Importin b4 Mediates Nuclear Import of GRF-InteractingFactors to Control Ovule Developmentin Arabidopsis1[OPEN]

Hai-Hong Liu,2 Feng Xiong,2 Cun-Ying Duan, Ya-Nan Wu, Yan Zhang,3 and Sha Li3,4

State Key Laboratory of Crop Biology, College of Life Sciences, Shandong Agricultural University, Tai’an271018, China

ORCID IDs: 0000-0003-3797-4602 (H.-H.L.); 0000-0002-3501-5857 (Y.Z.); 0000-0002-7197-0181 (S.L.).

Ovule development is critical for seed development and plant reproduction. Multiple transcription factors (TFs) have beenreported to mediate ovule development. However, it is not clear which intracellular components regulate these TFs during ovuledevelopment. After their synthesis, TFs are transported into the nucleus a process regulated by karyopherins commonly knownas importin alpha and b. Around half of Arabidopsis (Arabidopsis thaliana) importin b-coding genes have been functionallycharacterized but only two with specific cargos have been identified. We report here that Arabidopsis IMPORTIN b4 (IMB4)regulates ovule development through nucleocytoplasmic transport of transcriptional coactivator growth regulatingfactors–interacting factors (GIFs). Mutations in IMB4 impaired ovule development by affecting integument growth. imb4mutants were also defective in embryo sac development, leading to partial female sterility. IMB4 directly interacts with GIFsand is critical for the nucleocytoplasmic transport of GIF1. Finally, functional loss of GIFs resulted in ovule defects similar tothose in imb4 mutants, whereas enhanced expression of GIF1 partially restored the fertility of imb4. The results presented hereuncover a novel genetic pathway regulating ovule development and reveal the upstream regulator of GIFs.

Ovule development is critical for seed developmentand plant reproduction. Mature ovules contain sporo-phytic integuments and gametophytic embryo sacs andare formed through two processes, i.e. megasporogen-esis andmegagametogenesis (Schneitz et al., 1995, 1997;Drews et al., 1998). During megasporogenesis, ovulesestablish proximal-distal polarity. The distal part ofdeveloping ovules, i.e. the nucellus, forms megasporemother cell (MMC), which produces a tetrad of haploidspores through meiotic division. The most proximal

one survives to form the functional megaspore (FM),whereas the other three degenerate (Schneitz et al.,1995, 1997; Christensen et al., 1997; Drews et al.,1998). During megagametogenesis, FM eventuallyforms a seven-cell eight-nuclei female gametophyte, i.e.embryo sac (Christensen et al., 1997; Drews et al., 1998).In Arabidopsis (Arabidopsis thaliana), the growth ofouter and inner integuments initiates and eventuallyenvelops the embryo sac during megagametogenesis.Integuments grow asymmetrically, resulting in ana-tropy of mature ovules, i.e. mature ovules bend so thatthe micropyle is close to the funiculus (Schneitz et al.,1995, 1997; Drews et al., 1998). Because mutants de-fective in integument cells often result in defective for-mation of female gametophyte (Bencivenga et al., 2011;Chevalier et al., 2011; Wang et al., 2016), it is generallyconsidered that integuments control embryo sac de-velopment through unknown signaling molecules.

Multiple transcription factors (TFs) were reported tomediate ovule development (Colombo et al., 2008).INNER NO OUTER (INO) regulates abaxial-adaxialpatterning of Arabidopsis ovules (Villanueva et al.,1999). Mutations at ABERRANT TESTA SHAPE resultin the formation of a single integument layer due tocongenital fusion of the two integuments (McAbeeet al., 2006). Functional loss of AINTEGUMENTAabolishes the growth of both integuments (Elliott et al.,1996; Klucher et al., 1996). Some homeodomain pro-teins such as BELL1 (Reiser et al., 1995), PHABULOSA(Sieber et al., 2004), and WUSHEL (Gross-Hardt et al.,2002; Lieber et al., 2011) are also mediators of

1This work was supported by the National Natural ScienceFoundation of China (NSFC) (31871422 and 31625003 to Y.Z. and31771558 to S.L.), by Major Research Plant from the Ministry of Sci-ence and Technology of China (grant 2013CB945102), and by theNatural Science Foundation of Shandong Province (ZR2014CM027to S.L.). Y.Z.’s laboratory is partially supported by the Tai-ShanScholar Program of the Shandong Provincial Government.

2These authors contributed equally to this work.3Senior authors.4Author for contact: shali@sdau.edu.cn.The author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Sha Li (shali@sdau.edu.cn).

Y.Z. and S.L. conceived and supervised the project; H.-H.L. andF.X. performed most of the experiments with assistance from C.-Y.D.and Y.-N.W.; S.L. designed the experiments and analyzed the datawith the assistance of H.-H.L., F.X., and Y.Z.; Y.Z. and S.L. wrote thearticle, with contributions from all the authors.

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integument growth and ovule development. Althoughextensive studies have been performed to TF regula-tion of ovule development, it is not yet clear which in-tracellular components regulate their targeting andactivities.TFs are transported into the nucleus through the

formation of the nuclear pore complex (NPC), a pro-cess mediated by karyopherins, often called importinsor exportins, depending on the direction of transport(Meier and Brkljacic, 2009; Tamura and Hara-Nishimura, 2014). There are two subfamilies of kar-yopherins: importin alpha and importin beta (Tamuraand Hara-Nishimura, 2014). It is generally believedthat importin alpha recognizes a short positivelycharged nuclear localization signal, whereas importinbeta mediates their interaction with the NPC and Ran-GTP to complete cargo transport (Tamura and Hara-Nishimura, 2014). However, importin beta may playroles in cells other than merely cargo transport (Hareland Forbes, 2004; Tamura and Hara-Nishimura, 2014),such as mediation of microRNA activity as reported inplants (Wang et al., 2011; Cui et al., 2016).The Arabidopsis genome encodes 18 importin bs

(IMB), half of which were functionally characterized.IMB1/ATKPNB1 participates in responses to abscisicacid and drought (Luo et al., 2013); HASTY/XPO4wasinvolved in shoot meristem maintenance, floweringtime, and fertility (Telfer and Poethig, 1998; Bollmanet al., 2003); SAD2/EMA1/IPO8 not only mediates re-sponses to abscisic acid and UV but also regulates tri-chome development (Zhao et al., 2007; Wang et al.,2011); XPO1A and XPO1B are essential for gameto-phytic fertility (Blanvillain et al., 2008), whereasXPO1A also mediates heat responses (Wu et al., 2010);XPOT/PAUSED regulates leaf development andmeristem maintenance (Hunter et al., 2003; Li andChen, 2003); TRANSPORTIN1 (TRN1) and KETCH1/IMP3 are both involved in leaf development and fer-tility (Cui et al., 2016; Zhang et al., 2017). Despite theextensive functional studies on importin b, only twohave identified a cargo: one showed that SAD2 me-diates the nucleocytoplasmic transport of MYB4(Zhao et al., 2007), whereas another showed thatKETCH1/IMP3 mediates that of hyponastic leaves1 (Zhang et al., 2017).Here we report that Arabidopsis IMB4, an importin

b, regulates ovule development through nucleocyto-plasmic transport of transcriptional coactivator growthregulating factors (GRF)-INTERACTING FACTORS(GIFs). Functional loss of IMB4 compromised ovuledevelopment by impairing integument growth. Con-sequently, embryo sacs were not properly developedin imb4 mutants, leading to partial female sterility.We showed that GIFs directly interact with IMB4.Furthermore, through performing cell fractionationand examining fluorescence distribution, we foundthat nucleocytoplasmic transport of GIF1 relies onIMB4. Finally, functional loss of GIFs resulted in ovuledefects similar to those in imb mutants. Our resultshave uncovered a genetic pathway regulating ovule

development and the upstream regulator of the im-portant transcriptional coactivator GIFs.

RESULTS

Functional Loss of IMB4 Reduces Fertility

To identify novel components involved in fertility,we characterized Arabidopsis IMB4 (At4g27640),which encodes importin b, whose function has not yetbeen understood. Two T-DNA insertion mutant lineswere isolated (Fig. 1A). Transcript analysis showedthat no full-length transcript was detected in eithermutant, although imb4-2 did express a partial transcript(Fig. 1B). The twomutants displayed similar pleiotropicdevelopmental defects (Supplemental Fig. S1). In thisstudy, we focused on their reduced fertility for furtheranalysis (Fig. 1A, 1I). Unlike wild type in which siliquescontained fully developed seeds (Fig. 1, C and I), imb4siliques contained white and wrinkled ovules amongdeveloping seeds (Fig. 1, D and E). Embryogenesisstarted in wild type at 24 h after pollination (HAP), asjudged from whole-mount clearing of pistils (Fig. 1J).However, although 66.8% 6 3.7% (means 6 SD, n = 10)ovules were fertilized in imb4 pistils, as judged from thepresence of developing embryos (Fig. 1K), 33.2% 68.6% ovules of imb4 showed no pollen tube entrance(Fig. 1M) or occasionally a nondischarging pollen tube(Fig. 1L), suggesting that the reduced fertility in imb4was because of failed fertilization. Crosses betweenimb4-1 and imb4-2 yielded F1 plants with a reducedseed set comparable with either parent (Fig. 1, F and I),suggesting that imb4-1 and imb4-2 were allelic. Thefollowing results were mostly observed for imb4-1, butwere also observed for imb4-2.Reciprocal crosses between the homozygous imb4-

1 mutant and wild type indicated that the reducedseed set of imb4-1 was because of defects from the fe-male side, whereas pollen of the imb4mutants were notaffected (Fig. 1, G–I). Indeed, pollen developmentwas comparable between imb4 and the wild type(Supplemental Fig. S2). Next, we performed reciprocalcrosses between the wild type and the heterozygousimb4 mutants to determine whether defects of imb4were gametophytic. The segregation ratio showed thatfemale and male transmission of imb4 was comparablewith that of the wild type (Supplemental Table S1),indicating a sporophytic defect in female tissues due toIMB4 loss-of-function.

Functional Loss of IMB4 Compromises Ovule-ControlledPollen Tube Guidance

To determine the cause of failed fertilization whenthe imb4 mutants were used as the female parent,we first pollinated wild-type or imb4-1 pistils withProLAT52:GUS pollen and performed histochemicalb-glucuronidase (GUS) staining at 12 HAP. At 12HAP, all wild-type ovules were targeted by a pollentube (Fig. 2, A and G), indicating proper pollen tube

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guidance by wild-type ovules. By contrast, 78 out of293 imb4-1 ovules failed to attract pollen tubes (Fig. 2, Band H). To determine whether failed pollen tubeguidance led to reduced fertility in imb4-1, we polli-nated imb4-1 pistils with wild-type pollen and analyzedpistils by aniline blue staining at 48 HAP when wild-type ovules were fertilized, and found rapid sizeincrease (Fig. 2, C and E). Indeed, 118 out of 413 imb4-1 ovules did not grow as a consequence of failed fer-tilization (Fig. 2, D and F). To provide further evidenceof defective pollen tube guidance in imb4-1, weanalyzed wild-type and imb4-1 pistils pollinated bywild-type pollen at 12 HAP by scanning electron mi-crographs (SEMs). Pollen tubes efficiently targeted tothe micropyle of wild-type ovules as expected (Fig. 2I).In comparison, imb4-1 pistils contained not only ovulesnormally targeted by a pollen tube (Fig. 2J), but thosethat failed to attract a pollen tube at the micropyle(Fig. 2K). These results all point to a key role of IMB4 inovule-guided pollen tube targeting.

Functional Loss of IMB4 Impairs Integument Growth

To determine how IMB4 loss-of-function causedovule defects, we first performed SEMs on mature

unpollinated ovules. In the wild type, mature ovuleswere asymmetric, whose abaxial-adaxial polarity led toovule bending such that the micropyle was close to thefuniculus (Supplemental Fig. S3), as well known(Schneitz et al., 1995, 1997). By contrast, 21.4% 6 3.7%of imb4-1 ovules were abnormal such that outer integ-uments did not fully extend to enclose the inner integ-ument (Supplemental Fig. S3). As a consequence of thedisrupted polarity, the micropyle of imb4-1 often wasnot clearly formed and perpendicular to the funiculus(Supplemental Fig. S3). These results suggested thatintegument development was impaired in imb4-1.

To determine the exact stage when imb4-1 showeddefective ovule development, we performed opticalsections of ovules over entire developmental stages.Initiation of outer and inner integument was compa-rable between the wild type and imb4-1 (Fig. 3, A andE). At these stages, FMs were formed, i.e. three proge-nies of megasporocytes degenerated and only the oneproximal to the funiculus persisted (Schneitz et al.,1995, 1997; Christensen et al., 1997). At stage 3-I,when rapid differentiation and growth of the outer in-tegument began in the wild type, especially at the ab-axial side of ovules (Fig. 3B), the growth of the outerinteguments in imb4-1 was much delayed and oftensymmetric (Fig. 3F). At stage 3-III, wild-type ovules

Figure 1. Functional loss of IMB4 reduces fer-tility. A, Schematic illustration of the IMB4 ge-nomic region. Boxes indicate exons; short linesindicate untranslated regions or introns. Trian-gles point at the insertion sites of two T-DNAmutants. Primer binding sites are indicatedwitharrows. B, Transcript analysis demonstratingthat neither imb4-1 nor imb4-2 expresses thefull-length IMB4 transcript, whereas imb4-2expresses a partial IMB4 transcript. The com-plemented line, IMB4g-YFP imb4-1, expressedonly exogenous IMB4. C to H, a representativesilique from the wild type (C), imb4-1 (D),imb4-2 (E), imb4-1/+ imb4-2/+ (F), or from across between the wild type and imb4-1 whenimb4-1 was used as the male (G) or as the fe-male (H) parent. Arrowheads point at unfertil-ized ovules. I, Quantification of seed set fromdesignated genetic background. Results shownare means 6 SD (SD). Twenty siliques were an-alyzed for each genotype. Means with differentletters are significantly different (one-wayANOVA, Tukey-Kramer test, P , 0.05). J to M,Whole-mount clearing of wild-type (J) or imb4-1 (K to M) ovules at 24 HAP. Pink-highlightedregions are developing embryos (J and K) or anondischarging pollen tube (L). The dotted lineillustrates a protruding embryo sac with tra-cheary element-like structure inside (M).Bars = 1 mm (C to H) and 20 mm (J to M).

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established the abxial-adaxial polarity by bendingtoward the funiculus through differential growth ofouter integuments (Fig. 3C). At this stage, a centralvacuole in embryo sac separated the chalazal nucleusand the micropylar nucleus (Fig. 3C). In contrast, theouter integuments in imb4-1 showed symmetric

growth and ovules did not show the classic anatropy(Fig. 3G).Subsequently, wild-type ovules maintained the

bending status and embryo sacs developed (Fig. 3, Dand I), whereas in 63 out of 210 imb4-1 ovules, embryosac development was defective: either no embryo sac

Figure 2. Functional loss of IMB4 compromises female-controlled pollen tube guidance. A and B, Histochemical GUS staining ofwild-type (A) or imb4-1 (B) pistils at 12 HAP with ProLAT52:GUS pollen. Arrowheads point at ovules that failed to attract pollen.Two to three overlapping high-magnification images were taken for one pistil. The images were then overlaid with Photoshop(Adobe) to show the whole pistil. Numbers at the bottom: ovules targeted by a ProLAT52:GUS pollen tube versus ovules examined.C and D, Aniline blue staining of wild-type (C) or imb4-1 (D) pistils at 48 HAP with wild-type pollen. Two to three overlappinghigh-magnification images were taken for one pistil. The images were then overlaid with Photoshop (Adobe) to show the wholepistil. Arrowheads point at ovules that did not develop as a result of fertilization failure. Numbers at the bottom: fertilized ovulesversus total ovules examined. E and F, A close-up of aniline blue stained wild-type (E) or imb4-1 (F) pistils at 48 HAP with wild-type pollen. Arrowheads point at the unfertilized ovules. G and H, A close-up of histochemical GUS stained wild-type (G) orimb4-1 (H) pistils at 12 HAP with ProLAT52:GUS pollen. Arrowheads point at the micropyles that failed to attract pollen tubes. I toK, SEMs of wild-type (I) or imb4-1 (J-K) pistils at 12 HAP with wild-type pollen. Incoming pollen tubes are highlighted in pink. Apollen tube enters through the micropyle of a normal-developing ovule of imb4-1 (J) but not of a defective ovule of imb4-1 (K).Bars = 200 mm (A to D), 100 mm (E and F), 50 mm (G and H), and 20 mm (I to K).

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structures could be seen or embryo sacs did notcontain central cell, egg cell, and synergid cells(Fig. 3, H and J). Because of defective integumentgrowth, the embryo sacs in imb4-1 were often pro-truding (Fig. 3J). To provide further evidencethat embryo sac development was defective inimb4-1, we performed whole-mount ovule clearingon unfertilized mature pistils. In contrast withwell-enclosed embryo sacs in wild-type ovules(Fig. 3K), 67 out of 239 imb4-1 ovules containedprotruding embryo sacs, occasionally with trache-ary element-like structures inside (Fig. 3L), suggesting

that functional loss of IMB4 results in defective embryosac development.

Functional Loss of IMB4 Compromises Auxin andCytokinin Responses during Ovule Development

Because auxin and cytokinins (CK) play importantroles in ovule development (Benková et al., 2003;Bencivenga et al., 2012; Kelley et al., 2012; Ceccato et al.,2013), we examined whether auxin and CK responseswere compromised in imb4-1 by analyzing the

Figure 3. Functional loss of IMB4 impairs integument growth. A to J, Confocal laser scanningmicroscopy (CLSM) of wild-type (AtoD, I) or imb4-1 (G, H, J) ovules at stage 2-III (A, E), 3-I (B, F), 3-III (C, G), 3-V (D, H), or 3-VI (I, J). Midoptical sections of ovules areshown.Only imb4-1 ovuleswith visible nuclei are documented. cc, Central cell; cn, chalazal nucleus; ec, egg cell; fm, functionalmegaspore; F, funiculus; ii, inner integument; oi, outer integument; mmc, microspore mother cell; mn, micropylar nucleus; sc,synergid cell; V, vacuole. Numbers at the bottom of (J): displayed ovules versus total ovules examined. K to L,Whole-mount ovuleclearing of wild type (WT; K) or imb4-1 (L). Lilac-shaded areas indicate embryo sacs. Arrowhead points at tracheary element-likestructure in the embryo sac of imb4-1. Bars = 10 mm.

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distribution of their respective fluorescence reporters,DR5:GFP (Benková et al., 2003) and TCS:yellow fluo-rescence protein (YFP; Bencivenga et al., 2012). In thewild type, GFP signals showed a polar distribution inthe micropylar end of ovules from stage 2-III to 3-III(Fig. 4, A–C), which was consistent with the polardistribution of PIN-FORMED1 (PIN1; Fig. 4, I and K),the auxin efflux carrier mediating auxin distributionduring ovule development (Benková et al., 2003;Bencivenga et al., 2012; Ceccato et al., 2013). At mat-uration, strong GFP signals were detected only in thefuniculus (Fig. 4D). By contrast, GFP signals werehardly detectable in DR5:GFP;imb4-1 ovules overvarious developmental stages (Fig. 4, E–H). Corre-spondingly, PIN1 was mis-targeted in imb4-1 ovulessuch that PIN1 showed irregular membrane distribu-tion and was often detected in vacuoles or cytoplasmicaggregates both in the nucellus and in the funiculus(Fig. 4, J and L). Strong signals of PIN1 were also ec-topically detected in the plasma membrane of outerinteguments (Fig. 4L).Because CK is another important phytohormone for

ovule development and was reported to show a dis-tribution reciprocal to that of auxin (Bencivenga et al.,2012), we introduced TCS:YFP in imb4-1 to determineCK responses in the mutant. Indeed, strong CK signalswere detected at the chalazal ends of ovules dur-ing development (Supplemental Fig. S4), reciprocal tothat of DR5:GFP (Fig. 4). By contrast, YFP signals wereenhanced and expanded at early stages while beingirregularly distributed at the micropylar end at matu-ration in imb4-1 (Supplemental Fig. S4). The enhancedand ectopic CK responses were consistent with reduced

auxin signaling in imb4-1 ovules, providing furtherevidence that IMB4 is critical for ovule development.

IMB4 Is Expressed Constitutively during OvuleDevelopment and its Protein Localizes in Both theCytoplasm and the Nucleus

To determine the expression pattern and subcellularlocalization of IMB4, we generated IMB4g-YFP, inwhich the coding sequences of IMB4 fused with YFP-coding sequences were driven by the native promoterof IMB4, and introduced it into imb4-1 (Fig. 1). Thetransgene fully complemented the mutant defects ofimb4-1, including seed set and the ability to attractpollen tubes (Supplemental Fig. S5), indicating that YFPfusion did not interfere with its functionality. Consis-tent with the observation of its mutant phenotype inovules, IMB4 was expressed in all stages of ovule de-velopment (Fig. 5, A–D). YFP signals were detected inall cells during ovule primordial formation (Fig. 5A). Atstage 2-III, when MMC were formed, IMB4 was detec-ted in both outer and inner integument cells but wasabsent in MMC (Fig. 5B). At stage 3-I, when FM wasestablished, IMB4 was also detected in outer andinner integuments but not in FM (Fig. 5C). In matureovules, IMB4 was expressed in all sporophytic cells ofovules but not in the embryo sacs (Fig. 5D). Consistentwith the pleiotropic defects of the imb4 mutants(Supplemental Fig. S1), IMB4was detected in all tissuesby reverse transcription-quantitative PCRs (RT-qPCRs;Supplemental Fig. S6). YFP signals were detected inboth the cytoplasm and the nucleus in ovules and roots

Figure 4. Distribution of auxin maximumand PIN1 is compromised by IMB4 loss-of-function. A to H, CLSM of DR5:GFP;wildtype (WT; A to D) or DR5:GFP;imb4-1 (E toH) at stage 2-III (A, E), 3-I (B, F), 3-III (C, G),or 3-V (D, H). I to L, CLSM of PIN1:GFP(I, K) or PIN1:GFP;imb4-1 (J, L) at stage 2-I(I, J) or 3-I (K, L). Arrowheads point at DR5:GFP signals. Arrows point at funiculus (f).Dotted lines highlight nucellus (A, B) or de-veloping embryo sacs (C, D). dm, Degener-ating megaspores; oi, outer integument.Cells were stained with Lysotracker red(magenta). Bars = 10 mm.

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(Fig. 5, A–E), an observation in accordwith its role as animportin.

To exclude the possibility that compromised vege-tative growth of imb4 (Supplemental Fig. S1) resulted inovular defects, we expressed IMB4 specifically in outerinteguments of ovules (Supplemental Fig. S7) by usingthe promoter of INO (Wang et al., 2016). Indeed, theexpression of IMB4 in outer integuments was sufficientin complementing the ovular defects of the imb4 mu-tants (Supplemental Fig. S7). This result strongly sup-ported a specific role of IMB4 in ovules, in addition toits roles during vegetative development.

IMB4 Interacts with GIF1/AN3

Because importins shuttle specific cargo proteinsbetween the cytoplasm and the nucleus (Merkle, 2011;Tamura and Hara-Nishimura, 2014), we reasonedthat the defects of imb4were because of mis-localizationof specific IMB4-cargos. Although a classic role ofimportin beta is to facilitate cargo transport indirectly,there were cases when importin beta directly mediatesnucleocytoplasmic transport (Harel and Forbes, 2004;Tamura and Hara-Nishimura, 2014; Zhang et al.,2017). A study of high-throughput mass spectrometryreported that a transcriptional coactivator GIF1/ANGUSTIFOLIA3 (AN3) may interact with importinbs (Vercruyssen et al., 2014). Interestingly, a recent re-port showed that functional loss of Arabidopsis GIF1

and its two homologs GIF2, GIF3 (Kim et al., 2003; Kimand Kende, 2004; Omidbakhshfard et al., 2015) affectsfertility (Lee et al., 2014, 2018).

To test whether IMB4 interacted with GIFs directly,we applied bimolecular fluorescence complementa-tion (BiFC) and yeast two-hybrid (Y2H) assays. IMB4interacted with all three GIFs and most strongly withGIF1/AN3 (Fig. 6, A-C), suggesting that GIFs arecargos of IMB4.

Efficient Nucleus Import of GIF1/AN3 Requires IMB4

To examine whether nucleocytoplasmic transportof GIF1/AN3 depended on IMB4, we adopted twoapproaches. First, we introduced a GFP-GIF1/AN3translational fusion into imb4-1 and analyzed its sub-cellular distribution in stable transgenic plants. Byconfocal laser scanning microscopy (CLSM), we detec-ted strong nuclear signals of GFP-GIF1/AN3 in wild-type ovules but enhanced cytoplasmic signals in imb4-1ovules (Fig. 7A). Quantitative analysis supported asignificant difference on the ratio of nucleus-associatedversus cytoplasmic-associated GFP-GIF1/AN3 distri-bution between wild type and imb4-1 (Fig. 7C). Wealso examined the nucleus-cytoplasmic distributionof GIF1 in root epidermal cells. The results were con-sistent such that the nuclear accumulation of GIF1 wasreduced in imb4-1 (Fig. 7, B and D). Second, we per-formed cell fractionation on inflorescences from the

Figure 5. IMB4 is expressed constitutivelyduring ovule development and protein local-izes at both cytoplasm and the nucleus. A to D,CLSM of IMB4g-YFP imb4-1 ovule at develop-mental stage 1-II (A), 2-III (B), 3-I (C), or 3-V (D).D, inset is a stage 3-V ovule at a different angleshowing the embryo sac (illustrated by a dottedline). F, funiculus; fm, functional megaspore; ii,inner integument; mmc, megaspore mothercell; oi, outer integument. E, CLSM of IMB4g-YFP imb4-1 root at 4 DAG. Lysotracker red (A toD) or FM4-64 (E) in magenta was used to showcell silhouette. Bars = 10 mm.

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ProUBQ10:GFP-GIF1/AN3 or ProUBQ10:GFP-GIF1/AN3;imb4-1 plants. The successful separation of nucleiand cytoplasmwas demonstrated by using antihistone3 (H3) and anticytosolic Fru-1,6-bisphosphatase (cFBPase;Fig. 7E). Indeed, the signals of anti-GFP in the nucleusfraction of imb4-1were substantially reduced comparedwith that in the wild type (Fig. 7E). Thus, cell fraction-ation assays also support cytoplasmic retention ofGIF1/AN3 in imb4-1. Interestingly, we detected a sizeincrease in GIF1 in the nuclear fraction but not in thecytoplasmic or total fraction (Fig. 7E), implying thatGIF1 might be modified posttranslationally in the nu-cleus and de-modified in the cytoplasm.To further confirm that GIFs were cargos of IMB4

during ovule development, we examined the gif1;gif2;gif3 triple mutant based on the rationale that functionalloss ofGIFswould display ovule defects similar to thosein imb4-1. Although the gif1;gif2;gif3 triple mutant wasreported to be defective in both male and female re-productive development, integument growth duringvarious developmental stages was not closely exam-ined (Lee et al., 2014). Because we could not obtain thehomozygous triple mutant probably due to its severegrowth defects, we examined ovule development of thegif1;gif2;gif3/+ plants by CLSM of optical sections overdifferent developmental stages. Indeed, the gif1;gif2;gif3/+ mutant showed integument growth defectssimilar to those in imb4-1, albeit more pronounced(Supplemental Fig. S8). At stage 3-I, outer integumentsof the triple mutant developed slow and symmetric; atstage 3-III to 3-V, outer integument cells of the triple

mutant proliferated only slightly; at maturation, i.e.stage 3-VI and above, outer integuments of the triplemutant could not enclose inner integuments, and noembryo sac structure could be detected in these ovules(Supplemental Fig. S8). On the other hand, over-expressing GIF1 in imb4-1 partially restored its fertility(Fig. 8). These results suggested genetic epistasis be-tween IMB4 and GIF1 in ovule development.Because the imb4 mutants also displayed defects in

vegetative tissues, such as reduced primary roots anddwarfism (Supplemental Fig. S1), we wonderedwhether those defects were also because of reducednuclear accumulation of GIF1. By examining ProUBQ10:GFP-GIF1;imb4-1 plants, we determined that leafdevelopment, but not primary root growth or stemelongation, in imb4-1 was partially rescued by over-expressing GIF1 (Supplemental Fig. S9). Thus, the re-duced nuclear accumulation of GIF1 explains some butnot all developmental defects of imb4-1.

DISCUSSION

Although many studies on NPC components andimportins have suggested the importance of nucleocy-toplasmic transport in plant reproduction (Blanvillainet al., 2008; Tamura and Hara-Nishimura, 2014; Borucet al., 2015; Cui et al., 2016), no mechanistic insightshave been obtained. We show here that ArabidopsisIMB4, an importin b, is critical for ovule developmentthrough mediating the nucleocytoplasmic transport of

Figure 6. IMB4 interacts with the GIF1 familyproteins. A, CLSM of BiFC assays showing theinteraction between IMB4 and the GIF1 familyproteins. Magenta signals indicate the nuclear-localized U1-70K-mCherry. The bottom showsmerged images of the YFP, RFP, and transmis-sion channels. B, Quantification of BiFC intensity.a.u., Arbitrary fluorescence units. Results aremeans 6 SD. Sixteen to twenty-five cells weremeasured. No signals were detected for the neg-ative controls. C, Representative yeast two-hybridassays. Selection of interaction was performed onYSD medium lacking Trp (-W), Leu (-L), and Ade(-A), together with X-a-Gal. Bars = 50 mm.

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GIFs. The imb4 mutants showed reduced seed set onlyas the female parents during crosses (Fig. 1), suggestingthat the reduced fertility of imb4 mutants was becauseof female sporophytic defects. Indeed, optical sectionsduring ovule development showed that integumentgrowth in imb4 mutants was compromised (Fig. 3). Asin most mutants defective in integument growth(Villanueva et al., 1999; McAbee et al., 2006; Chevalieret al., 2011; Vaddepalli et al., 2011; Kelley et al., 2012;Wang et al., 2016), embryo sac development in imb4was impaired because of defects in the sporophytic

integuments. It is supported by three lines of evidence:(1) female transmission of imb4 was not affected(Supplemental Table S1); (2) IMB4was not expressed inFM or embryo sacs (Fig. 5); (3) heterozygous imb4plants showed full seed set (Fig. 1). Ovule developmentdefects in imb4 were not full penetrant (Fig. 1). It islikely that other importin bs play functionally redun-dant roles (Tamura and Hara-Nishimura, 2014). In-deed, functional loss of GIFs resulted in a much severedefect than that of IMB4 (Supplemental Fig. S7), sug-gesting redundancy by other importins.

Figure 7. Nucleocytoplasmic transport ofGIF1/AN3 requires IMB4. A and B, CLSM ofmature ovules (A) or root epidermal cells (B)from ProUBQ10:GFP-GIF1;wild type (WT) orProUBQ10:GFP-GIF1;imb4-1 stained with DAPI.Overlays of fluorescent (G for GFP, D for DAPI)and transmission (T) images are shown at theright. C and D, Quantification of intensity ratiobetween nuclear- (Nuc) and cytoplasmic- (Cyt)associated signals in mature ovules (C) or inroot epidermal cells (D) from ProUBQ10:GFP-GIF1;wild type or ProUBQ10:GFP-GIF1;imb4-1.Results are means 6 SD (n = 30). Asterisk indi-cates significant difference (t test, P , 0.05). E,Cell fractionation and Western blot analysis.Inflorescences from ProUBQ10:GFP-GIF1;wildtype or ProUBQ10:GFP-GIF1;imb4-1 were usedfor protein extraction, followed by subcellularfractionation and Western blotting. C, cyto-plasm; N, nucleus; T, total protein extract.Histone H3 and cFBPase were used for thenuclei and cytoplasmic markers, respectively.Numbers at the bottom are the values of GFP-GIF1 in the nuclear fraction (N) or cytoplasmicfraction (C) relative to that in total proteinfraction (T), which is set to 1.0. Main inflores-cences from 30 plants of each genotype werecollected for protein extraction used in oneexperiment. Results shown are representative ofthree biological replicates. Bars = 10 mm.

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According to classic thoughts, the role of importinbeta in nucleocytoplasmic transport is indirect, throughinteraction with the NPC and RAN-GTP (Harel andForbes, 2004; Merkle, 2011; Tamura and Hara-Nishimura, 2014). However, we show here that IMB4directly interacts with GIFs and mediates the nucleo-cytoplasmic transport of GIF1 (Figs. 6 and 7). Usingboth fluorescence imaging and cell fractionation, wedemonstrated that nuclear accumulation of GIF1 re-quires IMB4 (Fig. 7). In addition, functional loss of GIFsresulted in similar ovule developmental defects to imb4(Supplemental Fig. S8). All these results supported adirect role of IMB4 in the nucleocytoplasmic transportof GIF1.As transcriptional coactivators, GIFs function

through other TFs, amongwhich nine GRFs are the bestcharacterized GIF interactors (Kim et al., 2003; Kim andKende, 2004; Kim and Tsukaya, 2015). As transcrip-tional coactivators (Kim and Kende, 2004), GRFs

participate in several developmental processes inplants such as leaf development and leaf primordialproliferation (Kim et al., 2003; Horiguchi et al., 2005).Although it is unclear whether GRFs mediate ovuledevelopment, overexpression of microRNA396, anupstream regulator of GRFs, affected carpel develop-ment and caused seed set reduction (Liang et al., 2014;Omidbakhshfard et al., 2015). It is thus likely that GRFsare interactors of GIFs during ovule development. Re-cently, it was reported that GIFs also interact withchromatin remodelers (Vercruyssen et al., 2014;Nelissen et al., 2015), suggesting that GIFs regulatechromatin dynamics for growth regulation.In addition to ovule development defects, imb4 mu-

tants also displayed pleiotropic defects (SupplementalFig. S1). Some defects might have resulted fromcompromised nuclear transport of GIFs such as leafsize because mutations at GIFs resulted in similardefects (Kim et al., 2003; Kim and Kende, 2004;Kawade et al., 2013; Vercruyssen et al., 2014;Omidbakhshfard et al., 2015; Ercoli et al., 2018).However, other phenotypes of imb4, such as dwarf-ism (Supplemental Fig. S1), are not reported formutants of GIFs, suggesting the presence of otherIMB4 cargos regulating these processes. Indeed,enhancing the expression of GIF1 failed to restoreprimary root growth or stem elongation in imb4-1 (Supplemental Fig. S9), hinting at the presence ofother IMB4 cargos whose defects in nucleocytoplas-mic shuttling caused pleiotropic growth defects inimb4-1. On the other hand, the gif1;gif2;gif3 triplemutant failed to develop anthers (Lee et al., 2014,2018), whereas imb4 has no anther developmentaldefects (Supplemental Fig. S2), indicating that otherimportins might mediate the nucleocytoplasmictransport of GIFs in tissues such as anthers.We also showed that auxin and CK responses were

altered in imb4-1 ovules (Fig. 4; Supplemental Fig. S4).Both auxin and CK are instrumental for ovule devel-opment (Benková et al., 2003; Bencivenga et al., 2012;Ceccato et al., 2013). Auxin responses were substan-tially reduced in imb4-1, which correlates with thedisrupted polar distribution of PIN1 (Fig. 4). The ab-normal auxin responses at the micropyle (Fig. 4) mightexplain defective embryo sac development in imb4-1 because auxin is critical for embryo sac patterningand gamete specification (Pagnussat et al., 2009). As areciprocal phytohormone to auxin during ovule de-velopment (Bencivenga et al., 2012; Ceccato et al.,2013), CK signals were enhanced correspondingly(Supplemental Fig. S4). Because there is no evidencesupporting a direct role of IMB4 on auxin andCK signaling, it is more likely that defective auxin andCK signaling in imb4-1 ovules is associated with ab-normal ovule development. Alternatively, GIFs maymediate transcriptional changes through their inter-actors CYTOKININ RESPONSE FACTOR2 and ARA-BIDOPSIS RESPONSE REGULATOR4 (Vercruyssenet al., 2014) to influence hormonal responses duringovule development.

Figure 8. Enhanced expression of GIF1 partially restores the fertility ofimb4-1. A, A representative silique from wild type (WT), imb4-1, twoProUBQ10:GFP-GIF1 transgenic plants, and two ProUBQ10:GFP-GIF1;imb4-1 transgenic plants that were generated by crossing the ProUBQ10:GFP-GIF1 transgenic lines with imb4-1. B, Quantification of seed setfrom designated genetic backgrounds. Results shown are means 6 SD.Twenty-four siliques were analyzed for each genotype. C, Quantitativereal-time PCRs showing the expression of GIF1 in designated geneticbackgrounds. Results are means 6 s.e.m. (SEs, n = 3). Means with dif-ferent letters in (B and C) are significantly different (one-way ANOVA,Tukey-Kramer test, P , 0.05). Bars = 1 mm.

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MATERIALS AND METHODS

Plant Growth and Transformation

The T-DNA insertion lines SALK_049564 (imb4-1) and SALK_078852 (imb4-2) were obtained from the Arabidopsis Biological Resource Center (The OhioState University, Columbus; http://www.Arabidopsis.org). Other materials,including ProLAT52:GUS (Li et al., 2013), DR5:GFP (Ulmasov et al., 1997), PIN1:GFP (Benková et al., 2003), TCS:YFP (Bencivenga et al., 2012), and gif1;gif2;gif3/+ (Lee et al., 2014), have been previously described. Columbia-0 was used as thewild type. Plant growth, transformation, and selection were as previously de-scribed (Zhou et al., 2013).

DNA Manipulation

All constructs were generated using the Gateway technology (Invitrogen)unless noted otherwise. Entry vectors were generated using pENTR/D/TOPO(Invitrogen). For plant-expressing vectors, ProIMB4 containing the upstream1200-bp sequences before the start codon of IMB4 was amplified with theprimer pair ZP3899/ZP3900. PCR fragments were digested with HindIII/SpeIand replaced the Pro35S of the destination vector Pro35S:GW-YFP (Karimi et al.,2002) to generate the destination vector ProIMB4:GW-YFP. The IMB4 genomicsequence containing 4963 bp from 59-untranslated region to the nucleotidebefore stop codon was amplified with the primer pair ZP3605/ZP3549 andintroduced into ProIMB4:GW-YFP to generate IMB4g-YFP. Coding sequences ofIMB4, GIF1, GIF2, and GIF3 were amplified with the primer pair ZP3504/ZP4475, ZP6904/ZP6907, ZP6671/ZP6672, and ZP6673/ZP6674, respectively.ProUBQ10:GFP-GIF1 was generated by combining the GIF1 entry vector andProUBQ10:GFP-GW (Zhang et al., 2015) in an LR reaction. ProINO:red fluores-cence protein (RFP)-IMB4 was generated by combining the IMG genomic entryvector and ProINO:RFP-GW (Wang et al., 2016) in an LR reaction.

For vectors used in Y2H assays, entry vectors for IMB4,GIF1,GIF2, andGIF3were used in LR reactions with the destination bait vector pDEST-GBKT7 or thedestination prey vector pDEST-GADT7 (Clontech) to generate expressionvectors pGBKT7-IMB4, pGADT7-GIF1, pGADT7-GIF2, and pGADT7-GIF3.For vectors used in BiFC assays, entry vectors were used in LR reactions withthe destination vector pSITE::cEYFP-C1 or pSITE-nEYFP-C1 (Martin et al.,2009) to generate pSITE-nEYFP-IMB4, pSITE::cEYFP-GIF1, pSITE::cEYFP-GIF2, or pSITE::cEYFP-GIF3.

All PCR amplifications were performed using Phusion TM hot-start high-fidelity DNA polymerase, at the annealing temperature and extension timesrecommended by the manufacturer (Finnzyme). All entry vectors were se-quenced, and sequences were analyzed using Vector NTI (Invitrogen). TheBioneer PCR purification kit and the Bioneer Spin miniprep kit were used forPCR product recovery and plasmid DNA extraction, respectively. All primersare listed in Supplemental Table S2.

RNA Extraction, RT-PCRs, and qPCRs

Genotyping PCRs of imb4-1 and imb4-2were performed using the followingprimers: ZP4342/ZP4343 and ZP3917/ZP3918 for the wild-type copy of IMB4,ZP1/ZP4342 for imb4-1,ZP1/ZP3918 for imb4-2. Genotyping PCRs of theIMB4g-YFP;imb4-1 were performed using the following primers: ZP3915/ZP4438 for the endogenous IMB4 and ZP3915/ZP1848 for the transgene. ACT2was amplified with the primer pair ZP16/ZP17.

For RT-qPCRs analyzing the expression pattern of IMB4 and for RT-PCRs,total RNAs were isolated from open flower, seedlings of 3 d after germination(DAG) and 7 DAG, leaves of 14 DAG, stems of 25 DAG, inflorescence, matureovules, and mature pollen using a Qiagen RNeasy plant mini kit according tothe manufacturer’s instructions. Oligo(dT)-primed cDNAs were synthesizedusing Superscript III reverse transcriptase with on-column DNase II digestion(Invitrogen).

Expression analysis of IMB4 by RT-qPCRs was performed with the Bio-RadCFX96 real-time system using SYBR Green real-time PCR master mix (Toyobo)as described (Zhou et al., 2013). The specific primers used for IMB4, ACT2, andGAPDHwere ZP4492/ZP4493, ZP313/ZP314, and ZP687/ZP688, respectively.All primers are listed in Supplemental Table S2.

Phenotypic Analysis

Pollen development by Alexander staining, 4’,6-diamino-phenylindole(DAPI) staining, SEM, examination of the pollen tube in vivo growth by

histochemical GUS staining of ProLAT52:GUS-pollinated pistils and aniline bluestaining were performed as previously described (Li et al., 2013). Whole-mountovule clearing and CLSM of ovules were performed as described (Wang et al.,2016). For each assay on ovules, including the ProLAT52:GUS pollination assay,aniline blue staining, whole-mount ovule clearing, and optical sectioning byCLSM, 10 pistils were analyzed. Results are shown either as means 6 SD, orshown as the number of given category/total number of ovules (n = 210 to 469).

Protein Interaction Assays

Y2H assays were performed as previously described (Park et al., 2014), withslight modifications. Briefly, different combinations of bait and prey vectorswere cotransformed into the Y2HGold yeast strain (Clontech). Protein-proteininteractions were determined based on the growth of transformants after 3 d onYSD-WLA containing 80 mg/l X-a-Gal. Agrobacterium infiltration of expressionvectors used in BiFC was performed as described, in which a P19 protein wasused to suppress gene silencing (Park et al., 2014). The nuclear-localized proteinU1-70K-mCherry has been previously described (Wang et al., 2012). Confocalimaging was performed 48 h after infiltration.

Cell Fractionation and Western Blot

Cell fraction was performed according to a previously described method(Wierzbicki et al., 2008), with slight modifications. Specifically, two grams ofinflorescences were ground into powder in liquid nitrogen, suspended in 20 mlof Honda buffer (20 mM HEPES-KOH [pH 7.4], 0.44 M Suc, 1.25% [v/v] ficoll,2.5% [w/v] Dextran T40, 10 mM MgCl2, 0.5% [v/v] Triton X-100, 5 mM dithi-othreitol, 1 mM phenylmethylsulfonyl fluoride, 1% [w/v] plant protease in-hibitors), and filtered through two layers of Miracloth and centrifuged at 5000 gfor 20 min. The supernatant was used as the cytosolic fraction. The pellets werewashed five times, each with 1 ml of Honda buffer. Total nuclear protein wasobtained by adding 3 volumes of 1% (w/v) SDS directly to the nucleus pellet,followed by boiling at 95°C for 10 min. Equal volumes of each fraction weremixed with loading buffer, boiled, gel-separated, and subjected to protein gelblot analysis. Immunolabeling of GFP-GIF1/AN3was performed using an anti-GFP antibody (Beyotime, 1:1000 dilution). Anti-H3 (Beyotime, 1:1000 dilution)and AnticFBPase (Agrisera, 1:5000 dilution) were used as the nuclear and cy-toplasmic marker, respectively. Quantification of total proteins was performedusing Gel Image System 4.1.2 (www.bio-tanon.com.cn).

Fluorescence Microscopy and Pharmacological Treatment

Lysotracker red staining or FM4-64 staining was used to show cell silhou-ettes as previously described (Wang et al., 2016). CLSM of fluorescence mate-rials was performed with an LSM 880 (Zeiss) with the excitation and emissionwavelengths set to 488 nm/505–550 nm for YFP and GFP signals and 561 nm/600 nm for RFP signals. For the quantification of fluorescence intensity betweennuclear and cytoplasmic fractions in ovules, a region of interest of the same sizewas defined either in the nucleus or in cytoplasm within an outer integumentcell. For the quantification of fluorescence intensity between nuclear and cy-toplasmic fractions in roots, a region of interest of the same size was defined ineither the nucleus or the cytoplasm within a root epidermal cell. The ratio offluorescence intensity between the nuclear and cytoplasmic reactive oxygenspecies (Nucleus/Cytoplasm) was calculated using ImageJ (http://rsbweb.nih.gov/ij/).

Accession Numbers

Arabidopsis Genome Initiative locus identifiers for the genes mentioned inthis article are as follows: At4g27640 for IMB4; At5g28640 for GIF1/AN3;At1g01160 for GIF2; At4g00850 for GIF3; At1g23420 for INO.

SUPPLEMENTAL DATA

The following materials are available in the online version of this article.

Supplemental Figure S1. Functional loss of IMB4 causes pleiotropic devel-opmental defects.

Supplemental Figure S2. Functional loss of IMB4 does not affect pollendevelopment.

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Supplemental Figure S3. Integument growth is compromised by func-tional loss of IMB4.

Supplemental Figure S4. CK signaling during ovular development is af-fected by functional loss of IMB4.

Supplemental Figure S5. Phenotypic defects in IMB4 loss-of-function aresuppressed by IMB4g-YFP.

Supplemental Figure S6. IMB4 is constitutively expressed.

Supplemental Figure S7. Specific expression of IMB4 in outer integumentssuppresses ovular defects of imb4-1.

Supplemental Figure S8. GIFs are critical for ovule development.

Supplemental Figure S9. Leaf development, but not primary root growthor stem elongation, is partially rescued by ProUBQ10:GFP-GIF1 in imb4-1.

Supplemental Table S1. IMB4 loss-of-function does not compromise maleor female transmission.

Supplemental Table S2. Oligos used in this study.

ACKNOWLEDGMENTS

We thank Prof. Xian Sheng Zhang for DR5:GFP, PIN1:GFP, and TCS:YFP.We thank Prof. Jeong Hoe Kim for the kind gift of the gif1;gif2;gif3 mutant.

Received September 12, 2018; accepted January 8, 2019; published January 18,2019.

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1092 Plant Physiol. Vol. 179, 2019

Importin b4 Regulates Ovule Development

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