A Robust Auxin Response Network Controls Embryo andSuspensor Development through a Basic Helix Loop HelixTranscriptional Module[OPEN]

Tatyana Radoeva,a,1 Annemarie S. Lokerse,a,1,2 Cristina I. Llavata-Peris,a Jos R. Wendrich,a Daoquan Xiang,b

Che-Yang Liao,a Lieke Vlaar,a Mark Boekschoten,c,d Guido Hooiveld,c Raju Datla,b and Dolf Weijersa,3

a Laboratory of Biochemistry, Wageningen University & Research, Stippeneng 4, 6708WE Wageningen, the Netherlandsb Plant Biotechnology Institute, National Research Council, 110 Gymnasium Place, Saskatoon, Saskatchewan, Canada S7N 0W9cDivision of Human Nutrition, Wageningen University & Research, Stippeneng 4, 6708WE Wageningen, the Netherlandsd Top Institute Food & Nutrition, Nieuwe Kanaal 9A, 6709 PA Wageningen, the Netherlands

ORCID IDs: 0000-0002-7742-7817 (T.R.); 0000-0002-0986-2219 (C.I.L.-P.); 0000-0002-6198-3763 (J.R.W.); 0000-0002-2139-0795(M.B.); 0000-0003-1954-3542 (G.H.); 0000-0003-0790-5569 (R.D.); 0000-0003-4378-141X (D.W.)

Land plants reproduce sexually by developing an embryo from a fertilized egg cell. However, embryos can also be formedfrom other cell types in many plant species. Thus, a key question is how embryo identity in plants is controlled, and how thisprocess is modified during nonzygotic embryogenesis. The Arabidopsis (Arabidopsis thaliana) zygote divides to produce anembryonic lineage and an extra-embryonic suspensor. Yet, normally quiescent suspensor cells can develop a second embryowhen the initial embryo is damaged, or when response to the signaling molecule auxin is locally blocked. Here we used auxin-dependent suspensor embryogenesis as a model to determine transcriptome changes during embryonic reprogramming. Wefound that reprogramming is complex and accompanied by large transcriptomic changes before anatomical changes. Thisanalysis revealed a strong enrichment for genes encoding components of auxin homeostasis and response amongmisregulated genes. Strikingly, deregulation among multiple auxin-related gene families converged upon the re-establishment of cellular auxin levels or response. This finding points to a remarkable degree of feedback regulation to createresilience in the auxin response during embryo development. Starting from the transcriptome of auxin-deregulated embryos,we identified an auxin-dependent basic Helix Loop Helix transcription factor network that mediates the activity of thishormone in suppressing embryo development from the suspensor.

INTRODUCTION

In many land plants, including Arabidopsis (Arabidopsis thali-ana), zygotic embryogenesis begins with an asymmetric celldivision, generating two cells with distinct fates. The smallapical cell is the founder of the pro-embryo andwill formmost ofthe plant body. The larger basal cell divides several times andgives rise to the suspensor, a filamentous support structure ofwhich the topmost cell generates part of the seedling root (Lauxand Jurgens, 1997; Mayer and Jürgens, 1998). Hence, thesuspensor is largely an extra-embryonic, yet zygote-derivedstructure. In contrast with the pro-embryo, the suspensor isalready fully developed at the globular stage and plays animportant role in embryo development (Schwartz et al., 1997).The suspensor provides mechanistic and nutritional supportrequired for the growing embryo as well as a connection

between the pro-embryo and the maternal endosperm(Raghavan, 2006).Despite their mitotic quiescence under normal conditions,

suspensor cells in several species have the potential to gen-erate a new embryo (Lakshmanan and Ambegaokar, 1984). InArabidopsis, mutations that impair growth of the pro-embryo(raspberry [rsy], suspensor [sus], and twin [twn]) can causesuspensor proliferation, eventually recapitulating embryo-genesis and generating a new pro-embryo (Schwartz et al.,1994; Vernon and Meinke, 1994). Suspensor-derived em-bryogenesis can also be induced by experimental ablation ofthe pro-embryo through radiation or chemicals, or throughgenetic ablation (Haccius, 1955; Weijers et al., 2003) or laserirradiation (Gooh et al., 2015; Liu et al., 2015). These ob-servations reveal the developmental potential of the suspensorto undergo embryonic transformation and proves that its po-tential is limited by normal growth of the embryo proper. Al-though regulatory mechanisms underlying this switch in fateare largely unknown, we reported that this process involves thesignaling molecule auxin (Rademacher et al., 2012). Compo-nents of the auxin response are expressed in suspensor cells(Rademacher et al., 2011, 2012), and when these are inhibited,suspensor cells proliferate, express embryo markers, anddevelop a second embryo, which can ultimately give rise totwin-like seedlings (Rademacher et al., 2012). At present, it isunclear how direct the involvement of auxin response in

1 These authors contributed equally to this work.2 Current address: Rijk Zwaan Breeding B.V. Eerste Kruisweg 9, Fijnaart,The Netherlands.3 Address correspondence to dolf.weijers@wur.nl.The author responsible for distribution of materials integral to the findingspresented in this article in accordance with the policy described in theInstructions for Authors (www.plantcell.org) is: Dolf Weijers (dolf.weijers@wur.nl).[OPEN]Articles can be viewed without a subscription.www.plantcell.org/cgi/doi/10.1105/tpc.18.00518

The Plant Cell, Vol. 31: 52–67, January 2019, www.plantcell.org ã 2018 ASPB.

embryonic fate conversion is, but it provides a good entrypoint into studying the regulatory mechanisms underly-ing suspensor-to-embryo transformation (abbreviated S>Ehenceforth).

The auxin response is mediated by transcription factors of theAUXIN RESPONSE FACTOR (ARF) family, which are inhibited byinteracting Auxin/indole-3-Acetic Acid (Aux/IAA) proteins. In thepresence of auxin, Aux/IAA proteins are ubiquitinated by theSKP1-CULLIN1-F-BOX -TRANSPORT INHIBITORRESISTANT1/AUXIN F-BOX auxin receptor complex (Wang and Estelle, 2014)and marked for degradation. This releases ARFs to regulate thetranscriptionof primary target genes (LokerseandWeijers, 2009;Wang and Estelle, 2014). Although several target genes medi-ating the auxin response during embryonic root initiation(Schlereth et al., 2010; Crawford et al., 2015), lateral root de-velopment (Okushima et al., 2007; De Rybel et al., 2010), andflower development (Zhao et al., 2010; Yamaguchi et al., 2013)have been isolated, their role inmaintaining suspensor identity isnot yet clear. Here, we used auxin-dependent, suspensor-derived embryogenesis (Radoeva and Weijers, 2014) to iden-tify molecular components that mediate this important cell fatetransformation.

Through genome-wide transcriptional profiling upon local in-hibition of the auxin response, we discovered a convergentmisregulation of 39 genes involved in auxin homeostasis andresponse that collectively re-establish auxin activity. Following anexpression pattern analysis during embryogenesis of a selectedsubset of differentially expressed genes, we identified a set ofbasic Helix Loop Helix (bHLH) genes that is regulated duringsuspensor-derived embryogenesis. Previously, bHLH transcrip-tion factors have been identified as direct ARF target genes(Schlereth et al., 2010; De Rybel et al., 2013;). Our work suggestsa role for bHLH49 as a key mediator in controlling the de-velopmental potential of the suspensor.

RESULTS

Transcriptional Analysis of Suspensor Reprogramming

Expression of the mutated, stabilized transcriptional auxin re-sponse inhibitor protein iaa12/bodenlos (bdl) in suspensor cellsefficiently induces a switch from extra-embryonic to embryonicidentity (Rademacher et al., 2012). We used this predictable,uniform response to identify genes whose expression changesduring this fate transition. IAA12/BDLprotein isnormallydegradedin response to auxin (Dharmasiri et al., 2005), but a Pro74Sermutation in the BDL protein prevents degradation and leadsto accumulation of this ARF inhibitor (Hamann et al., 2002;Dharmasiri et al., 2005). We expressed mutant bdl protein usingthe two-component Galactose-induced gene 4/Upstream Acti-vation Sequence (GAL4/UAS) system (Weijers et al., 2006). TheGAL4 driver line M0171 is active in suspensor cells (Figure 1)until the heart stage (Figure 1A), after which GAL4 expressionexpands to include cells in the pro-embryo (Rademacher et al.,2012; Radoeva et al., 2016). By crossing homozygous M0171and UAS-bdl plants, suspensor proliferation could be inducedin most embryos at the heart stage (83%; n = 126). For whole-genome transcriptomic analysis, a time point after pollinationshould be selected, which is after the onset of M0171 expressionand before the appearance of phenotypic abnormalities. Wefound that by 72 h, about one-third of suspensors showeda first aberrant division (Figures 1A and 1B; 31%; n = 149) andselected this as the first time point for transcriptomic profiling.Wealso included a 96-h time point because by this time, mostsuspensors vigorously proliferated (Figures 1C and 1D; 86%;n = 109).M0171 plants were pollinated withUAS-bdl pollen (M0171>>bdl),

embryos were manually dissected after 72 and 96 h, and 300to 400 individuals per biological replicate were pooled for

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RNA extraction. In parallel, M0171 plants were pollinated withColumbia-0 (Col-0) wild-type pollen to serve as isogenic wild-type controls (M0171>>Col-0) in whole-genome transcriptprofiling. After statistical analysis, we identified 621 and 349genes that were robustly up- or downregulated in M0171>>bdl72 h embryos compared with the control, respectively (>2-foldchange in gene expression; significance at false discovery rate[FDR]<0.05; Figure 1E;SupplementalDataSet 1). Bycontrast, inM0171>>bdl 96-h embryos, 3421 genes were upregulated and2144 genes were downregulated (Figure 1E). Because thenumber ofmisregulated genes in theM0171>>bdl 96-h embryosequals one-quarter of the Arabidopsis genome and probablyincludes secondary and tertiary transcriptional responses, ouranalysis was focused on the 72-h M0171>>bdl transcriptome.

Due to the large number of misregulated genes, we did notidentify obvious enrichment of functional categories or knownregulators in Gene Ontology analysis. Nonetheless, this tran-scriptome analysis revealed massive genome-wide transcriptionalreprogramming associated with the induction of proliferation and

embryogenesis in the suspensor well before its morphologicalmanifestation.

Convergent Regulation of Auxin Homeostasis

Initial inspection of the M0171>>bdl transcriptome confirmed anexpected (10.6-fold) upregulation of BDL/IAA12. ConverselyIAA30, whose suspensor-specific expression (at earlier stages ofembryogenesis) is lost in the pARF13:iaa10 background(Rademacher et al., 2012), is 3.5-fold downregulated, confirmingthe validity of the M0171>>bdl data set. Further analysis revealedthat many other genes involved in auxin homeostasis and sig-naling were misregulated because of the targeted inhibition of theauxin response in the suspensor. In addition to BDL and IAA30,several other members of the Aux/IAA family (IAA19, 20, 26) werealso strongly downregulated in the M0171>>bdl data set (Fig-ure 2). A strong enrichment for genes encoding core factors andregulators of auxin biosynthesis, transport, (de)conjugationpathways, and response was observed as well (Figure 2A).

Figure 1. Selection of Time Points for Transcriptional Analysis of M0171>>bdl Embryos.

(A) to (D) Embryos from crosses between M0171 and wild type [(A) and (C)] or M0171 and UAS-bdl [(B) and (D)] prepared 72 h [(A) and (B)] or 96 h afterpollination [(C) and (D)]. GFP expression reflects the activity of theM0171 enhancer trap,which drives the expression of GAL4:VP16, which in turn activatesthe linkedUAS-erGFP gene. Images show overlay of GFP and cell wall (counterstained with Renaissance RS2200-magenta) signals. Scale bar represents10 mm in all panels. Arrowheads in (B) and (D) indicate aberrant cell divisions in suspensor cells.(E)UpSet plot showing overlap betweendifferentially expressedgenes (up- or downregulated relative towild type) inM0171>>bdl embryos at 72 h and96hafter pollination. Numbers indicate the number of common genes in each comparison.

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Figure 2. Convergent Regulation of Auxin Homeostasis Genes.

(A) Misregulated auxin homeostasis and core signaling genes in the M0171>>bdl data sets (in fold change M0171>>bdl/M0171>>wild type). Genes aregrouped per category [auxin biosynthesis, (de)conjugation, transport, and signaling].

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Interestingly, the expected effect of expression changes of singlegenes would be an increase in intracellular auxin levels. Specifi-cally, family members of three of the four Trp-dependent auxinbiosynthesis pathways (YUCCA1 [YUC1], 4, 10; TRYPTOPHANAMINOTRANSFERASE OF ARABIDOPSIS1; NITRILASE2) werestrongly upregulated in the M0171>>bdl data set (Figure 2A),suggesting an increase in auxin concentration as a result of theinhibited auxin response. Furthermore, fourGRETCHENHAGEN3(GH3) family members (GH3.3, 3.4, 3.5, 3.17) encoding auxin-conjugating enzymes were downregulated, whereas two genesthat encoding hydrolases involved in auxin deconjugation (IAA-amino acid hydrolase-IRL1-like 3 and ARABIDOPSIS THALIANAMETHYL ESTERASE16) were upregulated. The net effect of thelatter would also likely be elevated free auxin levels. This in-terrelationship between positive and negative misregulation isfurther toned up by the downregulation of 10 auxin transport-associated genes found in our data set (Figure 2A).

To address the spatial aspects of the apparent convergentregulation of auxin homeostasis and response upon targetedinhibition of the auxin response, we investigated the expressionpatterns of genes encoding the auxin biosynthesis enzymeYUC1,the Aux/IAA family member IAA30, and two auxin efflux carriers(PIN-FORMED1 [PIN1] andPIN4) in theM0171>>bdl background.Normally, YUC1 is expressed in the protoderm at the globularstageof embryogenesis, but inM0171>>bdl embryos, itwasmorebroadly expressed, also including the inner cells of the proembryo(Figures2Band2C). These results indicate that auxinbiosynthesisincreases after the perceived depletion of auxin, and they confirmthe upregulation of YUC1 in the M0171>>bdl data set. Duringembryogenesis, as previously described (Rademacher et al.,2012), IAA30 is expressed in the suspensor cells as well as thelower tier of the pro-embryo, which are known sites of the auxinresponse. InM0171>>bdlembryos, no IAA30expressioncouldbedetected (Figures 2D and 2E), which confirms that the tran-scriptional auxin response is indeed inhibited in these embryos.Similarly, the expression of the two auxin efflux carrier genesPIN1and PIN4, which is normally found in both the suspensor and thelower tier of the pro-embryo, was also lost from both embryonicexpression domains (Figures 2F and 2I). These results suggestthat an inhibited auxin response in the suspensor results in im-paired auxin transport as well as an impaired auxin responsethroughout the embryo.

Collectively, the misregulation of more than 10 gene familiesinvolved in auxin homeostasis occurs in suchaway that it appearsthat thesystem is responding to inhibitionof theauxin responsebytriggering increased auxin biosynthesis, decreased auxin trans-port, and altered auxin responsiveness (Figure 2J). Based on themisregulation of auxin homeostasis and transport genes, wepredicted that auxin levels should be higher in M0171>>bdl

embryos versus the wild type. We tested this prediction experi-mentally using the ratiometric auxin sensor R2D2 (Ratiometricversionof twoDomain II’s; Liao et al., 2015). This sensor combinesauxin-sensitive pRPS5A-driven DII-n3Venus and auxin-insensitive pRPS5A-driven mDII-ntdTomato (nuclear tandem di-meric Tomato) as an internal control (Figure 3; Liao et al., 2015).The mDII/DII ratio provides a measure of cellular auxin levels ir-respective ofwhether auxin triggers gene expression changes. Aspredicted, the R2D2-derived auxin levels strongly increased inM0171>>bdl embryos compared with wild type (Figures 3A, 3B,3G, and 3H). We next testedwhether the excess auxin leads to anincreased auxin response, which would be counterintuitive. Forthis,weused thedual auxin-responsive reporterDR5/DR5v2. Thisreporter harbors the classical DR5 and highly sensitive DR5v2reporters, eachdriving a different fluorescent protein, on the sametransgene locus (Liao et al., 2015). Normally, DR5v2 is expressedin the first suspensor cells at the globular stage of embryogenesisand isactivated in thecotyledon tipsand thevasculature lateronatthe heart stage (Figures 3C to 3F). Instead,DR5v2 expressionwascompletely absent from M0171>>bdl embryos up to the heartstage (Figures 3C to 3E and 3I to 3K), confirming that the phe-notypic embryos lacked an auxin response. Strikingly, later onduring embryogenesis, at the torpedo stage, DR5v2 expressionwas re-established and observed throughout the embryo (Figures3F and 3L). Thus, local suppression of the auxin response causessystemic changes in auxin homeostasis at many levels and in-volving many gene families. This systemic change inducesa strong increase in cellular auxin levels, but not in the auxin re-sponse. These results point to genetic wiring of the auxin networkaimed at high resilience to perturbation. The results also indicatethat inhibiting the auxin response in the context of the embryo hasstrong noncell autonomous effects.

Identification of Transcriptional Regulators duringSuspensor Reprogramming

Suspensor-specific inhibition of the auxin response inM0171>>bdl embryos induces transcriptional reprogrammingthat leads to systemic rewiring of auxin homeostasis, eventuallyleading to the proliferation of suspensors and the development ofembryo-like characteristics in proliferated suspensors. To ad-dresswhether the initiation of these embryo-like properties is alsodetectable at the transcriptional level,we surveyed the expressionofgenesknown topromote theacquisitionof embryonic identity inother systems (Radoeva and Weijers, 2014). Of these, two aredifferentially expressed in the M0171>>bdl data set. LEAFYCOTYLEDON1-LIKE, also known as NUCLEAR FACTOR-Y B6,encoding a subunit of the NUCLEAR FACTOR-Y transcriptionfactor complex, is able to induce embryo development in somatic

Figure 2. (continued).

(B) to (I)Expressionof promoter-n3GFP reporters forYUC1 [(B) and (C)], IAA30 [(D)and (E)],PIN1 [(F)and (G)], andPIN4 [(H)and (I)] inwild type [(B), (D), (G),(H)] andM0171>>bdl [(C), (E), (G), (I)] embryos;72hafterpollination.Cellwalls inall imagesarecounterstainedwithRenaissanceRS2200 (magentasignal).Scale bar represents 10 mm in all panels.(J)Schematic overview of convergent regulation of auxin homeostasis genes. Blue arrows represent upregulation in theM0171>>bdl data set and positiveeffect on IAA concentration or the auxin response; orange bars represent downregulation in the M0171>>bdl data set and negative effect on IAA con-centration or the auxin response.

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cells when overexpressed (Lotan et al., 1998; Kwong et al., 2003).LEAFY COTYLEDON1-LIKE is 2.5-fold upregulated in our dataset. Another factor implicated in somatic embryogenesis that is1.9-fold upregulated in our data set is FUSCA3, encoding a B3domain transcription factor that induces late embryo properties insomatic cells after ectopic expression (Gaj et al., 2005).Moreover,SHOOT MERISTEMLESS, an important embryo marker (Longet al., 1996) that is expressed in proliferating suspensor cells(Rademacher et al., 2012), is 2.3-fold upregulated in theM0171>>bdl data set, whereas the CLAVATA3 gene, which isexpressed in the shoot apicalmeristem (Fletcher et al., 1999), was45-fold upregulated. Hence, even before morphological changesare evident, the M0171>>bdl transcriptome reveals aspects ofreprogramming toward embryo identity.

To identify new components in the control of suspensor pro-liferation and/or the induction of embryo identity, we selected 68genes that were robustly misregulated in M0171>>bdl embryos(Supplemental Table). Selectionwasbasedon (1) the amplitude ofmisregulation (Fold-changeM0171>>bdl versusM0171>>Col-0),(2) functional annotation (transcription factors, signaling com-ponents), and (3) known auxin-responsiveness (using public mi-croarray data). All genes were at least twofold misregulated, and19geneswereamong the10%most stronglymisregulatedgenes.Twenty-two of the selected genes were identified as auxin-responsive in the Arabidopsis Hormone Database 2.0 (includingone Aux/IAA), and 33 encode transcription factors (Plant Tran-scription Factor Database v2.0; Zhang et al., 2011), 20 of whichbelong to auxin-responsive transcription factor families (Paponovet al., 2008).

We generated promoter-reporter lines for these 68 genes usinga sensitive nuclear-localized 3x green florescent protein (n3GFP)to assess their embryonic expression domains. We analyzed thepatterns of expression in T2generationwild-type embryos in up toeight independent primary transformants for all genes, but ex-pression in the embryo and/or suspensorwasonly detected for 40genes. The expression pattern of these 40 genes was further

confirmed in the T3 generation in at least two representative lines.Because our microarray analysis was aimed at identifying genesthat are misregulated as part of suspensor proliferation and/orembryo identity induction, we predict that, ideally, upregulatedgenes are normally expressed in embryo cells and downregulatedgenes in suspensor cells. Of the 40 genes that were expressedduring embryogenesis, 24 had broader expression domains, in-cluding both the pro-embryo and suspensor, which were in-consistent with our predictions (Figures 4H and 4J; SupplementalTable). The 16 remaining genes conformed to the aforementionedcriteria (Figure 4). Of these, eightwere upregulated and expressedin the pro-embryo (Figures 4A to 4F and 4M), whereas eight weredownregulated and expressed in the suspensor (Figures 4G, 4I,and 4K to 4M). Notably, this subset of validated genes containedfour genes encoding bHLH transcription factors (Figures 4D to 4Fand 4L). bHLHs are well-known regulators of cell identity inmulticellular organisms (Murre et al., 1994), including plants (Felleret al., 2011). Importantly, other bHLHs were previously shown tomediate auxin-dependent development (Chandler et al., 2009;Schlerethetal., 2010;DeRybelet al., 2013). Inparticular, thebHLHgenes TARGET OF MONOPTEROS5 (TMO5) and TMO7 are bothactivated by auxin in an ARF5-dependent manner and contributeto auxin-dependent vascular tissue and embryonic root de-velopment (Schlereth et al., 2010; De Rybel et al., 2013). Wetherefore focused our analysis on these genes as potential reg-ulators of suspensor proliferation.

bHLH Genes Are an Output of the EmbryonicAuxin Response

The bHLH genes identified here (bHLH49, 60, 63, and 100; Fig-ure 5) belong to two different clades, 12 and 25 (Figure 5A;Supplemental File). Although a postembryonic function has beendescribed for bHLH63, also known as CRYPTOCHROME-INTERACTINGbHLH1 (Liu et al., 2008) andbHLH100 (Wanget al.,2007; Sivitz et al., 2012; Andriankaja et al., 2014), no embryonic

Figure 3. Auxin Levels and Response upon Inhibition of the Suspensor-Specific Response.

(A), (B), (G), and (H)Ratio of ntdTomato / n3xVenus fluorescence of theR2D2 reporter in wild type [(A) and (B)] andM0171>>bdl embryos [(G) and (H)]. Thisratio is displayed as false color according to scale in the bottom, reflecting low to high auxin levels.(C) to (F) and (I) to (L) Expression of DR5-n3GFP/DR5v2-ntdTomato reporter in wild type [(C) to (F)] and M0171>>bdl embryos [(I) to (L)]. Cell walls in allimages are counterstained with Renaissance RS2200 (gray signal). Scale bar represents 10 mm in all panels.

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function has been reported for any of these genes. bHLH49 wasexpressed in the basal tier of the embryo, and weak expressionwas also detected in the suspensor (Figure 4D). bHLH60 wasexpressed in theouter cells of thepro-embryo, andweak transientexpression was detected in the inner cells of the pro-embryo andsuspensor (Figure 4E). bHLH63 had a very specific expressionpattern, limited to theprotoderm in the junctionbetween theapicaland basal tier of the pro-embryo (Figure 4F). These largely pro-embryo–enriched expression patterns are consistent with theirupregulation in the microarray data set. Consistent with itsdownregulation in theM0171>>bdlmicroarray,bHLH100showedsuspensor-specific expression until the globular stage, afterwhich the domain extended to protodermal cells in the basal tier(Figure 4L).

Although some bHLH proteins act cell autonomously in the cellswhere their underlying gene is transcribed (e.g., TMO5; Schlerethetal.,2010),othersactnoncellautonomouslybymovingtoadjacentcells (e.g., TMO7; Schlereth et al., 2010; UPBEAT1; Tsukagoshiet al., 2010). To determine whether the bHLH proteins encoded bythe genes identified here are mobile, we generated translationalfusions of genomic fragments fused to sensitive sYFP protein.Consistentwith their function as transcription factors, all four bHLHproteins localized to the nucleus. Protein localization domainsexactly matched the promoter expression domains (SupplementalFigure 1), demonstrating that these proteins likely do notmove andthat protein accumulation is transcriptionally controlled.These bHLH genes were identified based on their mis-

expression upon inhibition of the auxin response and were

Figure 4. Expression Patterns of Genes Misregulated in M0171>>bdl Embryos.

(A) to (L)Expression of promoter-n3GFP reporters forYUC1 (A),MYB82 (B),WRI1 (C),bHLH49 (D),bHLH60 (E),bHLH63 (F),PIN4 (G),bHLH153 (H), LBD4(I), KMD1 (J), AT4G18740 (K), and bHLH100 (L). All images show expression in the globular stage, except (C), which shows expression in the heart stage.Images (A) to (F) show expression of genes upregulated, whereas images (G) to (L) show expression of genes downregulated in theM0171>>bdl data set.Cell walls in all images are counterstained with Renaissance RS 2200 (magenta signal). Scale bar represents 10 mm in all panels.(M) Differential expression (in fold change M0171>>bdl/M0171>>wild type) of 16 genes conforming to the selection criteria discussed in the text.

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expressed in the relevant cell types within the embryo. We nextexaminedwhether the bHLHgeneswere indeed rapidly regulatedby auxin. We first tested the effect of exogenous auxin on tran-script levels in seedling roots. bHLH60, 63, and 100 were upre-gulated after 2 h of auxin treatment (Figure 5B). By contrast,bHLH49 was downregulated, suggesting that all four bHLHs areindeed regulated by auxin. In addition, to test whether this auxin-dependent bHLH regulation is direct, we performed a 2 h auxintreatment in the presence of cycloheximide (CHX). CHX is aninhibitor of protein biosynthesis and in its presence, only directtranscriptional regulation should occur (Franco et al., 1990).Auxin-dependent repression of bHLH49 expression was stillobserved in the presence of CHX, which strongly suggests thatbHLH49 is directly regulated by auxin (Figure 5B). By contrast, theactivation of bHLH60, 63, and 100 was suppressed by CHX,suggesting that the auxin-induced regulation of these genes doesnot represent a direct transcriptional response (Figure 5B).

The auxin-dependent expressionof these four bHLHgenes andtheir misregulation inM0171>>bdl embryos suggested that thesebHLH proteins might play a role in actual cell fate transformation.To determine whether this is indeed the case, we analyzed theexpression of all four bHLHs in M0171>>bdl embryos. The ex-pression of the downregulated genes in M0171>>bdl can only beaffected within their normal expression domain. Consistent withits 2.0-fold downregulation in the microarray, the expression ofbHLH100 was absent from almost all excessively dividing sus-pensor cells (Figures 5F and 5J), suggesting that these cells havelost their original identity. Upregulation in the M0171>>bdl tran-scriptome could either reflect enhanced or ectopic expression.Consistent with the transcriptome data, the expression ofbHLH49, bHLH60, and bHLH63 was strongly enhanced inM0171>>bdl embryos (Figures 5G to 5I). Strikingly, however, theirexpression was not activated in the suspensor, but it was insteadenhanced in the pro-embryo, notably in the L1 layer. Therefore, in

Figure 5. Auxin-Dependent Expression of bHLH Genes.

(A) Phylogenetic tree of Arabidopsis bHLH proteins, indicating the divergent positions of the TMO5, TMO7, and LHW clades relative to the misregulatedbHLH genes. The misregulation of genes in M0171>>bdl embryos is indicated in the top panels.(B)Relative expression levelsofbHLH49,60,63, and100 in roots upon treatmentwith 1mM2,4-D, 10mMCHXorboth, for 2 h. Expression levels in untreatedwild-type (ormock treated samples) were set to 1. Error bars indicate SE; t test: *P < 0.05, **P < 0.001. Reactionswere done in triplicate, with three biologicalreplicates (representing separate experiments).(C) to (J) Expressionofpromoter-nVenus reporters forbHLH49 [(C)and (G)],bHLH60 [(D)and (H)],bHLH63 [(E)and (I)], andbHLH100 [(F)and (J)] inwild type[(C) to (F)] andM0171>>bdl embryos [(G) to (J)]. Insets in (C) to (F) show the Venus signal at increased brightness. Magenta counterstaining in all images isRenaissance RS2200. Scale bar indicates 10 mm in all panels.

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contrast with the simple inference that suppression of auxin re-sponse in suspensor cells cell autonomously de-regulatesbHLH49, 60, and 63, their misregulation in M0171>>bdl em-bryos appears to be noncell autonomous and likely the conse-quenceofsystemiceffectsonauxinhomeostasisand response. Inthe case of bHLH60 and 63, the regulatory effect of auxin appearstobe indirect, becauseno regulationwasobserved in roots treatedwith auxin in the presence of CHX. bHLH49, however, is a directauxin response gene, because it was downregulated upon auxintreatment to roots. Therefore, rather than being locally regulatedby auxin in suspensor cells, bHLH49 is likely upregulated asa consequence of systemic suppression of the auxin response inM0171>>bdl embryos. Thus, the S>E transformation is a multi-step process that involves intensive communication between thesuspensor and pro-embryo and includes an auxin response inboth cell types.

bHLH Genes Help Establish theSuspensor-Embryo Junction

To determine whether bHLH49, 60, 63, and 100 contribute tozygote-derived embryogenesis, we identified and characterizedinsertionmutants (Figure 6)with strongly reduced transcript levels(Supplemental Figure 2). Strikingly, all single mutants showedembryo defects, with frequencies ranging from 7% to 15% (n >100; Figures 6A to 6E) that were distinguishable from the globularstage onward. Although these defects were incompletely pene-trant, these rates are highly significant given the 1.7% (n = 120) ofcomparable defects observed in the wild type. These phenotypesappear to be the consequence of bHLH mutations, rather thanunlinkedmutations, because the same phenotypes were found inmultiple independent alleles for bHLH49 and bHLH100 (bhlh49-1:15%, n = 137; bhlh49-2: 25%, n = 83; bhlh100-1: 6.8%, n = 118;bhlh100-2: 22%,n=101), and themutantphenotypeof thebhlh49alleles was rescued by introducing pbHLH49:bHLH49-tdTomato(bhlh49-1: 5.5%,n=199;bhlh49-2: 4.95%,n=202).Regardlessofthe rather broad expression domains of the genes, the phenotypeof the loss-of-functionmutantswasspecific to thesuspensor and/or the hypophysis (topmost cell of the suspensor), suggestingthese genes contribute to normal suspensor and/or hypophysis

development. In the bhlh49-1 and bhlh100-1 mutants, pro-liferative divisions were observed at the suspensor-embryojunction. It could not be unequivocally determined whether theprimary defect originated in the pro-embryo or the suspensor(Figures6A, 6B, and6E).bhlh60-3 showedabnormal hypophysealcell divisions, whereas marginal proembryo defects were ob-served in bhlh63 (Figures 6A, 6C, and 6D). Nonetheless, theseresults suggest that these bHLH genes, especially bHLH49 andbHLH100, are required for normal embryo development, partic-ularly for the embryo-suspensor junction.Although the loss-of-function phenotype suggests these genes

are required for normal embryo development, it is unclear whethertheir upregulation contributes to suspensor proliferation and/orembryo transformation in M0171>>bdl embryos. To determinewhether their upregulation contributes to abnormal suspensordevelopment, we misexpressed each gene individually(Supplemental Figure 2B) using the RPS5A promoter (Figure 7),which drives strong gene expression throughout the embryo,including thesuspensor (Weijerset al., 2001).Analysis ofup to fourindependent lines per construct revealed that bHLH49 andbHLH60 were able to induce severe suspensor phenotypes,whereas bHLH63 and 100 were less able to do so (Figures 7A to7E). In particular, pRPS5A-bHLH49 embryos showed proliferatedsuspensors with an embryo-like appearance (39%, n = 185); insome cases, three pro-embryo–like structures were stacked ontop of each other (arrowheads, Figure 7B). Consistent with thephenotypic appearance, the suspensor-specific expression ofpARF13-n3GFP (Rademacher et al., 2011) was lost in the pro-liferating suspensor cells (Figures 7F and 7G). It is thereforeconceivable thatbHLH49 is by itself sufficient to induce the lossofsuspensor identity along with proliferative cell divisions in thesuspensor. Thus, limiting the expression of bHLH49 is importantfor extra-embryonic cell identity specification.The analysis of bHLH49 expression in M0171>>bdl embryos

showed that its upregulation was restricted to pro-embryo cells(Figure 5G). We therefore addressed the spatial requirements ofbHLH49 misexpession for the induction of the misexpressionphenotype. Because of the lack of a reliable promoter that issolely expressed in the entire pro-embryo, yet absent fromthe suspensor, our analysis was limited to suspensor-specific

Figure 6. bHLH Genes Help Establish the Suspensor-Embryo Junction.

(A) to (E) Embryo phenotypes of wild-type (A) andmutants in bHLH49 (B), bHLH60 (C), bHLH63 (D), or bHLH100 (E). Percentages indicate penetrance ofphenotypes in homozygous mutants (bhlh49-1: 15%, n = 137; bhlh60-3: 13%, n = 265; bhlh63: 12%, n = 115; bhlh100-1: 6.8%, n = 118). Arrowheadsindicate abnormal cell divisions.

60 The Plant Cell

misexpression.Weused the suspensor-specificdriver lineM0171and another suspensor-specific promoter (pARF13; Figure 7F;Rademacher et al., 2011) to express bHLH49. We found thatsuspensor-specific expression of bHLH49 did not lead to ex-cessive divisions in the suspensor cells, as observed in pRPS5A-bHLH49 (M0171>>bHLH49: 0%, n = 181; pARF13-bHLH49: 0%,n=150). This result suggests that local regulationofbHLH49 in thesuspensor does not contribute to its misexpression phenotypeand is consistent with its upregulation in M0171>>bdl pro-embryos to be causal to altered development.

bHLH49 Mediates Postembryonic Auxin-Dependent Growth

Theauxin-dependentbHLH49gene is required for normal embryodevelopment, and its misexpression results in the loss of sus-pensor identity and the formation of embryo-like structures inthe suspensor. In contrast with the other three bHLHs, post-embryonic developmental phenotypes of plants overexpressingbHLH49were also observed (Supplemental Figure 3). The defectsobserved include short, almost empty siliques (SupplementalFigures3Aand3B), rootswithabnormalcell division, anddistortedroot apical meristems (Supplemental Figures 3C and 3D). Raredefects included the absence of flowers (Supplemental Figure 3E)and defective cotyledon development phenotypes such as cup-shaped or monocotyledonous seedlings or very distorted seed-lings with no cotyledons and a rudimentary root (SupplementalFigure3F to3H) reminiscent of auxin-relatedphenotypes (Hardtkeand Berleth, 1998; Hamann et al., 2002). To determine whetherbHLH49 functioncontributes to thedevelopmentaloutputof auxinsignaling, we first examined auxin sensitivity in bhlh49 mutantsand in the pRPS5A-bHLH49 misexpression lines (Figure 8). Be-cause no facile auxin sensitivity assay for embryos is currentlyavailable, this was performed in postembryonic roots, in whichbHLH49 is expressed in an auxin-dependent manner (Figure 5B).Changes in root growth inhibition by the synthetic auxin 2,4-D canbe quantified and used to determine the auxin sensitivity (Lincolnet al., 1990). Our results show that auxin sensitivity was greatlyenhanced in pRPS5A-bHLH49 roots (Figure 8A), whereas bhlh49

mutant roots did not showaclear change in sensitivity (Figure 8A).Hence,bHLH49 is not only regulatedbyauxin, but its repression isalso required for a normal auxin response during root growth. Todetermine whether auxin treatment affects the bHLH49 mis-expression phenotype, we observed root tips of the RPS5A-bHLH49 line grown on various auxin concentrations. We foundthat auxin treatment did not obviously modify the disorganizedprimary roots in this line (Supplemental Figures 4A to 4E, 4H,and 4I).Finally, because auxin activity is characterized by many feed-

back loops (Figure 2J; Benjamins and Scheres, 2008; Joneset al., 2010; Leyser, 2010), we determined whether bHLH49expression is only an output of auxin-induced regulation, orwhether it feeds back into auxin activity. We introduced thepDR5-GFP auxin response reporter (Ulmasov et al., 1997; Frimlet al., 2003) into pRPS5A-bHLH49 misexpression lines andfound no disruption in the DR5-GFP expression pattern (Figures8B to 8E), even after auxin treatment (Supplemental Figures 4F,4G, 4M, and 4N), despite the clear developmental defects in thisline, implying that there is no feedback from bHLH49 activity tothe auxin response.

bHLH49 Mediates Auxin-Dependent Regulation of GeneExpression in Suspensors

When overexpressed, bHLH49 is able to induce proliferative celldivisions and (subsequent) suspensor identity loss, resemblingthe suspensor-specific bdlmisexpression. To determine whetherincreasedbHLH49 expression contributes to the gene expressionprogram mediating embryo initiation, we performed whole-genome transcript profiling on bhlh49 mutant and pRPS5A-bHLH49 overexpression lines. Ideally, this should be done onembryonic tissue; however, given the severe embryo defects ofpRPS5A-bHLH49 (Supplemental Figure 3), we could not collectenough embryos and used root tips instead. We found 237 genesto be differentially expressedwhen comparing bhlh49 versuswildtype, and 1216 gene when comparing pRPS5A-bHLH49 versuswild type (>1.3-fold misregulated; FDR < 0.05).

Figure 7. Overexpression of bHLH49 Affects Suspensor and Embryo Development.

(A) to (E) Embryo phenotypes of wild-type (A), pRPS5A-bHLH49 (B), pRPS5A-bHLH60 (C), pRPS5A-bHLH63 (D), and PRPS5A-bHLH100 (E).(F) and (G) Expression of pARF13-n3GFP in wild-type (F) and pRPS5A-bHLH49 (G). Arrowheads in (B) indicate the pro-embryo and pro-embryo–likestructures. Percentages indicate penetrance of phenotypes in overexpression lines (pRPS5A-BHLH49: 39%, n = 185; pRPS5A-bHLH60: 28%, n = 128;pRPS5A-bHLH63:2.6%,n=117;pRPS5A-bHLH100:11%,n=175).Magentacounterstaining in (F)and (G) isRenaissanceRS2200.Scalebar510mminallpanels.

The Plant Cell 61

We then performed a meta-analysis to determine whetherthe upregulation of bHLH49 contributes significantly to auxin-dependent suspensor proliferation (Figure 9). This meta-analysis revealed a significant overlap between the genesthat were differentially expressed in either RPS5A-bHLH49roots or M0171>>bdl embryos. We found 85 and 91 genes tobe up- and downregulated, respectively, in both array ex-periments (>1.3-fold; P < 3.5e-06 for upregulated genes andP < 4.0e-14 for downregulated genes; Figures 9B and 9C).Interestingly, bHLH60 and bHLH100were 1.3-fold up- and 2.3-fold downregulated, respectively, in the RPS5A-bHLH49 dataset. This recapitulates their misregulation in M0171>>bdlembryos, and together with the finding that bHLH60 andbHLH100were not immediate auxin targets (Figure 5B) impliesthat auxin-dependent regulation of bHLH60 and 100 is medi-ated by bHLH49. The latter was also confirmed by quantitativeRT-PCR (qRT-PCR; Figure 9D). Thus, bHLH49 appears to be animportant mediator of the auxin-dependent suppression ofembryo identity in suspensor cells.

DISCUSSION

The plant suspensor plays a supportive role during embryogen-esis and is mitotically quiescent after three initial rounds of celldivision in Arabidopsis (Laux and Jurgens, 1997; Mayer andJürgens, 1998). This pattern of suspensor development is rep-resentative for a large number of plant species, although signifi-cant divergence is observed in the plant kingdom (Yeung andMeinke, 1993; Yeung and Clutter, 2011). The quiescence ofsuspensor cells does not reflect its developmental potential. Inseveral species, suspensor cells can be “reprogrammed” to forma second embryo (Yeung and Meinke, 1993). Although themechanisms of this conversion and its regulators are largelyunknown, we previously identified the transcriptional auxin re-sponse as a key pathway suppressing embryo identity in sus-pensor cells (Rademacher et al., 2012). Here, we further exploredthis systemas amodel for investigating the suspensor-to-embryotransformation. Our study identified a large degree of convergenthomeostatic control of many genes involved in auxin homeo-stasis, which cooperatively work to restore the auxin response.We also identified a number of genes whose misregulation uponinhibition of the auxin response correlates with embryo identityspecification. Importantly, we identified a genetic network in-volving several bHLH transcription factors that mediates auxinaction in controlling suspensor development and/or suspensorcell identity maintenance.Manygenesaremisregulated in the72-hM0171>>bdl data sets

even though the first visible morphological defects occurred atvery low frequency, and there was a relatively narrow window ofauxin response inhibition. This reflects the major role of auxin inthesecells andsuggests that reprogramming isacomplexgeneticresponse, and thus it may be difficult to isolate the first events. Apossible explanation for the latter is that although the pro-embryoand suspensor domains are well defined, they remain physicallyand symplastically connected. In fact, the suspensor serves asa conduit for delivering nutrients and growth regulators to the pro-embryo (KawashimaandGoldberg, 2010). Besides, impairment ofthe pro-embryo triggers developmental changes in the suspensor(Schwartz et al., 1994; Weijers et al., 2003; Liu et al., 2015).Therefore, changes in suspensor cells could alsobesensedby thepro-embryo and are expected to induce transcriptional changesand secondary consequences in the pro-embryo. The continuitybetween the pro-embryo and suspensor cells could also explainthe broader expression domains of 24 out of 40 tested genes,which is not in line with the maintenance of auxin-mediatedsuspensor cell identity and/or embryo transformation.Two readily apparent features of the transcriptomic and ex-

pressionanalysisdatasetsare (1) auxinhomeostasiscomponentsand (2) bHLH transcription factors involved in the suspensor auxinresponse. Our initial global analysis of M0171>>bdl data setsrevealed enrichment for genes involved in auxin homeostasis. Inparticular, genes thatbelong tomore than10 families representingeachof themain facetsof auxinhomeostasisweremisregulated insuch a way that the end result will be an increase in free cellularauxin levels or an increased auxin response. The level of free auxinin the cell is cooperatively defined by biosynthesis, (de)conju-gation, and transport and has a direct output, i.e., auxin signaling.Thus, inhibition of the auxin response might be sensed by the

Figure 8. bHLH49 Mediates Postembryonic Auxin-Dependent Growth.

(A) Root length of wild type (Col-0), pRPS5A-bHLH49, bhlh49-1, andbhlh49-2 seedlings upon treatment with 10 nM, 20 nM, 30 nM, and 40 nM2,4-D, comparedwith untreated control (t test: *P < 0.05; **P < 0.001). Errorbars indicate SD in three separate experiments where at least 30 and up to50 seedlings per genotype were used for the measurement.(B) to (E) Expression of DR5-n3GFP in wild-type [(B) and (D)] andpRPS5A-bHLH49 [(C) and (E)] globular stage embryos [(B) and (C)] androots [(D) and (E)]. Magenta counterstaining in (B) and (C) is RenaissanceRS2200 and in (D) and (E) it is Propidium iodide. Scale bar5 10 mm in allpanels.

62 The Plant Cell

systemasanauxinminimum.As a response to this process and toefficiently re-establish theauxin response, genes thatwill increaseintracellular auxin levels are upregulated and genes involved inauxin transport are downregulated (Figures 2A to 2J), resulting inincreased auxin levels in the embryo (Figures 3A, 3B, 3G, and 3H).Self-regulatory feedback loops from auxin signaling to the ex-pressionof auxinhomeostatic genesarewell known.For instance,the transcription of GH3 family members and most Aux/IAA in-hibitors is highly auxin inducible (Liu et al., 1994). Similarly, theexpression of both auxin efflux and influx carrier genes is upre-gulated by auxin (Vieten et al., 2005; Swarup et al., 2008). How-ever, all of these feedback loops have been independentlyobserved upon exogenous auxin treatment, and it is rather ob-scure to what degree they functionally converge. Our resultsdemonstrate a feedback loop represented by themisregulation ofgenes involved in auxin homeostasis and responses linked to

a biologically relevant output: suspensor proliferation and/orasubsequentswitchofcell identity.Thehighdegreeofconvergentregulation of 40 genes from 15 families shows how robust toperturbation the auxin system is.Because many bHLH superfamily members were misregulated

upon suspensor-specific inhibition of the auxin response, andbecause several bHLH transcription factors were previouslyshown to play a role in Arabidopsis embryo development, par-ticularly in cell specification events downstream of auxin, wedecided to further investigate a set of four bHLH genes in detail.Our results show that all are indeed regulated by auxin in an ARF-dependent manner. Their misregulation upon inhibition of theauxin response in the suspensor implies that the S>E trans-formation might not be a cell autonomous process involvinga single-step identity switch. Our results suggest that instead, theS>E transformation might be a multi-step process, where upon

Figure 9. bHLH49 Mediates Auxin-Dependent Regulation of Gene Expression in Suspensors.

(A) Venn diagram depicting the number of genes upregulated in pRPS5A-bHLH49 and downregulated in bhlh49 root tips.(B) Venn diagrams showing the overlap in genes either up- or downregulated in pRPS5A-bHLH49 seedling roots and M0171>>bdl embryos. Examples ofgenes present in the overlap are listed on the right.(C) Expression levels of selected genes in M0171>>bdl embryos and pRPS5A-bHLH49 seedling roots in micro-array experiments.(D) qRT-PCR validation of the expression levels of bHLH60 and bHLH100 in wild-type (Col-0) and pRPS5A-bHLH49 seedling roots. Expression levels inwild-type sampleswere set to 1. Error bars indicate SE. Three technical and three biological replicates (representing separate experiments) were performed.

The Plant Cell 63

inhibition of the auxin response, excessive cell division is turnedon, followed by the loss of suspensor identity (cells undergodedifferentiation) and subsequently (upon trigger) an installmentof embryo identity.

Based on phenotypic data, aswell as the dependence of auxin-regulation on de novo translation, bHLH49 appears to be a moredirect and biologically more important regulator than the otherbHLH proteins. Indeed, when probing the bHLH49-dependenttranscriptome,we found that theother bHLHgenesareamong thetargets. Thus, the auxin-repressed bHLH49 gene appears to bean important mediator of auxin-dependent suppression of pro-liferation in suspensor cells. Indeed, themisexpressionofbHLH49alone induced excess divisions and even the formation ofmultipleembryo-like structures in suspensors (Figure 7B), similar to theeffect of inhibition of the auxin response.

An important open question is what primary cellular processbHLH49 targets tobring about suspensor proliferation. Identifyingthe identity of the bHLH49-dependent genes did not directly re-veal a key cellular process that can explain its ability to triggerproliferative divisions in the suspensor. Nonetheless, the signifi-cantoverlapwith thegenesmisregulated inM0171>>bdl embryoshints toward the need for a defined set of genetic regulators re-quired to induce the switch from suspensor to embryonic cellfate. Interestingly, however, we detected an overlap betweenM0171>>bdl responsive and bHLH49-dependent genes, eventhough one process is performed in embryos and the other inroots. Thus, it is quite possible that bHLH49 regulates a rathergeneric cellular process, such as cell division. However, thepostembryonic misexpression phenotypes are not indicative ofexcess cell division. Postembryonic misexpression of bHLH49induces very strong defects (Supplemental Figures 3A to 3H),but these do not include the induction of ectopic embryo for-mation. Thus, unlike “embryo inducers” such as LEAFYCOTYLEDON1(Lotan et al., 1998) or BABYBOOM (Boutilier et al.,2002), bHLH49 does not appear to directly promote embryo-genesis. Its action is instead associated with the dedifferentiationof suspensor cells (erasure of their initial transcriptional program)into cells that can divide but cannot be transformed completelyinto functional embryo structures (installation of the embryonicprogram). In this regard, it will be interesting to test the potential ofbHLH49, using gain- and loss-of-function mutants, to induceembryogenesis in thecontext of somatic embryogenesisorduringplant regeneration from hypocotyl explants (Iwase et al., 2011).Furthermore, the suspensor cells of several plant speciesundergomany more divisions, yet they do not show features of embryoidentity (Yeung and Meinke, 1993; Kawashima and Goldberg,2010; Yeung and Clutter, 2011). Hence, unless Arabidopsis, withits minimal number or suspensor cells, represents an exceptionalsituation, triggering cell division in suspensor cells may not besufficient to induce embryogenesis.

METHODS

Plant Material

All Arabidopsis (Arabidopsis thaliana) plants used in this study were of theCol-0 ecotype except the M0171-GAL4 enhancer trap line, which is in theC24 background (made available by Dr. Jim Haseloff, Cambridge, UK).

T-DNA insertion lines bhlh49-1 (SALK_135188C), bhlh49-2 (SALK_087424C),bhlh60-3 (SAIL_1219_E01), bhlh63-2 (SAIL_1211_F11), bhlh100-1(SALK_150637C), and bhlh100-2 (SALK_074568C) and the M0171-GAL4 (Rademacher et al., 2012) enhancer trap line were obtained fromArabidopsis Stock Centers (NASC-ARBC) and genotyped using theprimers listed in Supplemental Data Set 2. The pARF13-n3GFP andDR5/DR5v2 and Ratiometric version of 2 D2’s (R2D2) lines were described(Rademacher et al., 2012; Liao et al., 2015).

All seeds were sterilized in 25% bleach/75% ethanol (v/v)solution for10 min and washed twice with 70% ethanol and once with 100% ethanol.Dried seeds were plated on half-strength Murashige and Skoog (MS)medium and the appropriate antibiotic (50 mg/l kanamycin or 15 mg/lphosphinothricin) for the selection of transgenic seeds. After 24 h in-cubation at 4°C, the plants were cultured under long-day conditions (16 hlight 110 mE m22 s21 [Philips Master TL-D HF 50W/840] and 8 h dark) at22°C.

Plant transformation was performed using the floral dip method, asdescribed in De Rybel et al. (2011).

Cloning

All cloning was performed using the LIC cloning system and previouslydescribed vectors (De Rybel et al., 2011). For transcriptional fusions,fragmentsup to3kbupstreamof theATGcodon including the59-UTRwereamplified from genomic DNA using Phusion Flash polymerase (ThermoScientific). For translational fusions of bHLH genes, the same promoterfragments were amplified along with the genomic coding sequences ex-cluding the stop codon. To generate constructs for pRPS5A-driven mis-expression, the coding sequences were amplified from complementaryDNA (cDNA) clones. All constructs were completely sequenced. The pri-mers used for cloning are listed in Supplemental Data Set 2. At least threeindependent lines were analyzed per construct.

Microscopy

Differential interferencecontrast andconfocalmicroscopywereperformedas described previously (Llavata-Peris et al., 2013). Cleared embryos wereobserved under a Leica DMR microscope equipped with differential in-terference contrast optics, and confocal imaging was performed usinga Leica SP5 II system (Hybrid detector). Cell outlines were generated bycounterstaining with SCRI Renaissance Stain 2200 (RenaissanceChemicals).

Phylogenetic Analysis

The sequences of all predicted ArabidopsisbHLH proteins (https://www.arabidopsis.org/browse/genefamily/blhm.jsp) were retrieved from TAIR(https://www.arabidopsis.org). Subsequently, multiple sequence align-ment was performed in ClustalW. An unrooted phylogenetic tree wasconstructed with TreeView (http://en.bio-soft.net/tree/TreeView.html).

Quantitative RT-PCR Analysis

qRT-PCR analysis was performed as described previously (De Rybel et al.,2010). RNA was isolated using TRIzol reagent (Invitrogen) and RNeasy kit(Qiagen). cDNAwasprepared from0.5mgof totalRNAwithan iScript cDNASynthesis Kit (BioRad). qRT-PCR was performed with iQ SYBR GreenSupermix (BioRad) and analyzed on a CFX384 Real-Time PCR detectionsystem (BioRad). The qRT-PCR cycling conditions were 95°C for 10 min;45cyclesof95°C for10s,55°C for20s,72°C for20s;95°C for10s;65°C for5 s, followed by dissociation curve analysis. Reactions were done in trip-licate, with three biological replicates (representing separate experiments).Data were analyzed with qBase as described in (Hellemans et al., 2007).

64 The Plant Cell

Primers were designed with Beacon Designer 8 (Premier Biosoft In-ternational). Gene expression levels were normalized relative toCDKA1;1,EEFa4, and GAPC. Primers for qRT-PCR are listed in Supplemental DataSet 2.

Auxin Sensitivity Assay

The auxin sensitivity assay was performed according to Lincoln et al.(1990). Seedswere first germinatedonstandardhalf-strengthMSmedium.Six-day-old seedlingswere transferred to freshMSmediumsupplementedwith10nM,20nM,30nM,and40nM2,4-Dor lacking2,4-D.After twodays,the plates were scanned, and the length of the newly grown roots wasmeasuredusing ImageJsoftware. Thepercentageof root growth relative toroot growth on MS without auxin was calculated.

Microarray Experiments

M0171>>bdl:

After crossing, embryos were isolated in 5%Suc solution containing 0.1%RNALater as described in (Xiang et al., 2011), and the isolated embryoswere pooled in a 1.5 mL Eppendorf tube on dry ice (300-400 embryos foreach biological replicate). Total RNA was extracted according to theprotocol of the RNAqueous-micro kit and amplified before labeling fol-lowing the protocol provided in the MessageAmp aRNA kit with minormodifications.

The Arabidopsis 70-mer oligo array slides prepared by University ofArizona were used in all the microarray experiments (version ATV 3.7.2;http://ag.arizona.edu/microarray). Antisense RNA was labeled accordingto the protocol of (Wellmer et al., 2004). The antisense RNA samplesrepresenting four biological replicates from experimental and controlsampleswere labeled (twowithCy3and twowithCy5)andhybridized to theslides following the protocol from http://eg.arizona.edu/microarray.Subsequently, the hybridized slides were scanned for Cy3- and Cy5-labeled mRNA targets with a ScanArray 4000 laser scanner (at a resolu-tionof10mm).TheQuantArrayprogram (GSILumonics)wasused for imageanalysisandsignalquantification.LimmaSoftware (Smyth,2004)wasusedfor normalization and to identify geneswithmodulated expression from themicroarraydata.Togenerate theplot inFigure1Eshowingoverlapbetweendifferentially expressed genes, UpSet plot was used (https://asntech.shinyapps.io/intervene/; Khan and Mathelier, 2017).

pRPS5A-bHLH49/bhlh49:

RNA from root tips was isolated as described above (see Quantitative RT-PCR), and total RNA (100 ng) was labeled using an Ambion Wild TypeExpression kit (Life Technologies). The RNA was then hybridized to Ara-bidopsis gene ST arrays (Affimetrix), which probe the expression of 27,827unique genes. Sample labeling and hybridization were performed ac-cording tomanufacturer’s instructions.Microarray analysiswasperformedas previously described (De Rybel et al., 2014).

Global Analysis of M0171>>bdl Data Sets

The data sets were initially subjected to the Biological Networks GeneOntology Tool to assess overrepresentation of gene ontology terms.Default settings were used (hypergeometric test, Benjamini and HochbergFDR for correction of multiple testing, significance level of 0.05), and thewhole genome annotation was used as a reference set together with geneontology-SLIM ontology terms for Arabidopsis. No strong over- or un-derrepresentation of specific functions was found.

Accession Numbers

The accession numbers of the major genes described in this study areshown in Supplemental Table.

All microarray data have been deposited in the Gene ExpressionOmnibus at the National Center for Biotechnology Information(M0171>>bdl: GSE69854; bHLH49 mutant and overexpression:GSE69700).

Supplemental Data

Supplemental Figure 1. Localization of bHLH proteins.

Supplemental Figure 2. Expression of bHLH genes in insertion andmisexpression lines.

Supplemental Figure 3. Post-embryonic phenotypes in pRPS5A-bHLH49 plants.

Supplemental Figure 4. Auxin treatment of pRPS5A-bHLH49 roots.

Supplemental Table. Sixty-eight genes selected for validation ofM0171>>bdl data set.

Supplemental Data Set 1. M0171>>bdl data set.

Supplemental Data Set 2. Primers used in this study.

Supplemental File. Alignment used to produce the phylogenetic treeshown in Figure 5.

ACKNOWLEDGMENTS

The authors thank Dr. Jim Haseloff and the NottinghamArabidopsis StockCenter for distributing the seedsandBertDeRybel andMaritza vanDop forcomments on the manuscript. This work was funded by grants from theNederlandse Organisatie voor Wetenschappelijk Onderzoek (NetherlandsOrganisation for Scientific Research) (ALW-NSFC grant 846.11.001; ALWOpenCompetition grant 816.02.014) and the EuropeanUnion ITN networkSIREN (contract no.214788 to D.W.).

AUTHOR CONTRIBUTIONS

T.R., A.S.L., C.I.L.-P., and D.W. designed the research; T.R., A.S.L.,C.I.L.-P., J.R.W., D.X., C.-Y.L., and L.V. performed research; all authorscontributed to data analysis; T.R. and D.W. wrote the paper with contri-butions from A.S.L. and C.I.L.-P. and with input from all other authors.

Received July 9, 2018; revised November 27, 2018; accepted December19, 2018; published December 20, 2018.

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The Plant Cell 67

DOI 10.1105/tpc.18.00518; originally published online December 20, 2018; 2019;31;52-67Plant Cell

Che-Yang Liao, Lieke Vlaar, Mark Boekschoten, Guido Hooiveld, Raju Datla and Dolf WeijersTatyana Radoeva, Annemarie S. Lokerse, Cristina I. Llavata-Peris, Jos R. Wendrich, Daoquan Xiang,

Basic Helix Loop Helix Transcriptional ModuleA Robust Auxin Response Network Controls Embryo and Suspensor Development through a

 This information is current as of July 4, 2020

 

Supplemental Data /content/suppl/2018/12/24/tpc.18.00518.DC2.html /content/suppl/2018/12/20/tpc.18.00518.DC1.html

References /content/31/1/52.full.html#ref-list-1

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