang C, Fan H, Li D, Dong L,Tao Y, et al (2013) Draft genome of the wheat A-genome progenitorTriticum urartu. Nature 496: 87–90

Liu C, Teo ZW, Bi Y, Song S, Xi W, Yang X, Yin Z, Yu H (2013) A con-served genetic pathway determines inflorescence architecture in Arabi-dopsis and rice. Dev Cell 24: 612–622

Liu J, Cheng X, Liu D, Xu W, Wise R, Shen QH (2014) The miR9863 familyregulates distinct Mla alleles in barley to attenuate NLR receptor-triggereddisease resistance and cell-death signaling. PLoS Genet 10: e1004755

Lu Q, Tang X, Tian G, Wang F, Liu K, Nguyen V, Kohalmi SE, Keller WA,Tsang EW, Harada JJ, Rothstein SJ, Cui Y (2010) Arabidopsis homologof the yeast TREX-2 mRNA export complex: components and anchoringnucleoporin. Plant J 61: 259–270

Lu Z, Yu H, Xiong G, Wang J, Jiao Y, Liu G, Jing Y, Meng X, Hu X, QianQ, et al (2013) Genome-wide binding analysis of the transcription acti-vator ideal plant architecture1 reveals a complex network regulating riceplant architecture. Plant Cell 25: 3743–3759

Luo L, Li W, Miura K, Ashikari M, Kyozuka J (2012) Control of tillergrowth of rice by OsSPL14 and Strigolactones, which work in two in-dependent pathways. Plant Cell Physiol 53: 1793–1801

Miura K, Ikeda M, Matsubara A, Song XJ, Ito M, Asano K, Matsuoka M,Kitano H, Ashikari M (2010) OsSPL14 promotes panicle branching andhigher grain productivity in rice. Nat Genet 42: 545–549

Nakagawa T, Kurose T, Hino T, Tanaka K, Kawamukai M, Niwa Y,Toyooka K, Matsuoka K, Jinbo T, Kimura T (2007) Development ofseries of gateway binary vectors, pGWBs, for realizing efficient con-struction of fusion genes for plant transformation. J Biosci Bioeng 104:34–41

Nakamura H, Xue YL, Miyakawa T, Hou F, Qin HM, Fukui K, Shi X, ItoE, Ito S, Park SH, et al (2013) Molecular mechanism of strigolactoneperception by DWARF14. Nat Commun 4: 2613

Paolacci AR, Tanzarella OA, Porceddu E, Varotto S, Ciaffi M (2007) Mo-lecular and phylogenetic analysis of MADS-box genes of MIKC type andchromosome location of SEP-like genes in wheat (Triticum aestivum L.).Mol Genet Genomics 278: 689–708

Pelaz S, Ditta GS, Baumann E, Wisman E, Yanofsky MF (2000) B and Cfloral organ identity functions require SEPALLATA MADS-box genes.Nature 405: 200–203

Peng J, Richards DE, Hartley NM, Murphy GP, Devos KM, Flintham JE,Beales J, Fish LJ, Worland AJ, Pelica F, et al (1999) ‘Green revolution’genes encode mutant gibberellin response modulators. Nature 400: 256–261

Peters L, Meister G (2007) Argonaute proteins: mediators of RNA silenc-ing. Mol Cell 26: 611–623

Ren D, Li Y, Zhao F, Sang X, Shi J, Wang N, Guo S, Ling Y, Zhang C, YangZ, He G (2013) MULTI-FLORET SPIKELET1, which encodes an AP2/ERF protein, determines spikelet meristem fate and sterile lemmaidentity in rice. Plant Physiol 162: 872–884

Ritter MK, Padilla CM, Schmidt RJ (2002) The maize mutant barren stalk1is defective in axillary meristem development. Am J Bot 89: 203–210

Schwab R, Palatnik JF, Riester M, Schommer C, Schmid M, Weigel D(2005) Specific effects of microRNAs on the plant transcriptome. DevCell 8: 517–527





Schwarz S, Grande AV, Bujdoso N, Saedler H, Huijser P (2008) ThemicroRNA regulated SBP-box genes SPL9 and SPL15 control shootmaturation in Arabidopsis. Plant Mol Biol 67: 183–195

Shan Q, Wang Y, Li J, Zhang Y, Chen K, Liang Z, Zhang K, Liu J, Xi JJ,Qiu JL, Gao C (2013) Targeted genome modification of crop plants usinga CRISPR-Cas system. Nat Biotechnol 31: 686–688

Shitsukawa N, Takagishi A, Ikari C, Takumi S, Murai K (2006) WFL, awheat FLORICAULA/LEAFY ortholog, is associated with spikelet for-mation as lateral branch of the inflorescence meristem. Genes Genet Syst81: 13–20

Si L, Chen J, Huang X, Gong H, Luo J, Hou Q, Zhou T, Lu T, Zhu J,Shangguan Y, et al (2016) OsSPL13 controls grain size in cultivated rice.Nat Genet 48: 447–456

Soundappan I, Bennett T, Morffy N, Liang Y, Stanga JP, Abbas A, LeyserO, Nelson DC (2015) SMAX1-LIKE/D53 family members enable distinctMAX2-dependent responses to strigolactones and karrikins in Arabi-dopsis. Plant Cell 27: 3143–3159

Sun J, Qi L, Li Y, Chu J, Li C (2012) PIF4-mediated activation of YUCCA8expression integrates temperature into the auxin pathway in regulatingarabidopsis hypocotyl growth. PLoS Genet 8: e1002594

Sun J, Qi L, Li Y, Zhai Q, Li C (2013) PIF4 and PIF5 transcription factorslink blue light and auxin to regulate the phototropic response in Ara-bidopsis. Plant Cell 25: 2102–2114

Takeda T, Suwa Y, Suzuki M, Kitano H, Ueguchi-Tanaka M, Ashikari M,Matsuoka M, Ueguchi C (2003) The OsTB1 gene negatively regulateslateral branching in rice. Plant J 33: 513–520

Umehara M, Hanada A, Yoshida S, Akiyama K, Arite T, Takeda-KamiyaN, Magome H, Kamiya Y, Shirasu K, Yoneyama K, Kyozuka J,Yamaguchi S (2008) Inhibition of shoot branching by new terpenoidplant hormones. Nature 455: 195–200

Wang J, Yu H, Xiong G, Lu Z, Jiao Y, Meng X, Liu G, Chen X, Wang Y, Li J(2017) Tissue-specific ubiquitination by IPA1 INTERACTING PROTEIN1 modulates IPA1 protein levels to regulate plant architecture in rice.Plant Cell 29: 697–707

Wang JW, Czech B, Weigel D (2009) miR156-regulated SPL transcriptionfactors define an endogenous flowering pathway in Arabidopsis thaliana.Cell 138: 738–749

Wang JW, Schwab R, Czech B, Mica E, Weigel D (2008) Dual effects ofmiR156-targeted SPL genes and CYP78A5/KLUH on plastochron lengthand organ size in Arabidopsis thaliana. Plant Cell 20: 1231–1243

Wang L, Wang B, Jiang L, Liu X, Li X, Lu Z, Meng X, Wang Y, Smith SM,Li J (2015) Strigolactone signaling in Arabidopsis regulates shoot de-velopment by targeting D53-Like SMXL repressor proteins for ubiq-uitination and degradation. Plant Cell 27: 3128–3142

Wang RL, Stec A, Hey J, Lukens L, Doebley J (1999) The limits of selectionduring maize domestication. Nature 398: 236–239

Wang S, Wu K, Yuan Q, Liu X, Liu Z, Lin X, Zeng R, Zhu H, Dong G, QianQ, Zhang G, Fu X (2012) Control of grain size, shape and quality byOsSPL16 in rice. Nat Genet 44: 950–954

Wu G, Park MY, Conway SR, Wang JW, Weigel D, Poethig RS (2009) Thesequential action of miR156 and miR172 regulates developmental timingin Arabidopsis. Cell 138: 750–759

Wu G, Poethig RS (2006) Temporal regulation of shoot development inArabidopsis thaliana by miR156 and its target SPL3. Development 133:3539–3547

Xie K, Shen J, Hou X, Yao J, Li X, Xiao J, Xiong L (2012) Gradual increaseof miR156 regulates temporal expression changes of numerous genesduring leaf development in rice. Plant Physiol 158: 1382–1394

Xie K, Wu C, Xiong L (2006) Genomic organization, differential expression,and interaction of SQUAMOSA promoter-binding-like transcriptionfactors and microRNA156 in rice. Plant Physiol 142: 280–293

Yamaguchi A, Wu MF, Yang L, Wu G, Poethig RS, Wagner D (2009) ThemicroRNA-regulated SBP-Box transcription factor SPL3 is a direct up-stream activator of LEAFY, FRUITFULL, and APETALA1. Dev Cell 17:268–278

Yamasaki K, Kigawa T, Inoue M, Tateno M, Yamasaki T, Yabuki T, AokiM, Seki E, Matsuda T, Nunokawa E, et al (2004) A novel zinc-bindingmotif revealed by solution structures of DNA-binding domains ofArabidopsis SBP-family transcription factors. J Mol Biol 337: 49–63

Yao R, Ming Z, Yan L, Li S, Wang F, Ma S, Yu C, Yang M, Chen L, Chen L,et al (2016) DWARF14 is a non-canonical hormone receptor for strigo-lactone. Nature 536: 469–473

Yu N, Cai WJ, Wang S, Shan CM, Wang LJ, Chen XY (2010) Temporalcontrol of trichome distribution by microRNA156-targeted SPL genes inArabidopsis thaliana. Plant Cell 22: 2322–2335

Zhang B, Liu X, Zhao G, Mao X, Li A, Jing R (2014) Molecular charac-terization and expression analysis of Triticum aestivum squamosa-promoter binding protein-box genes involved in ear development. JIntegr Plant Biol 56: 571–581

Zhou F, Lin Q, Zhu L, Ren Y, Zhou K, Shabek N, Wu F, Mao H, Dong W,Gan L, et al (2013) D14-SCF(D3)-dependent degradation of D53 regu-lates strigolactone signalling. Nature 504: 406–410

Zou J, Zhang S, Zhang W, Li G, Chen Z, Zhai W, Zhao X, Pan X, Xie Q,Zhu L (2006) The rice HIGH-TILLERING DWARF1 encoding an or-tholog of Arabidopsis MAX3 is required for negative regulation of theoutgrowth of axillary buds. Plant J 48: 687–698

Zuker M (2003) Mfold web server for nucleic acid folding and hybridiza-tion prediction. Nucleic Acids Res 31: 3406–3415


Liu et al.



mir156-targeted sbp-box transcription factors interact ... · mir156-targeted sbp-box transcription...

Documents