An Essential Role for miRNA167 in Maternal Control ofEmbryonic and Seed Development1[OPEN]

Xiaozhen Yao,a,2 Jilin Chen,b,2 Jie Zhou,a,2 Hanchuanzhi Yu,b Chennan Ge,b Min Zhang,a Xiuhua Gao,b

Xinhua Dai,b Zhong-Nan Yang,a and Yunde Zhaob,3,4

aShanghai Key Laboratory of Plant Molecular Sciences, College of Life Sciences, Shanghai Normal University,Shanghai 200234, ChinabSection of Cell and Developmental Biology, University of California San Diego, La Jolla, California 92093-0116

ORCID IDs: 0000-0002-8361-8345 (X.Y.); 0000-0003-0037-5112 (J.Z.); 0000-0001-8670-5324 (H.Y.); 0000-0002-9335-0186 (C.G.);0000-0002-5918-2385 (Z.-N.Y.); 0000-0002-7224-8449 (Y.Z.).

Maternal cells play a critical role in ensuring the normal development of embryos, endosperms, and seeds. Mutations thatdisrupt the maternal control of embryogenesis and seed development are difficult to identify. Here, we completely deleted fourMICRORNA167 (MIR167) genes in Arabidopsis (Arabidopsis thaliana) using a clustered regularly interspaced short palindromicrepeats (CRISPR)/CRISPR-associated protein9 (Cas9) genome-editing technology. We found that plants with a deletion ofMIR167A phenocopied plants overexpressing miRNA167-resistant versions of Auxin Response Factor6 (ARF6) or ARF8, twomiRNA167 targets. Both the mir167a mutant and the ARF overexpression lines were defective in anther dehiscence and ovuledevelopment. Serendipitously, we found that the mir167a (♀) 3 wild type (♂) crosses failed to produce normal embryos andendosperms, despite the findings that embryos with either mir167a+/2 or mir167a2/2 genotypes developed normally whenmir167a+/2 plants were self-pollinated, revealing a central role of MIR167A in maternal control of seed development. Themir167a phenotype is 100% penetrant, providing a great genetic tool for studying the roles of miRNAs and auxin in maternalcontrol. Moreover, we found that mir167a mutants flowered significantly later than wild-type plants, a phenotype that was notobserved in the ARF overexpression lines. We show that the reproductive defects of mir167a mutants were suppressed by adecrease of activities of ARF6, ARF8, or both. Our results clearly demonstrate that MIR167A is the predominantMIR167memberin regulating Arabidopsis reproduction and that MIR167A acts as a maternal gene that functions largely through ARF6and ARF8.

The formation of a normal Arabidopsis (Arabidopsisthaliana) seed requires the coordinated development ofthree genetically distinct components: seed coat, em-bryo, and endosperm (Ingram, 2010). The seed coatdevelops completely from maternal cells, whereas two-thirds of the genetic material in the endosperm is ma-ternal. Genetic screens for mutants defective in one of

the three components have identified many genes thataffect embryogenesis, endosperm development, andthe formation and integrity of the seed coat (Lafon-Placette and Köhler, 2014; Li and Li, 2015). Very fewgenes that exert maternal control of embryogenesis andseed development have been identified (Ray et al., 1996;Grossniklaus et al., 1998; Mizzotti et al., 2012; Zhanget al., 2017). The first reported maternal effect gene,SHORT INTEGUMENT, encodes DICER, which is anessential player in processing microRNA (miRNA)precursors and other small RNAs (Ray et al., 1996;Golden et al., 2002). A homozygous sin1 mutant em-bryo develops normally if it is generated from a self-pollinating sin1 heterozygous plant. About 90% ofembryos with sin1 or sin1+/2 genotypes are defective inembryogenesis when produced by pollination with asin1 homozygous plant with pollen from wild-type orsin1 heterozygous plants (Ray et al., 1996). The sin1studies suggest that small RNAs may play importantroles in maternal control of embryogenesis and seeddevelopment. Another example of maternal effects wasuncovered when either Mitogen activated Protein Ki-nase 6 or its upstream kinases MPK Kinase 4 (MKK4)/MKK5 are disrupted (Zhang et al., 2017). A significantfraction (6%–35%) of mpk6 or mkk4 mkk5 double mu-tants had embryos that burst out of the seed coats orhad wrinkled seeds. Overall, it is still very difficult to

1This work was supported by the National Institute of GeneralMedical Sciences (R01GM114660 to Y.Z.), the National Natural Sci-ence Foundation of China (31401030 to X.Y.), and the Science andTechnology Commission of Shanghai Municipality (18DZ2260500).

2These authors contributed equally to the article.3Author for contact: yundezhao@ucsd.edu.4Senior author.The author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Yunde Zhao (yundezhao@ucsd.edu).

Y.Z. conceived the original research plans; X.Y. and Y.Z. super-vised the experiments and analyzed the data; J.C. and J.Z. performedmost of the experiments; X.Y., H.Y., C.G., X.G., M.Z., and X.D. pro-vided some experimental materials and conducted some experi-ments. Z.-N.Y. provided assistance to X.Y. and J.Z. and supervisedsome of the experiments; X.Y. and Y.Z. wrote the article with contri-butions of all the authors.

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study maternal effects because of a lack of proper ge-netic materials.

MiRNAs are a class of ;21-nucleotide small RNAsthat regulate diverse developmental processes in bothplants and animals (Lee et al., 1993; Wightman et al.,1993; Bartel, 2004). It is generally believed that miRNAsdown-regulate the expression of target genes via cleav-age of target mRNA or inhibition of target mRNAtranslation (Bartel, 2009; Shukla et al., 2011). The biolog-ical functions of miRNAs were mainly inferred fromoverexpressing MICRORNA (MIR) genes or miRNA-resistant versions of the target genes. Whereas resultsfrom such experiments are informative, variations inphenotypes and stability are often encountered due todifferences in expression levels. Moreover, a miRNA of-ten hasmultiple targets, and overexpression of amiRNA-resistant version of a target may not lead to a completepicture of the miRNA functions. To unambiguously de-fine the physiological roles of miRNAs, characterizationof complete loss-of-function mirmutants is needed. MostMIR genes belong to gene families whose members pre-sumably have redundant functions, making it difficult toobtain plants that lack a particular miRNA. Furthermore,miRNAs are produced from small genes, and the chancesfor isolating T-DNA or transposon insertional mutants ofMIR genes are limited. In addition, miRNAs function bybase pairing with their target mRNAs. A point mutationin miRNAs may not completely abolish their functions.Therefore, very few studies in plants lacking a particularmiRNAhave been reported (Baker et al., 2005; Allen et al.,2007; Sieber et al., 2007; Liu et al., 2010). With the ad-vancement of clustered regularly interspaced short pal-indromic repeats (CRISPR)/CRISPR-associated protein9(Cas9) gene-editing technology, it is now feasible to sys-tematically generate mir knockout mutants to study theirroles in regulating plant growth and development.

We are interested in understanding how auxincontrols various plant developmental processes.Auxin is perceived by the TRANSPORT INHIBITORRESPONSE1/AUXIN SIGNALING F-BOX PRO-TEIN (TIR1/AFB) and AUXIN/INDOLE-3-ACETICACID (Aux/IAA) complexes (Dharmasiri et al., 2005;Kepinski and Leyser, 2005). Degradation of Aux/IAArepressors frees Auxin Response Factors (ARFs) fortranscriptional activities. Interestingly, several keycomponents of auxin signaling pathways are targets ofmiRNAs. The miRNA393 targets the mRNAs encodingthe auxin receptors TIR1/AFBs (Jones-Rhoades andBartel, 2004; Vidal et al., 2010; Si-Ammour et al., 2011;Windels et al., 2014). The miRNA160 targets themRNAs for several ARFs, including ARF10, ARF16,and ARF17 (Mallory et al., 2005; Wang et al., 2005; Liuet al., 2007). ARF3 and ARF4 are targets of trans-actingshort-interfering RNAs, which require miRNA390 fortheir biogenesis (Fahlgren et al., 2006; Marin et al.,2010). The miRNA167 has been reported to directlyregulate auxin signaling and auxin homeostasis inArabidopsis. The miRNA167 targets the mRNAs en-coding the ARF6 and ARF8 transcription factors (Ruet al., 2006; Wu et al., 2006; Yang et al., 2006). The

miRNA167 also regulates the expression levels ofIAA-ALA RESISTANT3 (IAR3), which encodes an auxin-amidohydrolase and plays a role in auxin conjugation/deconjugationwith amino acids inArabidopsis (Kinoshitaet al., 2012). Regulation of auxin signaling by miRNAsis evolutionarily conserved across plant species. Forexample, the interaction betweenmiRNA167 andARF6/8is conserved among Arabidopsis, tomato (Solanumlycopersicum), and soybean (Glycine max; Yang et al.,2006; Gutierrez et al., 2012; Liu et al., 2014; Wang et al.,2015). An understanding of the molecular mechanismsof miRNAs in auxin signaling may provide guides forimproving agriculturally important traits.

The Arabidopsis genome harbors fourMIR167 genes(MIR167A, MIR167B, MIR167C, and MIR167D). Over-expression of MIR167A using the Cauliflower mosaic vi-rus 35S promoter mimicked the phenotypes of arf6 arf8double mutants (Wu et al., 2006). Interestingly, over-expression ofMIR167B andMIR167C only caused mildphenotypes, whereas 35S:MIR167D plants were verysimilar to wild-type plants (Wu et al., 2006). Over-expression of miRNA167-resistant versions of ARF6 orARF8 caused pleiotropic phenotypes, including smallleaves and sterile flowers, suggesting that MIR167s areimportant for Arabidopsis development (Wu et al.,2006). Herein, we report the construction of knockoutmutants of the four MIR167 genes in Arabidopsis. Wefound that mir167a plants were defective in anther de-hiscence, ovule development, and seed development,phenotypes that were observed in plants that over-express the miRNA167-resistant versions of ARF6 orARF8 (referred to as mARF6 or mARF8, respectively; Wuet al., 2006). However, unlike the mARF overexpressionlines, which had small leaves and normal flowering time,mir167a single mutants had normal leaf size but floweredmuch later than wild-type plants. More importantly, wefound that the mir167a (♀) 3wild type (♂) crosses failedto produce normal embryos and endosperms. It was veryclear that embryos with either mir167a+/2 or mir167a2/2

genotypes resulting from the selfing of mir167a+/2 plantsdeveloped normally, revealing a central role ofMIR167Ain maternal control of seed development. The mir167aphenotype is 100% penetrant, providing a valuablegenetic material for studying the roles of miRNAs andauxin in maternal control of embryo and seed develop-ment. Another unexpected finding was that mir167bcdtriple mutants were very similar to wild-type plants, in-dicating that MIR167A plays more predominant rolesduring plant reproductive development. MiRNA167 hasseveral targets, but we show that miRNA167 regulatesplant development mainly through controlling the ex-pression levels of ARF6 and ARF8.

RESULTS

Deletion of MIR167A Completely Abolishes Fertility

To assess the roles ofMIR167A in plant developmentand auxin signaling, we deleted the MIR167A gene

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using CRISPR/Cas9 gene-editing technology (Fig. 1A).We used two guide RNAs that targeted the promoterand the region downstream of the mature miRNA167A(Fig. 1A) in order to remove the entire MIR167A gene.Plants that harbored a deletion of a 1,170-bp fragment,including the region corresponding to the maturemiRNA167A, failed to produce transcripts of theMIR167Agene (Supplemental Fig. S1) and displayed a completelysterile phenotype (Fig. 1B).

Plants without MIR167A Produce Normal Pollen But Failto Dehisce

We analyzed the mir167a flowers to determine thecauses of the observed sterility. Flowers of mir167aplants contained all of their floral organs, and all of thefloral organs appeared normal except for the anthers(Fig. 1C). The anthers of the mir167amutants were muchlarger than those of the wild type (Fig. 1C). Moreover, themir167a anthers failed to dehisce (Fig. 1C) and no pollengrains were released (Fig. 1C; Supplemental Fig. S1C),which was probably the cause of the observed sterility ofmir167a plants. In contrast, anther dehiscence releasespollen to the pistils when wild-type flowers are open(Fig. 1C; Supplemental Fig. S1C).

We investigated whether mir167a plants were de-fective in male gametophytic development. Themir167a anthers produced normal-looking tricellularintegrated pollen grains (Supplemental Fig. S1). More-over, when we analyzed the morphology of mir167aanthers with a dissecting microscope, we discoveredthat the mir167a anthers dehisced 30 min after theywere detached from the flower (Fig. 1D). The pollengrains appeared normal. We conducted in vitro ger-mination assays to determine the viability of themir167a pollen. About 80% (n . 300) of wild-type pol-len grains germinated normally, whereas less than 40%(n. 300) of the mir167amutant pollen grains were ableto germinate (Fig. 1E). Our results indicated thatMIR167A is required for anther dehiscence and plays acritical role for pollen maturation.

MIR167A Affects Ovule Development

The seven-celled female gametophyte (embryo sac)consists of one egg cell, one central cell, two synergidcells, and three antipodal cells. During development,the three antipodal cells degenerate. Before pollination,the female gametophyte in wild-type plants consists ofone egg cell, one central cell, and two synergid cells

Figure 1. Plants without MIR167A are defec-tive in anther dehiscence, pollen germination,and female gametophyte development. A, Gen-eration of the deletion mutations of MIR167Ausing CRISPR/Cas9 gene-editing technology.Two Cas9 target sequences are shown, and theprotospacer adjacent motif sites are high-lighted in red. The green box represents theprimary sequence of MIR167A. The small redbox refers to the location of the matureMIR167A. The black line represents the pro-moter of the MIR167A gene. The locations ofthe genotyping primers are indicated. B, Thesterile phenotype of mir167a mutants. Wild-type (WT) Columbia-0 (Col-0; left) and themir167amutant (right) are shown at 43 d aftergermination. Bars = 5 cm. C, The floral organsof Col-0 and themir167amutant. Bars = 1mm.D, The anthers of Col-0 (left) and mir167a(middle [0 min] and right [30 min]) plantsdissected from opened flowers. Bars = 500 mm.E, In vitro germination of wild-type (left) andmir167a (right) pollen. Bars = 100 mm. F to H,Mature ovules from wild type Col-0 (F) andmir167a (G and H) plants. EN, Egg cell nu-cleus; SEN, secondary endosperm nucleus;SN, synergid nucleus. The yellow stars indi-cate the leakage of embryo sac. Bars = 50 mm.

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(Fig. 1F). However, only 27.2% (n = 33) of the mir167aembryo sacs contained the three cell types (Fig. 1G), andthe other embryo sacs lacked any nucleus and con-tained degenerated remains (n = 33; Fig. 1H). In wild-type ovules, the embryo sac was enclosed by two layersof integuments: the inner and outer integuments(Fig. 1F). In contrast, the integuments inmir167a ovulesfailed to completely enclose the embryo sac. Themir167a embryo sac appeared to extrude out from themicropylar end (Fig. 1, G and H).

The mir167a (♀) 3 Wild Type (♂) Crosses Fail to ProduceNormal Embryos

We pollinated mir167a pistils with wild-type pollento investigate the function of MIR167A in embryogen-esis and seed development. The wild-type pollen ger-minated on the stigmas of mir167a plants (SupplementalFig. S2). After manual pollination with wild-type pollen,the siliques of mir167a plants became elongated, as weexpected (Fig. 2A). However, the seeds that generatedfrom the pollination of mir167a pistils with wild-typepollen were small and shriveled (Fig. 2, A–C). Wild-type embryos developed into the globular stage at

2 or 4 d after pollination (Fig. 2D; Supplemental TableS1). Some embryos from the mir167a (♀) 3 wild type(♂) crosses developed normally, but a significantnumber of ovules did not contain embryos at all withinthe same time window (Fig. 2, E and F; SupplementalTable S1). By 5 to 6 d after pollination, wild-type em-bryos had already developed into the heart or torpedostage, whereas the observed embryos from the mir167a(♀)3wild type (♂) crosses were abnormal and arrestedat the globular stages (Fig. 2, G and H; SupplementalTable S1).

In the following days (7–16 d) after pollination, wild-type ovules developed into the cotyledon stage andthen developed into mature seeds, while the embryosfrom the mir167a (♀) 3 wild type (♂) crosses werearrested at different stages, such as the globular,heart-shaped, or cotyledon stage (Fig. 2, K and L;Supplemental Table S1). Few embryos from themir167a(♀) 3 wild type (♂) crosses reached the bent cotyledonstage, but such embryos were noticeably abnormal andwere phenotypically distinguishable from those of thewild type by their shorter and asymmetric cotyledons(Fig. 2L). Embryos from mir167a (♀) 3 wild type (♂)crosses had the mir167a+/2 genotype, yet such embryoswere not able to develop normally. The fact that both

Figure 2. Defects in seed development inmir167aplants after being pollinated with wild-type (WT)pollen. A, Pollination of mir167a flowers withwild-type pollen produced elongated siliques butonly produced shriveled seeds. White trianglesindicate the elongation of siliques resulting frompollination. mir167a (♀) 3 wild type (♂) crossesresulted in progeny with shriveled seeds in siliques(left), whereas wild-type seeds are full and smooth(right). Bars = 1 cm (left) and 1mm (right). B and C,Seeds produced from wild-type plants (B) andmir167a plants pollinated with wild-type pollen(C). Bars = 1 mm. D to M, Embryo development inwild-type plants (D, G, I, and K) and mir167aplants after being pollinated with wild-type pollen(E, F, H, J, L, and M). DAP, Days after pollination.Bars = 50 mm (D–J) and 100 mm (K–M).

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mir167a2/2 and mir167a+/2 embryos from selfingmir167a+/2 plants developed normally indicated thatmaternal tissues, such as the seed coat, also needMIR167A for normal functions.

MIR167A Acts as a Maternal Gene for Embryogenesis

Reciprocal crosses between wild-type plants andmir167a+/2 plants revealed that the mir167a alleletransmitted normally from male (102%) and female(79.4%) gametophytes (Table 1; Supplemental TableS2), suggesting that the pollen immaturity and theovule abortion phenotypes of mir167a plants weremainly due to sporophytic effects.We also analyzed theprogeny generated from selfing a mir167a+/2 plant. Thesegregation pattern (wild type:mir167a+/2:mir167a =36:64:31) also followed Mendelian genetics (Table 1).Selfingmir167a+/2 plants or reciprocal crosses betweenmir167a+/2 plants and wild-type plants produced nor-mal seeds (Supplemental Table S2), which could ger-minate and develop into normal adult plants. A seed ismade from the maternal sporophyte, zygote embryo,and endosperm. Endosperm is composed of two-thirdsmaternal genes and one-third paternal genes. The de-tailed genotypes of the three components of seedsproduced by each cross are shown in Table 1. Embryoscould develop normally as long as there was a func-tional MIR167A copy in the maternal sporophyte, re-gardless of the genotype of the endosperm and theembryo (crosses 1–8; Table 1). In cross 8, even themir167a2/2 embryo and mir167a2/2/- endosperm dis-played normal embryogenesis. However, the mir167a(♀) 3 wild type (♂) crosses only produced nonfunc-tional and shriveled seeds (Fig. 2). As shown in cross 9,mir167a+/2 heterozygous embryos developed abnor-mally when they were borne on a mir167a2/2 homo-zygous mother. The only difference between cross 4 (or7) and cross 9 was the genotype of the maternal spo-rophyte. These results unambiguously indicated thatMIR167A acts as a maternal gene in regulating embryodevelopment. The number of cell layers of the seed coatfrommir167a2/2 (♀)3wild type (♂) crosses is the same

as that of the wild type (Fig. 2, I and J). The most innerlayer of the seed coat from the seeds of mir167a (♀) 3wild type (♂) crosses seemed less vacuolated and hadless intense staining (Fig. 2, I and J). We noticed that themir167a integuments, which eventually develop intothe seed coat, were short and might also contribute tothe observed shriveled seed phenotype. Moreover, en-dosperm cellularization after double fertilization ofmir167a plants withwild-type pollen often failed (Fig. 2,I and J), suggesting that the developmental defects ofthe seeds frommir167a (♀)3wild type (♂) crosses maybe caused by a combination of defects in the seed coatand endosperm development.

Deletion of MIR167A Leads to Late Flowering

Flowering represents the transition from vegetativegrowth to reproductive growth, and flowering time isan important trait. We observed that mir167a plants(33 d after germination; n = 10) flowered significantlylater than wild-type plants (22 d after germination;n = 18). Under our growth conditions, wild-type plantsflowered when they had 12 rosette leaves. However,mir167a plants did not flower until they had at least 17rosette leaves (Fig. 3, A–C). To better understand themolecular basis of the late-flowering phenotype of themir167a mutants, we analyzed the expression levels ofseveral flowering time genes by reverse transcriptionquantitative PCR (RT-qPCR). Among these genes,FLOWERING LOCUS C (FLC), MADS AFFECTINGFLOWERING (MAF4), and MAF5, which encode floralrepressors, repress floral integrator genes such asFLOWERING LOCUS T (FT) and SUPPRESSOR OFOVEREXPRESSION OF CONSTANS (SOC1) to delayflowering. SQUAMOSA PROMOTER BINDING PRO-TEIN-LIKE3 (SPL3) and SPL5 not only act downstreamof FT but also function in parallel with FT to promoteflowering (Wang et al., 2009; Lal et al., 2011). MYB33activates components of the GA pathway to promoteflowering (Gocal et al., 2001; Achard et al., 2004).Consistent with the late-flowering phenotype observedin mir167a plants, the expression levels of FLC, MAF4,

Table 1. Maternal effects of MIR167A

For each cross group (except for mir167a2/2 3 Col-0), we genotyped all the germinated seedlings for each (zygote) genotype.

Cross Group ♀ 3 ♂ Cross Number Central Cell Egg Sperm Endosperm Zygote Maternal Sporophyte Seed Phenotype and Number

Col-0 3 mir167a+/2 1 +/+ + + +/+/+ +/+ +/+ Normal, n = 492 +/+ + 2 +/+/2 +/2 +/+ Normal, n = 50

mir167a+/2 3 Col-0 3 +/+ + + +/+/+ +/+ +/2 Normal, n = 344 2/2 2 + +/2/2 +/2 +/2 Normal, n = 27

mir167a+/2 selfinga 5 +/+ + + +/+/+ +/+ +/2 Normal, n = 366 +/+ + 2 +/+/2 +/2 +/2 Normal, n = 64b

7 2/2 2 + +/2/2 +/2 +/28 2/2 2 2 2/2/2 2/2 +/2 Normal, n = 31

mir167a2/2 3 Col-0 9 2/2 2 + +/2/2 +/2 2/2 Abnormal, n = 132

aThe x2 value is calculated based on the expected zygote segregation ratio: AA:Aa:aa = 1:2:1. x2 = S (observed value-expected value)2/expectedvalue. x2 = 0.45, P . 0.5. The segregation ratio is consistent with the typical Mendelian ratio (1:2:1). bIn the offspring of the mir167a+/2 selfcross, since cross 6 cannot be differentiated from cross 7 via genotyping, n shows the total number of seeds of cross 6 and cross 7.

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and MAF5 were increased in mir167a plants, whereasthe expression levels of FT, SOC1, SPL5, SPL3, andMYB33 were reduced in mir167a plants (Fig. 3D).

MIR167A Is Expressed during Flower Development

Results from reciprocal crosses between wild-typeplants and mir167a+/2 plants indicated that the floraldefects of mir167a plants were mainly due to sporo-phytic effects (Supplemental Table S2). We con-structed transgenic plants that express the greenfluorescent protein (GFP) marker under the control of

the MIR167A promoter. Consistent with our geneticresults, we detected abundant expression of GFP inthe sporophytic cells of both the anther and the ovule(Fig. 3, E–J). The GFP signal was observed in severallayers of anther wall cells. This observation wasconsistent with the dehiscence defects of mir167aanthers. We also detected GFP signals in filamentsduring pollen development (Fig. 3, E and G). GFPwasvisible in ovules, but the signal was particularlystrong in the epidermis of ovules (Fig. 3, H–J). Theseexpression patterns correlated well with the observeddefects in the elongation of ovule integuments inmir167a plants.

Figure 3. The late-flowering phenotype ofmir167a plants and the expression pattern ofMIR167A. A and B, The number of rosette leavesthat wild-type (WT; A) and mir167a (B) plantshad when they started to flower. C, Quantifica-tion of the number of rosette leaves needed be-fore plants started to flower. The values of thewild type andmir167a represent means togetherwith SD (n $ 20). The asterisks indicate a statis-tical difference between wild-type andmir167aplants (**, P , 0.01 by two-sided Student’st test). D, Relative gene expression levels deter-mined by RT-qPCR analysis. RNAwas extractedfrom 20-d-old leaves. RT-qPCR results werenormalized to TUBULIN BETA CHAIN2. Rela-tive expression levels are shown as means to-gether with SD from three biological repeats. E toJ, The expression patterns of proMIR167A::GFPin the anther (E–G) and ovule (H–J). In E, anenlarged image (right) shows the expression ofGFP in the sporophytic cells of an anther.Bars = 1 cm (A and B), 100 mm (E–G), 20 mm(enlarged image of E), and 50 mm (H–J).

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Complementation of mir167a Mutants with the Wild-TypeMIR167A Gene

To provide genetic evidence that the deletion ofMIR167A was responsible for the observed develop-mental defects, we isolated a second mutant allele,mir167a-c2 (where c stands for CRISPR; SupplementalFig. S3), that harbored a deletion of 844 bp and a ran-dom insertion of 31 bp (Supplemental Fig. S3). Themir167a-c2 allele also lacked the entire region corre-sponding to the mature miRNA167A. The mir167a-c2plants displayed the same phenotypes as those ofmir167a described above, suggesting that the observedphenotypes of the mir167a plants were caused by de-letion of the MIR167A gene. We also carried out com-plementation experiments. A genomic fragment thatincluded both the 2.4-kb upstream sequence of theMIR167A gene and the primary sequence of MIR167Awas used for the complementation experiments. Theconstruct (proMIR167A::MIR167A) was transformed

into mir167a+/2 plants. Five independent lines of mir167athat contained the proMIR167a::MIR167a transgene dis-played normal fertility (Supplemental Fig. S3) and flow-ered at the same time as the wild type, furtherdemonstrating that themir167amutation was responsiblefor the observed developmental phenotypes.

MIR167A Functions through Regulating ARF6 and ARF8

Two ARFs, ARF6 and ARF8, have been shown to betargets of miRNA167 in several species (Wu et al., 2006;Glazi�nska et al., 2014; Wang et al., 2015). Over-expression of miRNA167-resistant versions of ARF6 orARF8 caused pleiotropic phenotypes, arrested growthof ovule integuments, and anther dehiscence defects,which were similar to what we observed in the mir167amutants (Fig. 1, C, D, G, and H; Wu et al., 2006). Wehypothesized that some of the developmental defects inmir167a plants might be caused by ectopic accumulation

Figure 4. Mutations in ARF6 and ARF8 partially rescue the defects of anther dehiscence, pollen germination, and sterility of themir167a mutant. A to F, The anther morphology of wild-type (WT; A), arf6 arf8 double mutants (B), mir167a (C), mir167a arf6arf8+/2 (D),mir167a arf6+/2 arf8 (E), andmir167a arf6 (F) plants. The yellow stars indicate the dehiscence of anthers. Note that theanther dehiscence defects ofmir167awere largely rescued inmir167a arf6 arf8+/2,mir167a arf6+/2 arf8, andmir167a arf6 plants.G, Quantification of the dehiscent anthers. All the anthers from eight opened flowers for each genotype were observed andcalculated. The values represent means together with SD. The asterisks indicate statistical differences between different genotypes(**, P, 0.01 by two-sided Student’s t test). Formir167a and arf6 arf8 plants, the percentages of dehiscent anthers were comparedwith that of the wild type. Formir167a arf6,mir167a arf6 arf8+/2, andmir167a arf8 arf6+/2 plants, the percentages of dehiscentanthers were compared with that of the mir167a mutant. H, Pollen germination rates of wild-type, mir167a, and mir167a arf6arf8+/2 plants. The data represent means together with error based on more than 300 pollen grains for each genotype per ex-periment from three independent experiments; the error bars indicate SD. The asterisks indicate statistical differences between thewild type andmir167a (**, P, 0.01 by two-sided Student’s t test) andmir167a andmir167a arf6 arf8+/ (*, P, 0.05 by two-sidedStudent’s t test). I, Opened siliques at 7 d after pollination in mir167a (left) and mir167a arf6 arf8+/2 (right) plants. Bars = 1 mm(A–F and I).

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ofARF6 andARF8. To test this hypothesis, we crossed themir167amutants to arf6 arf8 double mutants. As shown inFigure 4, the anther dehiscence defects of mir167a plantswere largely rescued in the arf6, arf6 arf8+/2, and arf6+/2arf8 backgrounds (Fig. 4, D–F). Both arf6 arf8 andmir167aplants were not able to undergo dehiscence (Fig. 4, B andC), but the underlying causes were different. The arf6 arf8double mutants did not make sufficient jasmonic acid,and the anther dehiscence defects of the arf6 arf8mutantswere rescued by exogenous jasmonic acid applications(Zhang et al., 2017). We observed that the anthers ofmir167a arf6 arf8 triple mutants did not dehisce, whichwas similar to that in the arf6 arf8 doublemutant (Fig. 4, Band G).

We conducted in vitro pollen germination assays todetermine the viability of the pollen released frommir167a arf6 arf8+/2 plants. The germination rate ofmir167awas less than 40% (n. 300; Fig. 1E), but it wasimproved to about 70% (n . 300) in the arf6 arf8+/2background (Fig. 4H). Moreover, we observed that thesiliques from self-pollination of mir167a arf6 arf8+/2plants always contained a few seeds (Fig. 4I), whichwas never observed in themir167amutants. Our resultsindicated that the developmental defects of anther,ovule, and seed maturation in mir167a mutants werelikely caused by overexpression of ARF6 and ARF8.

MIR167B, MIR167C, and MIR167D Play Minor Roles inArabidopsis Development

We used CRISPR/Cas9 technology to generate de-letion mutants of MIR167B, MIR167C, and MIR167D(Supplemental Fig. S4). Unlike the mir167a plants, sin-gle mutants mir167b, mir167c, and mir167d did notdisplay any obvious developmental defects under ourgrowth conditions. We generated mir167bcd triple mu-tants, but the triple mutants behaved very similarly towild-type plants (Fig. 5, A and E).

We analyzed mir167abcd quadruple mutants to de-termine whether MIR167B, MIR167C, and MIR167Dhave overlapping functions with MIR167A. Similar tomir167a plants, the mir167abcd quadruple mutantsflowered later (33 d after germination; n = 10) and weresterile (Fig. 5, B, D, and F). Compared with the mir167asingle mutants, the ovules of mir167abcd quadruplemutants displayed more severe defects. All of the em-bryo sacs we observed were abnormal, althoughsometimes one or two nuclei could be observed in someembryo sacs (Fig. 5G; n = 30). In addition to the celldifferential defects of the embryo sac, the outer integ-uments in mir167abcd plants were much shorter thanthose of mir167a mutants (Figs. 1, G and H, and 5G).When pollinated with wild-type pollen, mir167abcdgynecia did not elongate much compared with mir167aor wild-type plants (silique lengths were as follows: thewild type, 18–20 mm; mir167a pollinated with wild-type pollen, 16–18 mm; and mir167abcd pollinatedwith wild-type pollen, 15–16 mm [n. 20]; Figs. 2A and5C). These results suggested that MIR167B, MIR167C,

and MIR167D indeed have overlapping functions withMIR167A in regulating ovule and seed development.

DISCUSSION

In this study, we uncovered several functions ofMIR167s by analyzing the loss-of-function mutantsin Arabidopsis. MIR167s regulate anther dehiscence,ovule development, and fertility, as plants that over-express MIR167A or mARF6 and mARF8 are defectivein those processes (Wu et al., 2006). Our mir167a mu-tants largely phenocopied the mARF overexpressionplants in those processes (Wu et al., 2006). Moreover,we show that MIR167A is critical for flowering timecontrol, a process that was not observed in the mARFoverexpression lines.

An unexpected finding was that MIR167A has a truematernal effect. Thefirst reportedmaternal gene requiredfor embryo development in Arabidopsis is DICER-LIKE 1 (DCL1)/SHORT INTEGUMENT/SUSPENSOR1/CARPEL FACTORY (Ray et al., 1996). DCL1 is aprimary enzyme involved in miRNA biogenesisin plants (Kurihara and Watanabe, 2004; Fang andSpector, 2007). SERRATE, one of the accessory pro-teins of DCL1 required for miRNA biogenesis, was alsoreported to act as a maternal effector for embryo de-velopment (Prigge and Wagner, 2001). Although about100 families of miRNAs have been identified in Arabi-dopsis (Voinnet, 2009), it was not knownwhichmiRNAacts downstream of DCL1 in controlling the maternalsporophytic effect for embryogenesis. In this study, wefound thatMIR167A acts as amaternal gene for embryodevelopment. It has been reported that DCL1 is re-sponsible forMIR167 processing (Dong et al., 2008; Liuet al., 2012). Combinedwith the similar phenotypes andthe discovered function of DCL1 in miRNA processing,it is likely that miRNA167A is the key DCL1-processedmiRNA involved in controlling embryo developmentfrom maternal sporophytic tissues. Interestingly, thedefects in embryogenesis and seed development inmir167a mutants are 100% penetrant, whereas dcl1mutants were not 100% penetrant. The difference inpenetrance may be due to the nonnull nature of dcl1mutations. Alternatively, the difference may suggestthat other DCL proteins may also be involved in pro-cessing miRNA167 precursors. Nevertheless, the mir167amutants described here provide a valuable genetic ma-terial for studying maternal control.

The development of the maternal seed integument,the embryo, and the endosperm needs to be properlycoordinated (Ingram, 2010). Fertilization of the centralcell leads to the production of auxin, which is subse-quently transported to the maternal tissues to driveseed coat development (Figueiredo et al., 2016). Re-cently, it has also been reported that auxin biosynthesisincreased in the integuments at early embryogenesisand that this maternally produced auxin is required forembryo development (Robert et al., 2018). WithoutmiRNA167 in the integuments, it is expected that ARF6

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and ARF8 mRNAs will overaccumulate, causing animbalance of auxin signaling and disruption of seeddevelopment. In addition to the influence from mater-nal tissues, MIR167A and MIR167B were also detectedin the globular stage embryos (Armenta-Medina et al.,2017), suggesting that miRNA167 may also regulate theauxin response in embryos and contribute to normalembryo development. It is intriguing to explorewhether miRNA167 or auxin or both actually movefrom maternal cells to embryos and endosperms, giventhat both miRNAs and auxin are mobile. The mir167amutants reported here will be a valuable resource forstudying auxin signaling in seed development as wellas for studying how the development of the seed coat,embryo, and endosperm is coordinated.It is interesting that MIR167A plays a more pre-

dominant role in Arabidopsis reproductive develop-ment than the other three MIR167 genes. Thephenotypic differences among the mir167 mutantscould result from their different expression patterns.We observed significant expression of MIR167A in the

anther wall and ovule epidermis (Fig. 3), whereas theother three MIR167 genes did not show strong expres-sion in those cells (Wu et al., 2006). Differences in reg-ulatory elements in the primary MIR167 sequences,which may impact transcription efficiency or affect thematuration of miRNAs, may also partially account forthe observed phenotypic differences inmir167mutants.Consistent with this hypothesis, it was reported thatoverexpression of MIR167A, but not the other threeMIR167 genes, under the control of the sameCauliflowermosaic virus 35S promoter caused strong phenotypessimilar to those in arf6 arf8 double mutants (Wu et al.,2006).MiRNA167 has at least three target genes in Arabi-

dopsis: ARF6, ARF8, and IAR3. In rapeseed (Brassicanapus), the NRAMP1b gene, which encodes a metaltransporter, is also a target for miRNA167 (Meng et al.,2017). Our results suggest that ARF6 and ARF8 are themajor targets of miRNA167 in anther and ovule de-velopment. The phenotypes of mir167a mutants arevery similar to those ofmARF6/8 overexpression plants.

Figure 5. MIR167B, MIR167C, and MIR167Dplay minor roles in Arabidopsis reproductiondevelopment. A and B, mir167bcd (A) andmir167abcd (B) mutants 43 d after germina-tion. The mir167abcd mutants were sterile,while the development of mir167bcd wassimilar to that of the wild type. C, Morphologyof an adult shoot of mir167abcd pollinatedwith wild-type pollen. White arrows indicatethe pollinated siliques. D and E, The rosetteleaves of mir167abcd (D) and mir167bcd (E)when the plants started to flower. F, The num-bers of rosette leaves developed when theplants started to flower. The values ofmir167bcdandmir167abcd represent means togetherwithSD (n $ 20). The asterisks indicate a statisticaldifference betweenmir167bcd and mir167abcd(**,P,0.01by two-sided Student’s t test).G, Thesevere defects in the embryo sac of the mir167-abcd quadruplemutant. Bars = 5 cm (A–C), 1 cm(D and E), and 50 mm (G).

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Both mir167a mutants and mARF plants displayed in-dehiscent anthers and shorter integuments (Fig. 1; Wuet al., 2006). The anther indehiscent and sterility phe-notypes were partially rescued in mir167a arf6 arf8+/2and mir167a arf8 arf6+/2 mutants (Fig. 4), suggestingthat miRNA167s mainly function through the ARFs. Itis clear that the expression levels of ARF6 and ARF8should be tightly regulated. Too much (as in mir167aplants) or too little (as in arf6 arf8mutants) expression ofARF6/8 will lead to defects in anther dehiscence.

MATERIALS AND METHODS

Plant Materials and Growth Conditions

Arabidopsis (Arabidopsis thaliana) plants used in this study were in the Col-0genetic background. The mir167 mutants were generated using CRISPR/Cas9 gene-editing technology as described previously (Gao and Zhao, 2014;Gao et al., 2015, 2016). The two mir167a alleles harbor 1,170- and 844-bp dele-tions, respectively (Supplemental Fig. S3). The mir167a mutants were geno-typed by PCR using the 167A-Genotyping 1 (GT1) and 167A-GT2 primer pair(Supplemental Table S3), which amplify about a 1.6-kb fragment from the wildtype and a much smaller fragment from the mir167a mutants. The mir167bmutant had a 612-bp deletion, which completely removed theMIR167B codingregion (Supplemental Fig. S4). The mir167b mutant was genotyped using threeprimers, 167B-GT1, 167B-GT2, and 167B-GT3. The 167B-GT1 and 167B-GT2pair generates 1.6- and 1-kb fragments for the wild type and the mutant, re-spectively. To further clarify the homozygosity of themir167bmutants, we used167B-GT1 and 167B-GT3, which only amplify a fragment from the wild type.The mir167c mutant contained a 697-bp deletion (Supplemental Fig. S4), whichwas genotyped using 167C-GT1, 167C-GT2, and 167C-GT3 primers. The 167C-GT1 and 167C-GT2 pair produce 2- and 1.3-kb fragments for the wild type andthemir167cmutant, respectively. The 167C-GT1 and 167C-GT3 pair only amplythe wild type for about a 1.4-kb fragment. The mir167d mutant had a 1.6-kbdeletion (Supplemental Fig. S4). We used 167D-GT1 and 167D-GT2 to deter-mine whether a plant contained the mutation. We used 167D-GT2 and 167D-GT3 to determine the zygosity of the mir167d mutant. All of the genotypingprimers are listed in Supplemental Table S3.

Plants were grown at 22°C under long-day conditions (16 h of light/8 h ofdark).

Plasmid Construction and Complementation of themir167a Mutant

We amplified an ;1.5-kb upstream sequence from genomic DNA of Col-0into pEasy-Blunt for sequence verification (see Supplemental Table S3 forProMIR167A-F and ProMIR167A-R primers) and then subcloned it into themodified vector pCAMBIA1300-GFP to form the proMIR167a::GFP construct.The 3.8-kb MIR167A genomic sequence was amplified and recombined intopCAMBIA1300-nos to form the proMIR167A::MIR167A constructs (seeSupplemental Table S3 for MIR167A-F and MIR167A-R primers). The proM-IR167A::GFP and proMIR167A::MIR167A plasmids were transformed into Col-0 and mir167a+/2 plants by the floral dip method (Clough and Bent, 1998). TheT1 seeds were grown on one-half-strength Murashige and Skoog mediumcontaining 15 mg mL21 hygromycin for selecting transgenic plants. For proM-IR167A::MIR167A transgenic plants, we then genotyped the transgenic plantsfor the mir167a mutation using 167A-CGT3/CGT2 (see Supplemental Table S3for 167A-CGT3 and 167A-CGT2). For proMIR167A::GFP transgenic plants, aLSM 5 Pascal confocal laser scanning microscope (Zeiss)was used to visualizethe GFP expression pattern. Anthers and ovules at each stage were dissectedfrom flower buds. The fluorescence was excited at 488 nm and collected at 515to 530 nm.

Pollen Germination in Vitro and in Pistils

For invitropollengermination, openflowersweredehydrated for 2hat roomtemperature. Anthers were dissected and pollen grains were released onto agarmedium by dipping the anthers on the surface of agar plates, which contained

18% (w/v) Suc, 0.01% (w/v) boric acid, 1 mM MgSO4, 1 mM CaCl2, 1 mM

Ca(NO3)2, and 0.5% (w/v) agar. Six hours after germination at 24°C, pollengrains were examined and photographed using an Olympus BX51 digital mi-croscope. For the pollen germination ratio, more than 300 pollen grains of eachgenotypewere analyzedwith a lightmicroscope. This experimentwas carried outthree times. Themean values and SDwere calculated based on these three repeats.

For pollen germination in pistils, wild-type pollen was hand pollinated ontothe pistils of Col-0 and mir167 mutants in the afternoon of the first day. In themorning of the second day (18–19 h after pollination), the pollinated pistils werecollected and fixed in Carnoy’s solution (ethanol:acetic acid = 3:1) for 2 to 3 h.After rinsing in 0.01 mol L21 phosphate-buffered saline four times (each for5 min), 7 M NaOHwas used to soften the pistils for 4 h. After rinsing in 0.01 molL21 phosphate-buffered saline four times (each for 5 min), the pistils were in-cubated in 0.1% (w/v) Aniline Blue for 30min. Finally, we used aUV channel tomonitor the pollen tube growth and took photographs using an Olympus BX51digital microscope.

Late-Flowering Phenotype Analysis

The days to flowering after germination were calculated from more than 10plants of each genotype (thewild type,mir167a,mir167bcd, andmir167abcd). Fordetermining the rosette leaf numbers at bolting of wild-type, mir167a,mir167bcd, and mir167abcd plants, at least 20 plants for each genotype werescored.

RNA Extraction and qPCR

Twenty-day-old leaves of the wild type and mir167a mutants were used todetect the transcripts of flowering regulatory genes. For analyzing the tran-scriptional level ofMIR167 genes, inflorescences were collected fromwild-type,mir167a, and mir167bcd plants. Arabidopsis leaves or inflorescences were usedto extract total RNA via TRIzol Reagent (Invitrogen). Total RNAwas then usedfor first-strand cDNA synthesis according to the manufacturer’s instructions(TransGen Biotech). qPCR was performed using an ABI PRISM 7300 detectionsystem (Applied Biosystems) with the SYBR Green Realtime PCR Master Mix(Toyobo). The relative expression level of each gene was normalized to TU-BULIN BETA CHAIN2 and averaged over three biological repeats. The relevantprimer sequences are listed in Supplemental Table S3.

Microscopic Analysis

The protocol used to analyze female gametophyte development was mod-ified from the previously reported protocol (Christensen et al., 1997). Briefly,pistils were fixed in a solution of 4% (v/v) glutaraldehyde in 12.5 mM sodiumcacodylate buffer (pH 6.9) for 2 h at room temperature. Next, the pistils weredehydrated in a series of ethanol (v/v) solutions (10%, 20%, 40%, 60%, 80%, and95%), each for 10 min. The pistils were incubated in 95% (v/v) ethanol solutionovernight. The next day, the pistils were dehydrated in 100% ethanol two times,each for 10 min. Then the tissues were cleared in the benzyl benzoate:benzylalcohol (2:1) solution for 20 min. Ovules were dissected and mounted in im-mersion oil and examined using a Carl Zeiss LSM5 Pascal confocal laser scan-ning microscope. Excitation wavelength was 488 nm, and the emissionwavelength was 515 to 530 nm.

The modified pseudo-Schiff propidium iodide staining was performed asdescribed previously to observe the development of embryos (Truernit et al.,2008). Briefly, the tips of pistils were cut and fixed in fixative solution (50%[v/v] methanol and 10% [v/v] acetic acid) for 12 h at 4°C. Pistils were trans-ferred to 80% (v/v) ethanol solution for 5 min at 80°C. The materials were thenput back into the fixative solution for 1 h at 4°C. The pistils were retained in asolution of 1% SDS and 0.2 N NaOH overnight. After washing, the pistils wereincubated in bleach solution (2.5% NaClO), rinsed with water, and then incu-bated in 1% periodic acid for 40 min. After another wash, the pistils weretransferred into Schiff reagent with propidium iodide for 2 h (100 mM sodiummetabisulfite and 0.15 N HCl; PI concentration was 100 mg mL21). The pistilswere then cleared in chloral hydrate solution (4 g of chloral hydrate, 1 mL ofglycerol, and 2mL of water) overnight at room temperature. After being kept inHoyer’s solution for 3 d, pistils were observed using a Carl Zeiss LSM5 Pascalconfocal laser scanningmicroscope. Excitationwavelength was 488 nm, and theemission wavelength was 515 to 530 nm.

For the observation of endosperm cellularization in embryos, Col-0 andmir167a seeds (5 d after fertilization) were fixed and embedded as described

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(Zhang et al., 2007). Semithin sections (1 mm) of seeds were stained with To-luidine Blue and photographed using an Olympus BX51 digital microscope.

Accession Numbers

Sequence data from this article can be found in the GenBank/EuropeanMolecular Biology Laboratory data libraries under accession numbersMIR167A, AT3G22886; MIR167B, AT3G63375; MIR167C, AT3G04765; MIR167D,AT1G31173; ARF6, AT1G30330; ARF8, AT5G37020; FT, AT1G65480; SOC1,AT2G45660; SPL5, AT3G15270; SPL3, AT2G33810; MYB33, AT5G06100; CO,AT5G15840; FLC, AT5G10140; MAF4, AT5G65070; and MAF5, AT5G65080.

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Pollen mitosis I and II can be carried out inmir167a mutants.

Supplemental Figure S2. Wild-type pollen geminated and grew normallyon the pistils of mir167 mutants.

Supplemental Figure S3. Genetic complementation of the mir167amutants.

Supplemental Figure S4. The CRISPR mutants ofMIR167B,MIR167C, andMIR167D genes.

Supplemental Table S1. Various embryo developmental defects in mir167aplants pollinated with wild-type pollen.

Supplemental Table S2. The transmission efficiency of male and femalegametophytes of mir167a.

Supplemental Table S3. Primers used in this study.

Received February 1, 2019; accepted March 5, 2019; published March 13, 2019.

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464 Plant Physiol. Vol. 180, 2019

Roles of MIR167s in Embryo and Seed Development

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