RESEARCH ARTICLE

ATML1 activity is restricted to the outermost cells of the embryothrough post-transcriptional repressionsHiroyuki Iida, Ayaka Yoshida and Shinobu Takada*

ABSTRACTCell fate determination in plants relies on positional cues. Toinvestigate the position-dependent gene regulation in plants, wefocused on shoot epidermal cell specification, which occurs only inthe outermost cells. ATML1, which encodes an HD-ZIP class IVtranscription factor, is a positive regulator of shoot epidermal cellidentity. Despite the presence of a weak ATML1 promoter activity inthe inner cells, ATML1 protein was detected mostly in the outermostcells, which suggests that ATML1 accumulation is inhibited in theinner cells. ATML1 nuclear localization was reduced in the epidermisand therewas a positive, albeit weak, correlation between the amountof ATML1 in the nuclei and the expression of a direct target of ATML1.Nuclear accumulation of ATML1 was more strongly inhibited in theinner cells than in the outermost cells. Domain deletion analysesrevealed that the ZLZ-coding sequence was necessary andpartially sufficient for the post-transcriptional repression of ATML1.Our results suggest that post-transcriptional repressions contribute tothe restriction of master transcriptional regulator activity in specificcells to enable position-dependent cell differentiation.

KEY WORDS: Epidermal cell differentiation, ATML1, Transcriptionalactivation, Post-transcriptional repression, Embryogenesis,Arabidopsis thaliana

INTRODUCTIONFor proper pattern formation of multicellular organisms, celldifferentiation should be regulated spatiotemporally. In someanimals, cell lineage plays an important role in their developmentbecause cell fate is already determined during early embryogenesis(Sulston et al., 1983). Unlike animals, plants have a flexibility intheir cell-fate decision. Arabidopsis thaliana embryos show aregular well-organized cell division pattern. However, mutantembryos with disrupted division patterns can still acquire the correctbody organization (Torres-Ruiz and Jürgens, 1994). Lineage tracinganalyses have shown that the apical cell fate was not strictlydetermined in two-cell-stage embryos (Saulsberry et al., 2002).Moreover, cell ablation experiments in the root meristem haveshown that cortex and endodermis initial cells are regenerated fromthe neighboring pericycle cells (van den Berg et al., 1995). Thesereports suggest that cell specification in plants largely depends onthe cell position, rather than cell lineage. Namely, plant cells canrecognize where they are located and gene activities are changed in

response to their positions. However, the mechanisms that underlieposition-dependent cell-fate decisions are still unclear. To gainknowledge of these mechanisms, we focused on epidermal cell fate,which is acquired and maintained only in the outermost cells(Stewart and Dermen, 1975).

In A. thaliana, ARABIDOPSIS THALIANA MERISTEM L1LAYER (ATML1) and its closest homologue PROTODERMALFACTOR2 (PDF2) are necessary for epidermal cell identity. ATML1and PDF2 encode HD-ZIP class IV transcription factors, which arecomposed of four domains: a homeodomain (HD), a zipper-loop-zipper (ZLZ) motif, a StAR-related lipid-transfer (START) domain,and a START-associated domain (SAD) (Mukherjee and Bürglin,2006; Ariel et al., 2007). Weak and strong loss-of-function alleles ofATML1 have been reported; atml1-1;pdf2 can germinate but cannotform an epidermis on their leaves, whereas atml1-3;pdf2 arrestsdevelopment around the globular stage (Abe et al., 2003; San-Bentoet al., 2014; Ogawa et al., 2015). In addition, constitutive expressionof ATML1was shown to induce ectopic epidermal cell differentiationin the inner tissues of cotyledons and leaves (Takada et al., 2013).Taken together, these reports suggest that ATML1 is a positiveregulator of epidermal cell identity.

ATML1 mRNA and promoter activity are detected preferentiallyin the outermost cells during embryogenesis, which suggests thatATML1 transcription is promoted in the outermost cell layer (Luet al., 1996; Sessions et al., 1999; Takada and Jürgens, 2007). Inaddition to transcriptional activation, some reports imply thatATML1 activity is also under post-transcriptional repression. Forexample, ATML1 mRNA was not detected in suspensor cellsalthough the ATML1 promoter was active in these cells (Takada andJürgens, 2007; Nodine and Bartel, 2010). MicroRNA biogenesisappears to be necessary for ATML1 mRNA degradation in thesuspensor cells (Nodine and Bartel, 2010). In another report,GbML1, an ATML1 homologue in cotton, was not clearly localizedto the nuclei in the onion epidermis (Zhang et al., 2010). Thesereports imply that ATML1 mRNA accumulation and the nuclearlocalization of ATML1 may be decreased by post-transcriptionalmechanisms. However, it is still not clear whether ATML1 activity isindeed reduced post-transcriptionally and whether this reductioncontributes to the restriction of ATML1 activity to the outermostcells. In this paper, we show that ATML1 protein was rarelydetected in the inner cells of the embryos, although ATML1 wasweakly transcribed in these cells. Moreover, we demonstrate thatnuclear accumulation of ATML1 was attenuated, especially in theinner cells, during embryogenesis. We also performed domaindeletion experiments and found that the ZLZ-coding sequence wasnecessary and partially sufficient for the attenuation of ATML1nuclear accumulation and the suppression of ATML1 activity in theinner cells. Our study implies that spatial restriction of ATML1activity by post-transcriptional mechanisms may facilitate theformation of the single epidermal layer, a common feature inmany seed plants.Received 23 June 2018; Accepted 4 February 2019

Department of Biological Sciences, Graduate School of Science, Osaka University,1-1 Machikaneyama-cho, Toyonaka, Osaka 560-0043, Japan.

*Author for correspondence (shinobu_takada@bio.sci.osaka-u.ac.jp)

H.I., 0000-0001-7311-0700; A.Y., 0000-0002-7274-1858; S.T., 0000-0001-6587-2166

1

© 2019. Published by The Company of Biologists Ltd | Development (2019) 146, dev169300. doi:10.1242/dev.169300

DEVELO

PM

ENT

RESULTSReliable new reporters for ATML1 transcriptionin the embryosIn our previous reports, ATML1 transcription was visualized usingonly its promoter sequence (Takada and Jürgens, 2007). Therefore,the reporter expression pattern might not reflect the realtranscriptional activity of ATML1. To visualize the nativetranscriptional dynamics of ATML1, we generated transgenic plantsthat expressed the triple GFP reporter gene fused with the SimianVirus 40 (SV40) T-antigen nuclear localization signal (NLS) underthe control of the ATML1 regulatory sequence, which included a 3.4kb promoter sequence, exons, introns and a 3′ downstream sequence(gATML1-nls-3xGFP; Fig. 1A). gATML1-nls-3xGFP signals weredetected from as early as the one- or two-cell stage (12 of 12 lines)and were detected preferentially in the outermost cells from the earlyglobular stage (12 of 12 lines; Fig. 1D-H). These expression patternswere comparable with the 3.4 kb ATML1 promoter activity shown by

Takada and Jürgens (2007). These results suggest that the 3.4 kbATML1 promoter contains sufficient regulatory regions to drive thesame expression pattern as the 9.2 kb genomic sequence used ingATML1-nls-3xGFP.

ATML1 protein was not detected in the inner cellsof the embryos from the 32-cell stageDetailed observation of gATML1-nls-3xGFP plants revealed thatweak GFP signals were detected in the inner cells of the embryosfrom the 32-cell stage (Fig. 1E,F and Fig. S1A-C). Weak GFPsignals were still observed in the inner cells of the heart-stageembryos, especially in the L2 layer (12 of 12 lines), which suggeststhat ATML1 is weakly transcribed in the inner cells (Fig. 1G,H andFig. S1D-F). Our previous study has shown that ectopic ATML1expression induces epidermal cell identity in the inner tissues of thecotyledons and leaves (Takada et al., 2013). Therefore, ATML1activity should be repressed in the inner cells to avoid the formation

Fig. 1. Transcription of the ATML1gene and localization of ATML1protein during embryogenesis.(A-C) Constructs to visualize thetranscriptional activity of the ATML1 gene(A) and the localization of ATML1 protein(B,C) using the single (B) or triple GFP (A,C) reporter. In B and C, but not in A, GFPor triple GFP is translationally fused toATML1. Blue boxes indicate untranslatedregions; orange boxes indicate codingregions. ATG, start codon; NLS, SV40nuclear localization signal; NOSt,nopaline synthase terminator; STOP,stop codon. (D-R) GFP signals (green) ingATML1-nls-3xGFP (D-H), gATML1-1xGFP (I-M) and gATML1-3xGFP (N-R)embryos at the two-cell stage (D,I,N), the32-cell stage (E,F,J,K,O,P) and the heartstage (G,H,L,M,Q,R). In F,K and P, GFPsignals of the same embryos in E,J andO, respectively, are displayed accordingto the color map (bottom right). H,M andR show magnified views of thecotyledons in G,L and Q, respectively,colored with the color map. Scale bars:10 µm.

2

RESEARCH ARTICLE Development (2019) 146, dev169300. doi:10.1242/dev.169300

DEVELO

PM

ENT

of multiple epidermal layers. To test whether ATML1 activity issuppressed after its transcription, we observed ATML1 proteinlocalization by generating translational fusion lines, in which asingle or triple GFP reporter gene fused in-frame with the ATML1-coding sequence was expressed under the control of the nativeregulatory sequence of ATML1 (gATML1-1xGFP and gATML1-3xGFP; Fig. 1B,C). These constructs rescued the embryonic lethalphenotype of atml1-3;pdf2-1, which indicates that these fusionproteins were functional (Table S1). GFP signals were detected fromthe one- or two-cell stage in gATML1-1xGFP and gATML1-3xGFP(9 of 9 and 10 of 11 lines, respectively; Fig. 1I,N), as observed ingATML1-nls-3xGFP. Unlike in gATML1-nls-3xGFP, however,gATML1-1xGFP and gATML1-3xGFP signals were decreased ornot detected in the inner cells of some 16-cell-stage embryos (11 of 26and 28 of 69 embryos, respectively; Fig. 2A-C). From the 32-cellstage, GFP signals were detectable mostly in the outermost cells (10of 10 lines in gATML1-1xGFP and 10 of 10 lines in gATML1-3xGFP;Fig. 1J-M,O-R and Fig. S1G-L). These results suggest that ATML1protein accumulation is post-transcriptionally restricted to theoutermost cells from the 32-cell stage. Treatment of the gATML1-3xGFP embryos with a proteasome inhibitor MG132 did not affectthe 3xGFP-ATML1 protein accumulation pattern, which suggeststhat 26S proteasome-mediated protein degradation is not involved inthe inhibition of ATML1 accumulation in the inner cells (Fig. S2).Our results also suggested that ATML1 protein does not move

intercellularly, at least between the outermost cells and the innercells, because 3xGFP-ATML1 fusion protein, the cell-cellmovement of which should be limited due to the large triple GFPreporter, showed the same localization as 1xGFP-ATML1 protein(Fig. 1J-M,O-R; Kim et al., 2005).

Subcellular localization of ATML1 changed duringembryogenesisWe found that ATML1 protein showed a change in subcellularlocalization during embryogenesis. gATML1-1xGFP and gATML1-

3xGFP signals were detected only in the nuclei until the eight-cellstage.Whereas some 16-cell-stage embryos of gATML1-1xGFP andgATML1-3xGFP showed GFP signals only in the nuclei, otherembryos showed GFP signals not only in the nuclei but also in thecytoplasm of the embryo-proper cells (Fig. 2A-C). After the 32-cellstage, gATML1-1xGFP and gATML1-3xGFP signals were detectedin both the nuclei and cytoplasm in the outermost cells of theembryo proper (10 of 10 and 12 of 12 lines, respectively; Fig. 1J,K,O,P). The cytoplasm-to-nuclei signal ratios in gATML1-1xGFP andgATML1-3xGFP were significantly higher than those in gATML1-nls-3xGFP (Fig. S3A). Weak nuclear localization of GFP-ATML1was evident, particularly in the protoderm of the shoot apicalmeristem (SAM) region and the adaxial side of cotyledons at theheart stage (9 of 9 lines in gATML1-1xGFP and 11 of 11 lines ingATML1-3xGFP; Fig. 1L,M,Q,R). By contrast, the suspensor cellsdid not show a cytoplasmic GFP-ATML1 signal in all gATML1-1xGFP lines and most gATML1-3xGFP lines examined (10 of 10and 9 of 10 lines, respectively; Fig. 1I-K,N-P).

It is possible that these cytoplasmic gATML1-1xGFP and gATML1-3xGFP signals were caused by spontaneous cleavage ofGFP from thefusion proteins. To test this possibility, we fused an NLS sequence tothe N- or C-terminus of 3xGFP-ATML1 (NLS-3xGFP-gATML1 and3xGFP-gATML1-NLS, respectively). If the cytoplasmic GFP signalsindeed represented cleavage products of 3xGFP-ATML1, thesecytoplasmic GFP signals should be decreased in NLS-3xGFP-gATML1, but not in 3xGFP-gATML1-NLS. If the cytoplasmic signalswere not derived from cleavage products, GFP signals in NLS-3xGFP-gATML1 should show the similar localization as those in3xGFP-gATML1-NLS. Nuclear-to-cytoplasmic GFP signal intensityratios were increased in NLS-3xGFP-gATML1 compared withgATML1-3xGFP embryos, although GFP signals were still detectedin the cytoplasm (4 of 4 lines; Fig. 2D and Fig. S3B). This increasednuclear accumulation of GFP was also detected in 3xGFP-gATML1-NLS embryos (11 of 11 lines), which suggests that addition of a strongNLS sequence at either the N- or C-terminal end similarly enhancesthe nuclear import of 3xGFP-ATML1, and at least a large part ofcytoplasmic gATML1-3xGFP signals are not caused by the cleavageof 3xGFP-ATML1 (Fig. 2E and Fig. S3B). We also performedwestern blot analyses using antibodies against GFP and confirmedthat free GFP was not detected in protein extracts from gATML1-3xGFP (Fig. S4). These results suggest that nuclear localization ofATML1 is repressed in the outermost cells of the embryo proper fromthe 16-cell stage. We also found that gATML1-1xGFP and gATML1-3xGFP signals were detected in both the nuclei and cytoplasm in theepidermis of the post-embryonic root tip, which suggests that thenuclear localization of ATML1 tends to be inhibited in the youngepidermis, independently of organ identity (Fig. S5). To gainadditional mechanistic insights into ATML1 protein localization,gATML1-3xGFP plants were treated with a nuclear export inhibitor,leptomycin B (LMB; Fig. S6). We found that cytoplasmic gATML1-3xGFP signals were reduced in LMB-treated embryos and roots,which suggests that shuttling between the nucleus and cytosol isinvolved in determining ATML1 nuclear localization (Fig. S6).

A large portion of ATML1 protein was localized to thecytoplasm in the inner cells of the embryosAlthough ATML1 protein was detected mainly in the epidermis,gATML1-3xGFP signals were still detected in a small population ofsubepidermal cells. We found that 11.1% of the embryos showedGFP signals in an inner cell at the globular stage (n=9) and 60.0% atthe heart stage (n=15). In these ATML1-positive inner cells, thecytoplasmic-to-nuclei signal ratios of gATML1-3xGFP were higher

Fig. 2. ATML1 protein was localized to the nuclei and cytoplasm inembryos. (A-C) 3xGFP-ATML1 protein localization in the 16-cell-stageembryos of gATML1-3xGFP. GFP signals were detected, albeit weakly, in theinner cells in B but not in C. (D,E) Localization of 3xGFP-ATML1 with the SV40NLS fused to theN-terminus (D;NLS-3xGFP-gATML1) or to the C-terminus (E;3xGFP-gATML1-NLS), which is produced under the native regulatorysequence of ATML1. Scale bars: 10 µm.

3

RESEARCH ARTICLE Development (2019) 146, dev169300. doi:10.1242/dev.169300

DEVELO

PM

ENT

compared with those in the outermost cells and GFP signals showedless co-localization with a nuclear marker at the heart stage (7 of 9embryos), which implies that ATML1 is primarily localized to thecytoplasm in the inner cells of the embryos (Fig. 3A-F and Fig. S7).To further investigate the subcellular localization of ATML1 proteinin the inner cells, we generated transgenic plants in which GFP-ATML1 expression was induced in the whole embryo upon estradioltreatment (RPS5A>>GFP-ML1). TheGFP-ATML1-overexpressingglobular-stage embryos did not show clear nuclear localization ofGFP signals in most of the inner cells (14 of 15 embryos; Fig. 3Mand Fig. S8). GFP-ATML1 signals in some inner cells were strongin the nuclei, as seen in the outermost cells (Fig. 3M).Next, to examine whether the enhanced cytoplasmic accumulation

of ATML1 protein requires the inner cell lineage, we observed GFP-ATML1 localization in the inner daughter cells of the protoderm. Weutilized the fass ( fs) and hanaba taranu (han) mutant embryos thatexhibit aberrant cell division orientation. The FS gene encodes a B″regulatory subunit of PP2A, which is necessary for the properorientation of interphase cortical microtubules and the formation ofpreprophase bands (Camilleri et al., 2002). Mutations in the FS gene

cause abnormal cell division orientation during embryonic andpostembryonic development (Torres-Ruiz and Jürgens, 1994). Loss-of-function mutations in HAN, which encodes a GATA transcriptionfactor, cause pleiotropic developmental defects including abnormalcell division orientation during embryogenesis (Zhao et al., 2004;Nawy et al., 2010; Kanei et al., 2012). In fs mutant embryos, thenuclear-to-cytoplasmicATML1 signal ratioswere reduced in putativeinner daughter cells of the epidermis comparedwith those in the outerdaughter cells (Fig. 3G-I). Because cell arrangements were severelydisrupted in fs embryos, it was sometimes difficult to distinguishwhether cells were located at the surface or not, under a confocal laserscanning microscope. For this reason, we then observed gATML1-3xGFP signals in han, which showed much milder cell divisiondefects. hanmutant embryos often showed periclinal divisions in theepidermal layer, which enabled us to easily observe 3xGFP-ATML1localization in the inner daughter cells of the epidermis. In hanembryos, 3xGFP-ATML1 signals were detected throughout thecytoplasm and nuclei in the inner daughter cells of the epidermis(Fig. 3J-L). Although nuclear shapes were clearly visible in theoutermost cells, the outlines of nuclei were obscure in 13 of 16 inner

Fig. 3. ATML1 protein showed predominant cytoplasmic localization in the inner cells. (A-F) 3xGFP-ATML1 localization (green; A,B) and the ACR4promoter activity (magenta; C,D) in a heart-stage embryo carrying gATML1-3xGFP and proACR4-nls-TdTomato. B and D show magnified views of A and C,respectively. E and F show merged views of A,C and B,D, respectively. (G-I) gATML1-3xGFP reporter signals in the fs-1mutant embryo. H and I show magnifiedviews of G with or without the cell wall staining SR2200 signals (white), respectively. (J-L) Localization of 3xGFP-ATML1 protein in the han-30 mutant embryocarrying gATML1-3xGFP. K and L show magnified views of J with or without SR2200 signals (white), respectively. Arrows indicate the inner cells showingpredominantly cytoplasmic 3xGFP-ATML1 localization. (M) Constitutive expression ofGFP-ATML1 in the globular-stage embryo using an estradiol-inducible line.The ovule was cultured with 10 µM estradiol for 2 days. (N) Scatter plot of the intensity of gATML1-3xGFP signals in the nuclei (x-axis) and proACR4-nls-TdTomato signals (y-axis). The average TdTomato signal intensity in each embryo was set to one. The correlation is significantly different from zero (r=0.283,P<0.01; Pearson’s product-moment correlation). Scale bars: 10 µm.

4

RESEARCH ARTICLE Development (2019) 146, dev169300. doi:10.1242/dev.169300

DEVELO

PM

ENT

cells when observed under GFP channel. These results suggest thatnuclear localization of ATML1 is more strongly inhibited in the innercells compared with the outermost cells, irrespectively of the lineageof the inner cells.

The amount of ATML1 protein in the nuclei showed a weakpositive correlation with the promoter activity of ACR4Next, we investigated the biological significance of the inhibition ofATML1 nuclear localization. It has been reported that presence of aprotein in the cytoplasm is required for the intercellular movementof the protein (Crawford and Zambryski, 2000; Gallagher et al.,2004). However, as described above, gATML1-1xGFP andgATML1-3xGFP showed the same GFP localization pattern,which suggests that ATML1 protein accumulation in thecytoplasm does not facilitate ATML1 movement between theoutermost cells and the inner cells. Rather, we speculated thatreduction of ATML1 nuclear localization might decrease theATML1-mediated transcription in the nuclei. To examine thishypothesis, ATML1 protein localization and the promoter activityof ARABIDOPSIS THALIANA HOMOLOGUE OF CRINKLY4(ACR4), a direct target of ATML1, were visualized simultaneously(see Materials and Methods; Fig. 3A-F). Expression of ACR4,which encodes a leucine-rich repeat receptor-like kinase, is directlyactivated by ATML1 in the embryos (Tanaka et al., 2002; San-bentoet al., 2014; N. Takada, A.Y., H.I. and S.T., unpublished).Therefore, the ACR4 promoter activity is expected to reflect thetranscriptional activity of the ATML1 protein. We measured thefluorescence intensities of GFP and TdTomato signals in the nucleiof the outermost cells at the heart stage and found that there was aweak, but statistically significant, positive correlation between thesesignal intensities (Fig. 3N and Fig. S9H). We also examined thecorrelation between ATML1 nuclear accumulation and ATML1promoter activity in the heart-stage embryos using the nls-TdTomato reporter ( proATML1-nls-TdTomato) as ATML1 itself isalso a direct target of ATML1 (Takada et al., 2013; San-bento et al.,2014). The analysis showed that there was a statistically significantpositive correlation between these signal intensities (Fig. S9F,G).In addition, we also examined whether the nuclear localization of

ATML1 is correlated with the activation of downstream genesFIDDLEHEAD (FDH) and ATML1 in the overexpression line(Takada et al., 2013; Takada, 2013). We found that constitutive

expression of ATML1was able to induce ATML1 and FDH promoteractivity in the L3 layer of cotyledon primordia but not in the stele ofthe hypocotyl in the heart-stage embryos (Fig. S9A-D). Consistentlywith the absence of downstream gene activation, ATML1 proteinaccumulation was weak in the nuclei of the stele (Fig. S9E).

This result suggests that inhibition of nuclear localizationpotentially represses the transcriptional activity of ATML1protein. Considering the weak nuclear localization of ATML1 inthe inner cells, this inhibition might contribute to restrict ATML1activity to the outermost cells.

ZLZ domain was required for the inhibition of ATML1 nuclearlocalization during embryogenesisTo further gain the mechanical insights of the post-transcriptionalrepression of ATML1, we performed domain deletion analyses.Among the four domains of ATML1, we focused on ZLZ andSTART domains because of their potential to interact with otherregulatory molecules: the ZLZ domain is known as a dimerizationmotif and the START domain is predicted as a lipid/sterol-bindingdomain (Schrick et al., 2014). To assess the role of each domain inATML1 localization, we generated transgenic lines that expressed3xGFP-ATML1 fusion genes, with or without deletion of ZLZor START, under the control of the ATML1 promoter ( proML1-3xGFP-ML1, proML1-3xGFP-ML1ΔZLZ and proML1-3xGFP-ML1ΔSTART). GFP signals were detected in both the nuclei andcytoplasm at the heart stage in proML1-3xGFP-ML1 as observed ingATML1-3xGFP plants (8 of 8 lines; Fig. 4A,D,E and Fig. S10A,E).3xGFP-ATML1ΔSTART signals, although slightly decreased in oneline, showed cytoplasmic GFP signals in the epidermis of the heart-stage embryos, which suggests that the START domain is notessential for the inhibition of nuclear localization at this stage (10 of10 lines; Fig. 4C,H,I and Figs S3C and S10C,E). In contrast, 3xGFP-ATML1ΔZLZ signals were not detected in the cytoplasm of theepidermis at the heart stage (10 of 10 lines; Fig. 4B,F,G and Figs S3Cand S10B,E). The wild-type plants did not show fluorescence signalsabove background (Fig. S10D-F). This observation suggests thatthere is an active mechanism to attenuate nuclear localization ofATML1 and that the ZLZ domain is necessary for the inhibition ofATML1 nuclear localization in the epidermis.

Next, to examine the roles of ZLZ and START in subcellularlocalization of ATML1 in the inner cells, we generated estradiol-

Fig. 4. ZLZ domain-deleted ATML1 protein showed enhanced nuclear localization. (A-I) Localization of ATML1 protein variants in proML1-3xGFP-ML1(A,D,E), proML1-3xGFP-ML1ΔZLZ (B,F,G) and proML1-3xGFP-ML1ΔSTART (C,H,I) embryos at the heart stage showing GFP (green) and SR2200 (white)signals. Bottom panels showmagnified views of the top panel. (J,K) Constitutive expression ofGFP-ATML1ΔZLZ (J) andGFP-ATML1ΔSTART (K) in the globular-stage embryos using estradiol-inducible lines. Ovules were cultured with 10 µM estradiol for 2 days. Scale bars: 10 µm.

5

RESEARCH ARTICLE Development (2019) 146, dev169300. doi:10.1242/dev.169300

DEVELO

PM

ENT

inducible lines that expressed the GFP-ATML1ΔZLZ or GFP-ATML1ΔSTART fusion gene under the control of the RPS5Apromoter (RPS5A>>GFP-ML1ΔZLZ and RPS5A>>GFP-ML1ΔSTART). Two days after estradiol induction, GFP-ATML1ΔZLZ and GFP-ATML1ΔSTART signals were primarilyfound in the nuclei of the inner cells at the globular stage (19 of 20 and12 of 12 embryos, respectively), whereas no preferential nuclearaccumulation of ATML1 was observed in the inner cells ofRPS5A>>GFP-ML1 embryos at the same stage (Figs 3M and 4J,K).In addition, we noticed that RPS5A>>GFP-ML1ΔSTART showedweak GFP signals, which suggests that the START-coding sequencemay be required to increase the stability or amount of ATML1 mRNAor ATML1 protein (Fig. 4K). To clearly observe ATML1ΔSTARTlocalization, 3xGFP was fused to ATML1ΔSTART. Constitutiveexpression of 3xGFP-ATML1ΔSTART and 3xGFP-ATML1ΔZLZshowed stronger nuclear localization compared with that of 3xGFP-ATML1 even in the inner cells of the embryos (Fig. S8). These resultssuggest that both ZLZ and START domains are necessary for theinhibition of nuclear localization in the inner cells of the embryos.

Deletion of ZLZ caused accumulation of ATML1 in the innercells of the 32-cell-stage embryosAs GFP signals in proML1-3xGFP-ML1, proML1-3xGFP-ML1ΔZLZ and proML1-3xGFP-ML1ΔSTART were weak at theearly stages compared with those in gATML1-3xGFP, it wasdifficult to observe ATML1ΔZLZ and ATML1ΔSTART proteinlocalization in the early embryos (data not shown). Weak GFPsignals in these transgenic lines at the early stages imply that thereare enhancer sequences in the introns and/or the 3′ downstreamregion of ATML1. Because the ZLZ domain but not the STARTdomain is encoded within a single exon, we were able to delete theZLZ-coding sequence from the gATML1-3xGFP construct withoutaffecting the exon-intron boundary structure to express 3xGFP-ATML1ΔZLZ under the control of the native ATML1 regulatorysequence (gATML1ΔZLZ-3xGFP). gATML1ΔZLZ-3xGFP signalswere detected only in the nuclei even after the 32-cell stage (8 of 8lines), which is consistent with the observation in proML1-3xGFP-ML1ΔZLZ (Fig. 5A-D). Whereas gATML1-3xGFP signals wererestricted to the outermost cells from the 32-cell stage, weakgATML1ΔZLZ-3xGFP signals were detected also in the nuclei of theinner cells at the 32-cell stage (6 of 6 lines; Figs 1O,P and 5A,B).

However, at the heart stage, gATML1ΔZLZ-3xGFP signals werenot detected in the inner cells (8 of 8 lines; Fig. 5C,D). Theseobservations suggest that the ZLZ-coding sequence inhibitsATML1 protein accumulation in the early embryos. Moreover,absence of detectable gATML1ΔZLZ-3xGFP signals in the innercells of the heart-stage embryos implies the existence of ZLZ-coding sequence-independent mechanisms to prevent ATML1protein accumulation.

The ZLZ-coding sequence alone was not fully sufficientfor the post-transcriptional repressions of ATML1To further ascertain whether the ZLZ-coding sequence alone issufficient for the post-transcriptional repression of ATML1, NLS-3xGFP fused with the ZLZ-coding sequence was expressed underthe control of the native ATML1 regulatory sequence (gATML1-nls-3xGFP-ZLZ). gATML1-nls-3xGFP-ZLZ signals were detected onlyin the nuclei even after the 32-cell stage, which suggests that theZLZ domain is not sufficient for the inhibition of nuclearlocalization (10 of 10 lines; Fig. 5E-H and Fig. S3B). We alsoexamined whether GFP signals were detected in the inner cells ofthe gATML1-nls-3xGFP-ZLZ embryos. Whereas gATML1-nls-3xGFP-ZLZ signals were weakly detected in the inner cells of theembryos around the 32-cell stage (9 of 10 lines), GFP signals werenot observed in the inner cells of the heart-stage embryos (10 of 10lines; Fig. 5E-H). These results suggest that ZLZ is sufficient for theinhibition of protein accumulation only in the late-stage embryos.Taken together, we conclude that the ZLZ-coding sequence isnecessary, but not entirely sufficient, for the post-transcriptionalrepressions of ATML1.

DISCUSSIONPosition-dependent and position-independent repressionsof ATML1 activityDespite weak transcriptional activity of ATML1 in the inner cells,ATML1 protein was accumulated only in the outermost cell layer,which implies that ATML1 protein synthesis is inhibited ordegradation of ATML1 mRNA or ATML1 protein is enhanced inan inner cell-specific manner. In addition to position- or cell-type-dependent inhibition, it is also possible that ATML1 proteinaccumulation is reduced equally in both the outermost cells andinner cells. Because the expression of ATML1 mRNA was weak in

Fig. 5. ZLZ domain negatively influenced theprotein accumulation and nuclear localizationof ATML1. (A-D) Localization of 3xGFP-ATML1ΔZLZ in the 32-cell-stage (A,B) and theheart-stage (C,D) embryos in gATML1ΔZLZ-3xGFP. B shows GFP signals (green) in A coloredaccording to the color map (bottom right). D showsa magnified view of the cotyledon in C coloredaccording to the color map. (E-H) Localization ofnls-3xGFP-ZLZ protein, produced under thecontrol of the native ATML1 regulatory sequence,in the 32-cell-stage (E,F) and the heart-stage (G,H)embryos. F shows GFP signals in E coloredaccording to the color map. H shows a magnifiedview of the cotyledon in G, colored according to thecolor map. Scale bars: 10 µm.

6

RESEARCH ARTICLE Development (2019) 146, dev169300. doi:10.1242/dev.169300

DEVELO

PM

ENT

the inner cells, general attenuation of ATML1 protein accumulationin the whole embryo would result in the outermost cell-specificlocalization of ATML1 protein. Although we cannot exclude eitherof these two possibilities, the attenuation of protein accumulation inall cells appears to be more simple and feasible than the inner cell-specific inhibition. Equal accumulation of ATML1 protein in theinner and outermost cells of ATML1-overexpressing embryossupports the latter possibility. Therefore, we assume that thereduction of ATML1 protein accumulation might not be position-dependent.By contrast, nuclear localization of ATML1 in the inner cells

appeared to be strongly inhibited compared with that in theoutermost cells. Firstly, gATML1-3xGFP signals were occasionallydetected in subepidermal cells and these cells showed weakernuclear localization of ATML1 than those of the epidermis.Secondly, in the GFP-ATML1 overexpression experiments,ATML1 protein was present equally both in the nuclei and in thecytoplasm in most of the inner cells of the embryos. Lastly, in fs andhanmutant embryos, ATML1was not clearly localized to the nucleiin the epidermis-derived inner cells. These results suggest that thesubcellular localization of ATML1 is affected not by cell types orcell lineage, but by the position of the cells.

ZLZ might, in combination with other domains, facilitatethe post-transcriptional or post-translational repressionof ATML1Our domain deletion analyses showed that there are ZLZ-dependentmechanisms for post-transcriptional repression of ATML1. TheZLZ-coding sequence was necessary for the repression of ATML1protein accumulation in the inner cells of the 32-cell-stage embryos.Also, the ZLZ-coding sequence alone was sufficient to inhibitprotein accumulation in the inner cells of the heart-stage embryos.However, ATML1ΔZLZ protein accumulation was still restricted tothe outermost cells in the heart-stage embryos, which suggests that aZLZ-independent mechanism also exists to attenuate ATML1protein accumulation in the inner cells. This idea is consistentwith the observation that the ZLZ-coding sequence alone was notsufficient for the inhibition of the nls-3xGFP accumulation in theinner cells of 32-cell-stage embryos.We showed that ATML1ΔZLZ protein was clearly localized to

the nuclei of the outermost cells and the inner cells. However, ZLZdid not decrease the nuclear localization of nls-3xGFP, whichsuggests that ZLZ is necessary, but not sufficient, for the repressionof nuclear localization. This result implies that nuclear localizationis reduced through the combinatory effects of ZLZ and otherdomains. The START domain might be a candidate domain thataffects nuclear localization because ATML1ΔSTART was mainlylocalized to the nuclei in the inner cells of the ATML1ΔSTART-overexpressing embryos. These results suggest that the outermostcells and the inner cells use different mechanisms to reduce nuclearlocalization of ATML1, and that ATML1 nuclear localization in theinner cells was inhibited through ZLZ and START domains.ZLZ and START domains were shown to interact with other

molecules or act as target sites for post-translational modification(Tron et al., 2002; Schrick et al., 2014). It has been reported that theSTART domain of ATML1 stimulated transcription factor activityin yeast in response to increased sterol biosynthesis (Schrick et al.,2014). Therefore, HD-ZIP class IV START domains may beinvolved in post-translational activation of transcription factors.Finding interactors of ZLZ and START domains and analyzing theirfunctions should be promising approaches for future research toelucidate the position-dependent changes of ATML1 activity.

Post-transcriptional repressions may facilitate theposition-dependent cell fate decisionWe propose that both transcriptional activation and post-transcriptional repression are important to restrict ATML1 activityto the outermost cells. Our results suggest that outermost cell-specificlocalization of ATML1 might be explained by the combinatoryeffects of strong ATML1 transcription in the outermost cells and thereduction of ATML1 protein accumulation in the whole embryos.Even if a small amount of ATML1 protein were mistakenlyaccumulated in the inner cells, it would not induce ectopicepidermal cell identity because the inner cell-specific strongattenuation of ATML1 nuclear localization inhibits ATML1 activityin the inner cells. Transcriptional activation appears to play a majorrole in determining ATML1 activity because overexpression ofATML1 induced ectopic epidermal cell differentiation even in theinner cells of the leaves and cotyledons (Takada et al., 2013). Weassume that post-transcriptional repressions of ATML1 counteract anexcess of ATML1 activity.

As the ATML1 promoter contains an L1-box sequence, an ATML1binding site, it is expected that ATML1 directly enhances its ownexpression (Abe et al., 2001). This positive feedback suggests thatonce ATML1 is expressed in a cell, ATML1 expression is maintainedin its daughter cells. However, when epidermal cells accidentallyundergo periclinal cell divisions, inner daughter cells do not maintainepidermal cell identity (Stewart and Dermen, 1975; Takada and Iida,2014). Thus, we speculate that the position-dependent and position-independent post-transcriptional repressions of ATML1 activity arelikely to suppress the positive feedback loop ofATML1 and, therefore,ectopic epidermal cell specification in the inner cells, to enable singleepidermal layer formation.

In the present work, it was not possible to test whether enhancednuclear localization and stabilization of ATML1 affected singleepidermal layer formation. Although ATML1ΔZLZ showednuclear accumulation in the inner cells of the globular-stageembryos, deletion of the dimerization domain eliminates thefunction of ATML1. In addition, NLS-3xGFP-gATML1 and3xGFP-gATML1-NLS, which were able to rescue the embryoniclethal phenotype of atml1-3;pdf2-1, did not cause ectopicepidermal cell differentiation in the inner tissues (Fig. S11 andTable S1). Because reduction of ATML1 protein accumulation canalso counteract ectopic activation of ATML1 in the inner tissues,multiple layers of ATML1 repression mechanisms might mask thedefects of the cytoplasmic retention in NLS-3xGFP-gATML1 and3xGFP-gATML1-NLS.

MATERIALS AND METHODSPlant materials and growth conditionsMutants used in this experiment, atml1-3;pdf2-1, fs-1 and han-30, have beendescribed previously (Torres-Ruiz and Jürgens, 1994; Kanei et al., 2012;Ogawa et al., 2015). Plants were germinated and grown on Murashige andSkoog (MS) plates that contained 0.4% phytagel and 1% sucrose at 22°C.After 2-3 weeks, plants were transferred to soil and grown at 18°C or 22°C.

Ovules were cultured in liquid Nitsch medium (Duchefa Biochemie) thatcontained MES-KOH, vitamins and trehalose dihydrate, as describedpreviously (Gooh et al., 2015). For the estradiol induction, β-estradiol,dissolved in dimethyl sulfoxide (DMSO), was added to the liquid mediumto a final concentration of 10 µM. The same volume of DMSOwas added tothe control medium.

In inhibitor treatment experiments, seedlings or ovules were grown for 24 hin liquidmedia (MS for seedlings andNitsch for ovules) that contained 10 µMMG132 (Sigma-Aldrich) or 2 µM LMB (Santa Cruz Biotechnology). Thesame volume of DMSO (for MG132) or ethanol (for LMB) was added to thecontrol medium.

7

RESEARCH ARTICLE Development (2019) 146, dev169300. doi:10.1242/dev.169300

DEVELO

PM

ENT

Plasmid construction and transgenic plantsgATML1-nls-3xGFP, gATML1-1xGFP and gATML1-3xGFP in pBar vectorA 4.9 kb region including the ATML1 promoter sequence and the 5′untranslated region (from −3055 to +1926) was amplified by polymerasechain reaction (PCR) using primers 11218 and 11109 (primer sequencescan be found in Table S2) and cloned into the KpnI and SmaI sites of thepBluescript KS (pBKS) vector (Stratagene) (KpnI-AscI-gATML1-half-SmaI/pBKS). The ATML1-coding sequence and 3′ downstream sequence(from +1927 to +6190) was amplified by PCR using primers 11110 and11111 (Table S2) and cloned into the SmaI and SacI sites of the KpnI-AscI-gATML1-half-SmaI/pBKS (KpnI-AscI-gATML1-full-SacI/pBKS). Thenls-3xGFP-nost sequence was excised from the nls-3xGFP-nost/pBKSvector (Takada and Jürgens, 2007) and cloned into the SmaI site of theKpnI-AscI-gATML1-full-SacI/pBKS vector (KpnI-AscI-gATML1-nls-3xGFP-nost-SacI/pBKS). The gATML1-nls-3xGFP-nost fragment wasexcised from KpnI-AscI-gATML1-nls-3xGFP-nost-SacI/pBKS with AscIand cloned into the pBar-linker vector, in which an AscI-XhoI linker sequencewas added to the multiple cloning site of the pBar-A vector (GenBank:AJ251013), to generate gATML1-nls-3xGFP/pBar. The gATML1-nls-3xGFP/pBar was used to transform the wild-type Columbia.

Fragments of single and triple GFP sequences followed by three glycinecodons were generated by PCR and cloned into the SmaI site of the KpnI-AscI-gATML1-full-SacI/pBKS vector (KpnI-AscI-gATML1-1xGFP-SacI/pBKS and KpnI-AscI-gATML1-3xGFP-SacI/pBKS). The gATML1-1xGFP and gATML1-3xGFP fragments from the KpnI-AscI-gATML1-1xGFP-SacI/pBKS and KpnI-AscI-gATML1-3xGFP-SacI/pBKS vectorswere inserted into the AscI site of the pBar-linker vector (gATML1-1xGFP/pBar and gATML1-3xGFP/pBar). gATML1-1xGFP/pBar and gATML1-3xGFP/pBar were used to transform atml1-3;pdf2-1/+.

NLS-3xGFP-gATML1 and 3xGFP-gATML1-NLS in pBar vectorAn SV40 NLS sequence was fused to the 5′ end of the 3xGFP reporter genethat lacked the stop codon, and cloned into the pGEM-5zf vector (Promega)(nls-3xGFP-nonstop/5zf). The nls-3xGFP-nonstop sequence was clonedinto the BamHI site of the pBKS-Sma-Gly vector, in which SmaI sites werelocated both upstream and downstream of the BamHI site in pBKS and threeglycine codons were added to the 5′ end of the downstream SmaI site (SmaI-nls-3xGFP-nonstop-Gly-SmaI/pBKS). The triple GFP sequence in thegATML1-3xGFP/pBar vector was replaced with the nls-3xGFP-Glysequence that was excised from the SmaI-nls-3xGFP-nonstop-SmaI-Gly/pBKS vector using SmaI (NLS-3xGFP-gATML1/pBar). NLS-3xGFP-gATML1/pBar was used to transform atml1-3;pdf2-1/+.

A part of the ATML1-coding sequence from +3840 to +5590 relative tothe transcriptional start site was amplified by PCR using primers 11949 and11950 (Table S2). The amplified fragment was cloned into the SacI and SpeIsites of the pBKS vector (SacI-AflII-gATML1-SpeI/pBKS). An SV40 NLSsequence was fused in-frame to the 3′ end, i.e. immediately before the stopcodon, of the partial ATML1-coding sequence in SacI-AflII-gATML1-SpeI/pBKS by PCR-mediated insertion using primers 12004 and 12003(SacI-AflII-gATML1-NLS-SpeI/pBKS; Table S2). The AflII-gATML1-NLS-SpeI fragment that was excised from SacI-AflII-gATML1-NLS-SpeI/pBKS was cloned into the AflII and SpeI sites of the gATML1-3xGFP/pBar(3xGFP-gATML1-NLS/pBar). 3xGFP-gATML1-NLS/pBar was used totransform atml1-3;pdf2-1/+.

proML1-3xGFP-ML1, proML1-3xGFP-ML1ΔZLZ and proML1-3xGFP-ML1ΔSTART in the pBar vectorA BsrGI fragment was excised from the 2xGFP/5zf vector, in which twoGFP-coding sequences were translationally fused and cloned into thepGEM-5zf vector, and inserted into the BsrGI site of the GFP-ATML1/pBKS, at which the GFP-ATML1 fusion gene was cloned in pBKS.Insertion of two BsrGI-GFP fragments resulted in 3xGFP-ML1/pBKS.3xGFP-ML1 was excised from 3xGFP-ML1/pBKS with XhoI and SpeI andinserted into the XhoI and SpeI sites of the proATML1-35St/pBar vector, inwhich the 3.4 kb ATML1 promoter sequence and the SpeI-linker sequencefollowed by the cauliflower mosaic virus 35S terminator sequence (35St)were cloned upstream and downstream of the XhoI site of the pBar-linker,respectively (proML1-3xGFP-ML1/pBar; N. Takada, A.Y., H.I. and S.T.,

unpublished). proML-3xGFP-ML1/pBar was used to transform the wild-type Columbia.

GFP-ATML1without the ZLZ- or START-coding sequence was generatedby PCR amplifying GFP-ATML1/pBKS using primers 6422 and 6423, or6420 and 6421, respectively (Table S2; GFP-ATML1ΔZLZ/pBKS and GFP-ATML1ΔSTART/pBKS). 3xGFP-ML1ΔZLZ and 3xGFP-ML1ΔSTARTwere generated with the same procedure as 3xGFP-ML1/pBKS by addingtwo BsrGI-GFP fragments, and these coding regions were inserted into theXhoI and SpeI sites of the proATML1-35St/pBar vector (proML1-3xGFP-ML1ΔZLZ/pBar and proML1-3xGFP-ML1ΔSTART/pBar). proML-3xGFP-ML1ΔZLZ/pBar and proML-3xGFP-ML1ΔSTART/pBar were used totransform the wild-type Columbia.

RPS5A>>GFP-ML1/ER8, RPS5A>>GFP-ML1ΔZLZ/ER8, RPS5A>>GFP-ML1ΔSTART/ER8, RPS5A>>3xGFP-ML1/ER8, RPS5A>>3xGFP-ML1ΔZLZ/ER8 and RPS5A>>3xGFP-ML1ΔSTART/ER8GFP-ATML1, GFP-ATML1ΔZLZ, GFP-ATML1ΔSTART, 3xGFP-ATML1,3xGFP-ATML1ΔZLZ and 3xGFP-ATML1ΔSTART regions from theGFP-ATML1/pBKS, GFP-ATML1ΔZLZ/pBKS, GFP-ATML1ΔSTART/pBKS, 3xGFP-ATML1/pBKS, 3xGFP-ATML1ΔZLZ/pBKS and 3xGFP-ATML1ΔSTART/pBKS vectors were inserted into the XhoI and SpeI sitesof the RPS5A/ER8, which was described previously (RPS5A>>GFP-ML1/ER8, RPS5A>>GFP-ML1ΔZLZ/ER8, RPS5A>>GFP-ML1ΔSTART/ER8,RPS5A>>3xGFP-ML1/ER8, RPS5A>>3xGFP-ML1ΔZLZ/ER8 andRPS5A>>3xGFP-ML1ΔSTART/ER8; Takada et al., 2013). The wild-typeColumbia was transformed with the RPS5A>>GFP-ML1/ER8,RPS5A>>GFP-ML1ΔZLZ/ER8, RPS5A>>GFP-ML1ΔSTART/ER8,RPS5A>>3xGFP-ML1/ER8, RPS5A>>3xGFP-ML1ΔZLZ/ER8 andRPS5A>>3xGFP-ML1ΔSTART/ER8 vectors.

gATML1ΔZLZ-3xGFP and gATML1-nls-3xGFP-ZLZ in the pBar vectorA part of the ATML1 genomic sequence that included the ZLZ-codingsequence was amplified by PCR using primers 13238 and 13239(Table S2) and inserted into the BamHI and KpnI sites of the pBKS vector(BamHI-NruI-gATML1-AflII-KpnI/pBKS). The ZLZ-coding sequencewas removed from BamHI-NruI-gATML1-AflII-KpnI/pBKS by PCR-based deletion using primers 6422 and 13427 (Table S2; BamHI-NruI-gATML1ΔZLZ-AflII-KpnI/pBKS). The gATML1ΔZLZ fragment fromthe BamHI-NruI-gATML1ΔZLZ-AflII-KpnI/pBKS vector was insertedinto the NruI and AflII sites of the gATML1-3xGFP/pBKS vector usingthe SLiCE method (gATML1ΔZLZ-3xGFP/pBKS; Motohashi, 2015).The gATML1ΔZLZ-3xGFP fragment was inserted into the AscI site ofthe pBar-linker vector (gATML1ΔZLZ-3xGFP/pBar). gATML1ΔZLZ-3xGFP/pBar was used to transform the wild-type Columbia.

To generate gATML1-nls-3xGFP-ZLZ/pBar, the ZLZ-coding sequencewas amplified using primers 13907 and 13908 (Table S2) and inserted intothe BglII site of nls-3xGFP-nonstop/5zf (nls-3xGFP-ZLZ/5zf). The nopalinesynthase terminator (nost) sequence was amplified using primers 13909 and13910 (Table S2) and inserted after the ZLZ-coding sequence of nls-3xGFP-ZLZ/5zf to generate nls-3xGFP-ZLZ-nost/5zf. The nls-3xGFP-ZLZ-nostfragment was excised from nls-3xGFP-ZLZ-nost/5zf and inserted into theBamHI and NotI sites of the pBKS vector (XmaI-BamHI-nls-3xGFP-ZLZ-nost-XmaI-NotI/pBKS). The nls-3xGFP-ZLZ-nost fragment of the resultingconstruct was cloned into the XmaI sites of the gATML1-nls-3xGFP/pBarvector; so that the nls-3xGFP-nost region of gATML1-nls-3xGFP/pBar wasreplaced with nls-3xGFP-ZLZ-nost (gATML1-nls-3xGFP-ZLZ/pBar). Thewild-type Columbia was transformed with the gATML1-nls-3xGFP-ZLZ/pBar vector.

proACR4-nls-TdTomato, proFDH-nls-TdTomato and pATML1-nls-TdTomatoThe ACR4 promoter sequence (from −1222 to +616; gene structureAT3G59420.1 fromAraport 11) was inserted into the XhoI and SmaI sites ofthe pGTV-KAN-derived pHM3 vector (Becker et al., 1992). The TdTomatoreporter gene with an SV40 NLS sequence followed by the 35S terminatorwas cloned downstream of the ACR4 promoter sequence (proACR4-nls-TdTomato-35St/pHM3). The proACR4-nls-TdTomato-35St sequence wasexcised from the pHM3 vector and inserted into the XbaI and EcoRI sites ofthe BinHygTOp-derived pKH3 vector (proACR4-nls-TdTomato/pKH3;

8

RESEARCH ARTICLE Development (2019) 146, dev169300. doi:10.1242/dev.169300

DEVELO

PM

ENT

Höfgen et al., 1994). The proACR4-nls-TdTomato/pKH3 was used totransform the gATML1-3xGFP/pBar line T2#5-4.

The FDH promoter sequence (from −938 to +204; gene structureAT2G26250.1 from Araport 11) was inserted into the HindIII and SmaIsites of the pHM3 vector. The TdTomato reporter gene with an SV40NLS sequence followed by the 35S terminator was cloned downstream ofthe FDH promoter sequence (proFDH-nls-TdTomato-35St/pHM3). TheproFDH-nls-TdTomato/pHM3 was used to transform the RPS5A>>ATML1line #7-1.

The nls-TdTomato reporter genewas inserted intoXhoI and SpeI sites of theproATML1-35St/pBar vector to generate proATML1-nls-TdTomato/pBar.The proATML1-nls-TdTomato/pBar was used to transform the wild-typeColumbia. A T3 line #5-2, homozygous for the proATML1-nls-TdTomato/pBar transgene, was crossed with the gATML1-3xGFP/pBar line T2#5-4.

Confocal laser scanning microscope and imaging analysisFor GFP, TdTomato, SR2200, 4′,6-diamidino-2-phenylindole (DAPI) andpropidium iodide (PI) fluorescence observation, an LSM710 (Carl Zeiss) orFV1000 (Olympus) confocal laser scanning microscope was used. Embryoswere fixed in 4% paraformaldehyde and 5% glycerol solution in phosphate-buffered saline (PBS) (4% PFA) before observation. For SR2200 staining,SR2200 was added to 4% PFA (2 µl/ml). For PI staining, roots weremounted in 10 µg/ml PI solution.

Intensity of GFP or TdTomato signals was measured using the imagingsoftware Fiji (fiji.sc). To measure transcriptional activity of ATML1protein, a homozygous gATML1-3xGFP line was transformed with theproACR4-nls-TdTomato/pKH3 vector, and T2 generation plants carryingproACR4-nls-TdTomatowere used for embryo isolation and observation. Tonormalize the TdTomato signal intensity, the average of TdTomato signalintensity in each embryo was set to one.

The cytoplasmic-to-nuclear signal ratios shown in Fig. S3 were measuredusing Fiji. Nuclear regions were visualized by staining with DAPI. Anoptical section with the largest nuclear area from a confocal z-stack was usedfor quantification in each cell.

Western blot analysisProteins were extracted from ∼100 3 day-old seedlings in lysis buffer[125 mM Tris-HCl (pH 8.8), 1% sodium dodecyl sulfate (SDS), 10%glycerol, 50 mM sodium metabisulfite] and 5% of the total samples weresubjected to SDS-polyacrylamide electrophoresis using a 10% gel. Theseparated protein was blotted onto Hybond-P PVDF membrane(Amersham) using an electroblotter Mini-PROTEAN II Cell (Bio-Rad).The blotted membranes were blocked for 1 h in 4% skimmed milk in PBSwith 0.1% Tween-20 (PBST) (pH 7.0) at room temperature and incubatedwith a mouse monoclonal antibody against GFP (1:1000; Roche,11814460001) for 1 h at room temperature. Membranes were washed inPBST and incubated with an ECL peroxidase-labeled anti-mouse secondaryantibody (1:10,000; Amersham, NA931-100UL) for 1 h. After washing, thesignals were detected on ChemiDoc XRS (Bio-Rad) using SuperSignalWest Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific).

AcknowledgementsWe thank Prof. Nam-Hai Chua (Rockefeller University, USA), Prof. Taku Takahashi(Okayama University, Japan), Dr Ulrike Mayer (University of Tubingen, Germany)and Dr Gorou Horiguchi (RikkyoUniversity, Japan) for the ER8 vector, atml1-3;pdf2-1/+ seeds, fs-1 seeds and han-30 seeds, respectively. We also thank Dr ShunsukeMiyashima (Nara Institute of Science and Technology, Japan) and Hirofumi Ohmori(Osaka University, Japan) for sharing a method for cell wall staining and DNAsequencing work, respectively. We greatly acknowledge Prof. Tatsuo Kakimoto(Osaka University, Japan) for supporting our projects in his laboratory. We alsothank all other members of the plant growth and development laboratory for theirhelpful comments and discussions.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsConceptualization: H.I., A.Y., S.T.; Investigation: H.I., A.Y., S.T.; Writing - originaldraft: H.I.; Writing - review & editing: H.I., S.T.; Supervision: S.T.; Projectadministration: S.T.; Funding acquisition: H.I., S.T.

FundingThis work was supported by grants from the Japan Society for the Promotion ofScience to S.T. (JP20657012, JP22687003, JP23657036, JP26440142 andJP18K06286) and to H.I. (JP16J00702).

Supplementary informationSupplementary information available online athttp://dev.biologists.org/lookup/doi/10.1242/dev.169300.supplemental

ReferencesAbe, M., Takahashi, T. and Komeda, Y. (2001). Identification of a cis-regulatory

element for L1 layer-specific gene expression, which is targeted by an L1-specifichomeodomain protein. Plant J. 26, 487-494.

Abe, M., Katsumata, H., Komeda, Y. and Takahashi, T. (2003). Regulation ofshoot epidermal cell differentiation by a pair of homeodomain proteins inArabidopsis. Development 130, 635-643.

Ariel, F. D., Manavella, P. A., Dezar, C. A. and Chan, R. L. (2007). The true story ofthe HD-Zip family. Trends Plant Sci. 12, 419-426.

Becker, D., Kemper, E., Schell, J. and Masterson, R. (1992). New plant binaryvectors with selectable markers located proximal to the left T-DNA border. PlantMol. Biol. 20, 1195-1197.

Camilleri, C., Azimzadeh, J., Pastuglia, M., Bellini, C., Grandjean, O. andBouchez, D. (2002). The Arabidopsis TONNEAU2 gene encodes a putative novelprotein phosphatase 2A regulatory subunit essential for the control of the corticalcytoskeleton. Plant Cell 14, 833-845.

Crawford, K. M. and Zambryski, P. C. (2000). Subcellular localization determinesthe availability of non-targeted proteins to plasmodesmatal transport. Curr. Biol.10, 1032-1040.

Gallagher, K. L., Paquette, A. J., Nakajima, K. and Benfey, P. N. (2004).Mechanisms regulating SHORT-ROOT intercellular movement. Curr. Biol. 14,1847-1851.

Gooh, K., Ueda, M., Aruga, K., Park, J., Arata, H., Higashiyama, T. and Kurihara,D. (2015). Live-cell imaging and optical manipulation of Arabidopsis earlyembryogenesis. Dev. Cell 34, 242-251.

Hofgen, R., Axelsen, K. B., Kannangara, C. G., Schuttke, I., Pohlenz, H. D.,Willmitzer, L., Grimm, B. and von Wettstein, D. (1994). A visible marker forantisense mRNA expression in plants: inhibition of chlorophyll synthesis with aglutamate-1-semialdehyde aminotransferase antisense gene. Proc. Natl. Acad.Sci. USA 91, 1726-1730.

Kanei, M., Horiguchi, G. and Tsukaya, H. (2012). Stable establishment ofcotyledon identity during embryogenesis in Arabidopsis by ANGUSTIFOLIA3 andHANABA TARANU. Development 139, 2436-2446.

Kim, I., Kobayashi, K., Cho, E. and Zambryski, P. C. (2005). Subdomains fortransport via plasmodesmata corresponding to the apical–basal axis areestablished during Arabidopsis embryogenesis. Proc. Natl. Acad. Sci USA 102,11945-11950.

Lu, P., Porat, R., Nadeau, J. A. and O’Neill, S. D. (1996). Identification of ameristem L1 layer-specific gene in Arabidopsis that is expressed duringembryonic pattern formation and defines a new class of homeobox genes.Plant Cell 8, 2155-2168.

Motohashi, K. (2015). A simple and efficient seamless DNA cloning method usingSLiCE from Escherichia coli laboratory strains and its application to SLiP site-directed mutagenesis. BMC Biotechnol. 15, 47.

Mukherjee, K. andBurglin, T. R. (2006). MEKHLA, a novel domain with similarity toPAS domains, is fused to plant homeodomain-leucine zipper III proteins. PlantPhysiol. 140, 1142-1150.

Nawy, T., Bayer, M., Mravec, J., Friml, J., Birnbaum, K. D. and Lukowitz, W.(2010). The GATA factor HANABATARANU is required to position the proembryoboundary in the early Arabidopsis embryo. Dev. Cell 19, 103-113.

Nodine, M. D. and Bartel, D. P. (2010). MicroRNAs prevent precocious geneexpression and enable pattern formation during plant embryogenesis. GenesDev. 24, 2678-2692.

Ogawa, E., Yamada, Y., Sezaki, N., Kosaka, S., Kondo, H., Kamata, N., Abe, M.,Komeda, Y. and Takahashi, T. (2015). ATML1 and PDF2 play a redundant andessential role in Arabidopsis embryo development. Plant Cell Physiol. 56,1183-1192.

San-Bento, R., Farcot, E., Galletti, R., Creff, A. and Ingram, G. (2014). Epidermalidentity is maintained by cell–cell communication via a universally active feedbackloop in Arabidopsis thaliana. Plant J. 77, 46-58.

Saulsberry, A., Martin, P. R., O’Brien, T., Sieburth, L. E. and Pickett, F. B. (2002).The induced sector Arabidopsis apical embryonic fate map. Development 129,3403-3410.

Schrick, K., Bruno, M., Khosla, A., Cox, P. N., Marlatt, S. A., Roque, R. A.,Nguyen, H. C., He, C., Snyder, M. P., Singh, D. et al. (2014). Shared functions ofplant and mammalian StAR-related lipid transfer (START) domains in modulatingtranscription factor activity. BMC Biol. 12, 70.

Sessions, A., Weigel, D. and Yanofsky, M. F. (1999). The Arabidopsis thalianaMERISTEM LAYER 1 promoter specifies epidermal expression in meristems andyoung primordia. Plant J. 20, 259-263.

9

RESEARCH ARTICLE Development (2019) 146, dev169300. doi:10.1242/dev.169300

DEVELO

PM

ENT

Stewart, R. N. and Dermen, H. (1975). Flexibility in ontogeny as shown by thecontribution of the shoot apical layers to leaves of periclinal chimeras. Am. J. Bot.62, 935-947.

Sulston, J. E., Schierenberg, E., White, J. G. and Thomson, J. N. (1983). Theembryoniccell lineageof thenematodeCaenorhabditiselegans.Dev.Biol.100, 64-119.

Takada, S. (2013). Post-embryonic induction of ATML1-SRDX alters themorphology of seedlings. PLoS ONE 8, e79312.

Takada, S. and Iida, H. (2014). Specification of epidermal cell fate in plant shoots.Front. Plant Sci. 5, 49.

Takada, S. and Jurgens, G. (2007). Transcriptional regulation of epidermal cell fatein the Arabidopsis embryo. Development 134, 1141-1150.

Takada, S., Takada, N. and Yoshida, A. (2013). ATML1 promotes epidermal celldifferentiation in Arabidopsis shoots. Development 140, 1919-1923.

Tanaka, H., Watanabe, M., Watanabe, D., Tanaka, T., Machida, C. and Machida,Y. (2002). ACR4, a putative receptor kinase gene of Arabidopsis thaliana, that isexpressed in the outer cell layers of embryos and plants, is involved in properembryogenesis. Plant Cell Physiol. 43, 419-428.

Torres-Ruiz, R. A. and Jurgens, G. (1994). Mutations in the FASS gene uncouplepattern formation and morphogenesis in Arabidopsis development. Development120, 2967-2978.

Tron, A. E., Bertoncini, C. W., Chan, R. L. and Gonzalez, D. H. (2002). Redoxregulation of plant homeodomain transcription factors. J. Biol. Chem. 277,34800-34807.

van den Berg, C., Willemsen, V., Hage, W., Weisbeek, P. and Scheres, B. (1995).Cell fate in the Arabidopsis root meristem determined by directional signalling.Nature 378, 62-65.

Zhang, F., Zuo, K., Zhang, J., Liu, X., Zhang, L., Sun, X. and Tang, K. (2010). AnL1 box binding protein, GbML1, interacts with GbMYB25 to control cotton fibredevelopment. J. Exp. Bot. 61, 3599-3613.

Zhao, Y., Medrano, L., Ohashi, K., Fletcher, J. C., Yu, H., Sakai, H. andMeyerowitz, E. M. (2004). HANABA TARANU is a GATA transcription factor thatregulates shoot apical meristem and flower development in Arabidopsis. PlantCell 16, 2586-2600.

10

RESEARCH ARTICLE Development (2019) 146, dev169300. doi:10.1242/dev.169300

DEVELO

PM

ENT