The MYB107 Transcription Factor Positively Regulates ... · 1999; Graça et al., 2002; Li-Beisson et al., 2013). So far, several sets of analogous enzymes and/or proteins in-volved

The MYB107 Transcription Factor Positively RegulatesSuberin Biosynthesis1[OPEN]

Mingyue Gou, Guichuan Hou, Huijun Yang2, Xuebin Zhang, Yuanheng Cai, Guoyin Kai3, andChang-Jun Liu*

Biology Department, Brookhaven National Laboratory, Upton, New York 11973 (M.G., H.Y., X.Z., Y.C., G.K.,C.-J.L.); and Appalachian State University, Boone, North Carolina 28608-2027 (G.H.)

ORCID IDs: 0000-0001-8855-6617 (M.G.); 0000-0002-3856-056X (G.H.); 0000-0002-9012-8491 (X.Z.); 0000-0001-6189-8756 (C.-J.L.).

Suberin, a lipophilic polymer deposited in the outer integument of the Arabidopsis (Arabidopsis thaliana) seed coat, represents anessential sealing component controlling water and solute movement and protecting seed from pathogenic infection. Althoughmany genes responsible for suberin synthesis are identified, the regulatory components controlling its biosynthesis have notbeen definitively determined. Here, we show that the Arabidopsis MYB107 transcription factor acts as a positive regulatorcontrolling suberin biosynthetic gene expression in the seed coat. MYB107 coexpresses with suberin biosynthetic genes in atemporal manner during seed development. Disrupting MYB107 particularly suppresses the expression of genes involved insuberin but not cutin biosynthesis, lowers seed coat suberin accumulation, alters suberin lamellar structure, and consequentlyrenders higher seed coat permeability and susceptibility to abiotic stresses. Furthermore, MYB107 directly binds to the promotersof suberin biosynthetic genes, verifying its primary role in regulating their expression. Identifying MYB107 as a positiveregulator for seed coat suberin synthesis offers a basis for discovering the potential transcriptional network behind one of themost abundant lipid-based polymers in nature.

The seed coat is an outer covering of the seed thatprotects the embryo and other seed components againstdiverse environmental agents, including biotic and abi-otic stresses (Mohamed-Yasseen et al., 1994). Thematureseed coat of Arabidopsis (Arabidopsis thaliana) is com-posed mostly of five cell layers of maternal origin. Dur-ing development, its outer integument (the outer twocell layers) undergoes extensive secondary thickeningand becomes a sclerotic layer (Beeckman et al., 2000;

Moïse et al., 2005),where suberin, a lipophilic polymer, isdeposited and functions as the primary sealing compo-nent, contributing to the seed coat’s impermeability towater and nutrients (Molina et al., 2006, 2008). The defectof suberin biosynthesis in some mutant lines, such asglycerol-3-phosphate acyltransferase5-1 (gpat5-1), rendershighly permeable seed coats, which is correlated withdecreased dormancy and capacity of the seeds to ger-minate after storage (Beisson et al., 2007).

Suberin and cutin are two types of apoplastic poly-mers responsible for the sealing property of plant tis-sues. Chemically, both polymers are glycerol-based,aliphatic polyesters impregnated with waxes (Frankeet al., 2005; Molina et al., 2006). Suberin differs fromcutin mainly as it consists of more abundant aromaticcomponents (Bernards and Razem, 2001) and the lon-ger chain-length fatty acyl constituents (Moire et al.,1999; Graça et al., 2002; Li-Beisson et al., 2013). So far,several sets of analogous enzymes and/or proteins in-volved in suberin and cutin biosynthesis have beencharacterized, including the b-ketoacyl-CoA synthase(KCS) responsible for fatty acid elongation (Frankeet al., 2009; Lee et al., 2009), the cytochrome P450CYP77A, CYP86A, and CYP86B family members in-volved in fatty acid oxygenation (Wellesen et al., 2001;Xiao et al., 2004; Rupasinghe et al., 2007; Höfer et al.,2008; Compagnon et al., 2009; Li-Beisson et al., 2009),the long-chain acyl-CoA synthases for fatty acid acti-vation (Schnurr et al., 2004; Bessire et al., 2007; Lü et al.,2009), the fatty acyl-CoA reductases (FARs) for fattyacid reduction (Domergue et al., 2010), and theenzymes responsible for subsequent acyltransfer to

1 This work was supported by the Division of Chemical Sciences,Geosciences, and Biosciences, Office of Basic Energy Sciences, U.S.Department of Energy (grant no. DEAC0298CH10886 to C.-J.L.)and the National Science Foundation (grant no. MCB-1051675 toC.-J.L).

2 Present address: Department of Plant Pathology and Plant-Microbe Biology, Cornell University, Ithaca, NY 14853-4203.

3 Present address: College of Life and Environment Sciences,Shanghai Normal University, Shanghai 200234, People’s Republicof China.

* Address correspondence to [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Chang-Jun Liu ([email protected]).

M.G. and C.-J.L. designed the experiments, analyzed and inter-preted the data, and wrote the article; C-.J.L. conceived the initialidentification of the transcription factor; M.G. performed most ofthe experiments; G.H. conducted the electron microscopic studies;H.Y. conducted the RNA-seq data processing; X.Z., Y.C., and G.K.contributed to the experiments; all authors edited the article.

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glycerol-3-phosphate (i.e. the GPATs; Beisson et al.,2007; Li et al., 2007; Li-Beisson et al., 2009). In addi-tion, a few close homologs of BAHD acyltransfer-ases, namely HYDROXYCINNAMOYL-COENZYMEA:v-HYDROXYACIDO-HYDROXYCINNAMOYLTRANS-FERASE/ALIPHATIC SUBERIN FERULOYL TRANS-FERASE (HHT/ASFT; Gou et al., 2009; Molina et al.,2009), DEFECTIVE IN CUTIN FERULATE (DCF;Rautengarten et al., 2012), and FATTY ALCOHOL:CAFFEOYL-COENZYME A CAFFEOYL TRANS-FERASE (FACT; Kosma et al., 2012), were character-ized for incorporating aromatics into suberin, cutin,and/or some of suberin-associated waxes. Specifi-cally, HHT/ASFT acts as the key enzyme transfer-ring ferulate to aliphatic compositions of suberin,yielding suberin aromatic esters (Gou et al., 2009;Molina et al., 2009). Disrupting suberin biosyntheticgenes, as in the mutants gpat5-1, far1far4far5, or reducedlevels of wall-bound phenolics1-1 (rwp1-1)/asft, enhancesseed coat permeability (Beisson et al., 2007; Gou et al.,2009; Molina et al., 2009; Vishwanath et al., 2013),supporting the primary barrier function of suberin inthe seed coat.

Despite the core reactions in suberin and cutin bio-synthesis being comparable, the deposition of twopolyesters and the expression of their correspondingbiosynthetic genes are highly tissue/organ specific. InArabidopsis, cutin, as a major structural component ofcuticles, is presented on the outermost epidermal cellwalls of virtually every aerial organ, whereas suberin isfound primarily in the outer integument layer of the seedcoat and in the peridermal cells of mature root and theendodermal cells of young root (Pollard et al., 2008;Nawrath et al., 2013). Consistently, nearly the entire setof suberin biosynthetic genes identified in Arabidopsisdisplay preferential expression in the seed and/or root(Pollard et al., 2008; Schreiber, 2010; Beisson et al., 2012;Andersen et al., 2015), whereas the genes required forcuticle formation typically are expressed in the epider-mis of stem, leaf, and/or flower (Wellesen et al., 2001; Liet al., 2007; Li-Beisson et al., 2009). This strict tissue-specific distribution suggests the existence of a sophis-ticated regulation mechanism at the transcription levelthat governs the polyester’s synthesis and depositionprocesses. Several transcription factors (TFs) were im-plicated in the formation of cuticle (wax and cutin) inArabidopsis, including members of the ethylene-response factor family, WAX INDUCER1/SHINE1(WIN1/SHN1), SHN2, and SHN3 (Aharoni et al., 2004;Broun, 2004; Broun et al., 2004; Kannangara et al., 2007;Shi et al., 2011), and the MYB TFs MYB106 and MYB16(Aharoni et al., 2004; Broun, 2004; Cominelli et al., 2008;Yeats and Rose, 2013). However, the regulatory mecha-nism and the related TFs specifically involved in thetissue/organ-specific biosynthesis of suberin have notbeen definitively determined. In this study,we identifiedaMYBTF,MYB107, that functions as a positive regulatorspecifically controlling suberin gene expression in de-veloping seeds of Arabidopsis and, consequently, af-fecting seed coat permeability.

RESULTS

Altered Seed Coat Permeability of the myb107 Mutant

Previous studies from our and other groups (Gouet al., 2009; Rautengarten et al., 2012) and in silico geneexpression data (Supplemental Fig. S1) revealed thatArabidopsis HHT and DCF, the two phylogeneticallyrelated BAHD genes responsible for suberin and cutinaromatic synthesis, displayed distinct tissue-specific ex-pression patterns. HHT is expressed predominantly indeveloping seeds and roots, whereas DCF is expressedmainly in epidermis of aerial tissues. In attemptingto identify the regulatory components governingtheir tissue-specific expression, we used HHT/AFST(At5g41040) and DCF (At3g48720) genes as the baits tosearch for their coexpression partners. Among a set ofrecognized genes (Supplemental Table S1), we foundthat HHT exhibited high coexpression with a few an-notated TF genes, including one encoding a MYBdomain-containing protein,MYB107 (Dubos et al., 2010).Intriguingly, in silico gene expression data also indicatethat MYB107 expresses primarily in developing seeds(Supplemental Fig. S2) and coexpresses with most ofthe suberin biosynthetic genes (Supplemental Table S2),which indicates its involvement in suberin synthesis.

Quantitative real-time (qRT)-PCR further revealedthat the transcripts of both MYB107 and HHT werestrikingly high in siliques but nearly absent in shootsand leaves, confirming their tissue-specific expressionpatterns and their coexpression (Fig. 1A). To assessMYB107 functions, two homozygous T-DNA insertionmutants were isolated: SAIL_242_B04, with T-DNAinserted in the 59 untranslated region (designatedmyb107-1); and SALK_203615, with T-DNA in thefirst intron of the coding region (designated myb107-2;Fig. 1B). Those insertions led to substantial reduction ofMYB107 transcripts in both mutant lines (Fig. 1C;Supplemental Fig. S3A). Under normal growth condi-tions, the mutant (represented by myb107-1) was mor-phologically identical to the Columbia-0 (Col-0) wildtype throughout the entire developmental process(Supplemental Fig. S4, A and B); neither its fertility norseed setting was affected, except that its mature seedswere slightly darker than those of the wild type(Supplemental Fig. S4C), a feature reminiscent of thesuberin biosynthetic mutant gpat5-1 (Beisson et al.,2007). Interestingly, compared with the wild type andthe HHTmutant rwp1-1 (Gou et al., 2009), the seed coatpermeability of both myb107-1 and myb107-2 exhibitedsubstantial increase; much stronger red coloration wasdeveloped after their mature seeds were stainedwith tetrazolium salt, a cation dye that normallycannot penetrate the Arabidopsis seed coat (Fig. 1E;Supplemental Fig. S3B). Becausemyb107-1 andmyb107-2show similar phenotypes, only myb107-1 was furthercharacterized for the following studies (for simplicity,myb107-1 is referred to as myb107 hereafter).

To determine whether the increased permeability ofmyb107 is attributable to the compromised MYB107

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gene expression, we first examined the seed coat per-meability of a population of the segregated progeny of amyb107 heterozygous mutant line (myb107/MYB107)using Tetrazolium Red. Out of 22 progeny, only seedsfrom six homozygous plants exhibited enhanced redstaining, whereas heterozygous and non-T-DNA in-sertion seeds did not (Supplemental Fig. S5), demon-strating a strict cosegregation of the T-DNA insertionwith the enhanced seed coat permeability. Next,myb107 was complemented with a genomic DNAfragment spanning theMYB107 native promoter and itsfull-length gene. A higher abundance of MYB107 tran-scripts was detected in the siliques of the complemen-tation lines compared with myb107 (Supplemental Fig.S6A); correspondingly, the mature seeds of the com-plementation lines showed abrogated Tetrazolium Redstaining similar to the wild type (Fig. 1F). These resultsindicate that MYB107 is essential for maintaining seedcoat impermeability.We then examined whether the altered seed coat

permeability of myb107 leads to alteration of its seedphysiological property under salinity, osmotic, andoxidative conditions that mimic environmentalstresses. While no significant germination differenceswere observed for the seeds of myb107, the wild type,

and the complementation lines placed on plain one-half-strength Murashige and Skoog (MS) medium(Fig. 2, A and E), the percentage of seedling establish-ment ofmyb107was notably lower than that of the wildtype in all treatments (Fig. 2, B–E). Particularly, whenmyb107 seeds were grown on the medium supple-mented with 5 mM H2O2, the seedling establishmentwas reduced almost 80% compared with that ofthe wild type (Fig. 2, D and E). Conversely, thecomplementation lines exhibited a rescued seedlingestablishment similar to the wild type in all treatments(Fig. 2, B–E). These data indicate that down-regulationof MYB107 raises the susceptibility of seeds to abioticstresses.

Decrease of Suberin Monomers in the myb107 Seed Coat

We then testedwhether the enhanced permeability ofthe seed coat ofmyb107was due to the defect in suberinsynthesis. HPLC analysis of the depolymerized seedcoat cell wall revealed that the content of ferulate andcaffeate esters, the primary phenolics in seed coat su-berin (Gou et al., 2009; Molina et al., 2009; Kosma et al.,2012), was reduced up to 50% to 60% in myb107 seeds

Figure 1. Characterization of the myb107 mutant. A, Relative expression levels of MYB107 and HHT in different tissues. Theexpression levels of both genes in seedlings were set as 1. B, Diagram of the T-DNA insertion mutant of MYB107. The trianglesindicate the sites of T-DNA insertion. The dashed line marks the region for qRT-PCR analysis of MYB107 gene expression in D.C, Genotyping of the T-DNA insertion mutant by PCR using T-DNA left-border primer (LB1) and two pairs of MYB107-specificprimers (LPand RP). D, Relative expression levels ofMYB107 in siliques of the wild type (WT) andmyb107-1mutant revealed byqRT-PCR. E, myb107-1, rwp1-1, and wild-type seeds stained with tetrazolium salt for 12 h. F, Tetrazolium-stained seeds of thewild type,myb107-1, and three individual transgenic lines ofmyb107-1 harboring the pMYB107::MYB107 construct. The data inA and D represent means 6 SD from three experimental repeats.

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MYB107 Controls Seed Coat Suberin Biosynthesis






compared with those of the wild type (Fig. 3, A and B).Conversely, the accumulation levels of both ferulate andcaffeate were restored close to those of the wild type inthe complementation lines (Supplemental Fig. S6B).

GC-MS resolved a set of aliphatic monomers fromseed polyester methanolysates. In general, nearly all thedetected aliphatics in the seed polyesters of myb107showed substantial reduction compared with the wildtype; in particular, the dominant compositions of C24:0dicarboxylic acid and C24:0 v-hydroxy acid displayedup to a 50% decrease (Fig. 3C; Supplemental Fig. S7).These data suggest that MYB107 affects the synthesis ofboth suberin aromatics and aliphatics in the seed coat.

However, when suberin methanolysates from rootswere examined, there was no difference in the contentof resolved aromatics between myb107 and wild-typeplants (Supplemental Fig. S8A). Similarly, the abun-dance of all the detected aliphatic monomers remainedunchanged in myb107 root polyesters compared withthe wild type (Supplemental Fig. S8B). Consistently, nodifference was observed when wild-type and myb107roots were stained with Sudan Black B (SupplementalFig. S8C), a lipophilic dye used for the histochemicaldetection of suberin and associated waxes (Robb et al.,1991). These data suggest that disruptingMYB107 doesnot affect root suberin synthesis.

The Ultrastructures of Seed Coat Suberin andSurface Cuticles

Examining the epidermal surface of different organsof the wild type and myb107 by scanning electron

microscopy (SEM), we found that both wild-type andmyb107 seeds displayed the same preserved surfacestructure with the thick cell walls of columella(Supplemental Fig. S9, A and B). Similarly, there wereno notable morphological changes on the leaf sur-face, trichomes, the nanoridges of petals, and thecuticular wax crystals distributed on stem surfaces(Supplemental Fig. S9, C–E). Furthermore, transmis-sion electron microscopy (TEM) of the leaf epidermalcells of the wild type and myb107 revealed similarelectron-dense cuticular layers on the outer surfaces ofthe cells and the identical cuticular ledges (projections)surrounding the stomatal pores (Supplemental Fig. S9,F and G). Consistently, when myb107 and wild-typeplants were stained with Toluidine Blue, an aqueousdye monitoring plants with defective surface cuticles(Tanaka et al., 2004), no obvious difference was ob-served (Supplemental Fig. S10). These data suggest thatcuticular layers of the aerial epidermis were not defec-tive in myb107.

However, when the ultrastructures of the seed coatsof myb107 and the wild type were examined by TEM,the finely arranged and largely continuous lamellarstructures of suberin were found near the brown pig-ment layer of the wild-type seed coat, as reported pre-viously (Yadav et al., 2014), where they showed regularalternating light- and dark-staining bandswith the lightbands spaced around 7 to 10 nm apart (Fig. 4), whichwere predicted to represent the aromatic and aliphaticdomains of suberin, respectively (Bernards, 2002; Graçaand Santos, 2007). In contrast, the lamellation ofthe suberin layer was largely disordered, with seem-ingly shorter, thicker, but discontinuous light-staining

Figure 2. Characterization of themyb107mutant under abiotic stresses. A to D, Seven-day-old seedlings of the wild type (WT),myb107, and two complementation lines ofmyb107 harboring pMYB107::MYB107 (L1 and L3) germinated on a plain one-half-strength MS plate (A) and plates containing 150 mM NaCl (B), 300 mM mannitol (C), and 5 mM H2O2 (D). E, Percentage of theestablished seedlings in A to D. The data represent means 6 SD of three replicates with approximately 80 seeds for each repeat.Asterisks indicate statistical differences compared with the wild type (Student’s t test, ***P , 0.001).


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strands in the myb107 mutant seed coat (Fig. 4C).This observation suggests that knocking downMYB107 impairs the seed coat suberin ultrastructuralarrangement.

Alteration of Suberin Biosynthetic Gene Expression

The observed drastic effects of MYB107 on seed coatsuberin synthesis and assembly prompted us to assessthe potential function of MYB107 as a putative TF inregulating suberin biosynthetic gene expression. First,we explored the genes misregulated inmyb107 by RNAsequencing (RNA-seq) analysis. Since MYB107 isexpressed dominantly in siliques (Fig. 1A), RNAs frommixed siliques of different developmental stages wereextracted and used in the study. Analyzing RNA-seqdata sets revealed that most genes known to be in-volved in the biosynthesis of suberin and its associatedwaxes were down-regulated in the myb107 mutant tonear or above 2-fold; they include not only HHT but

also the genes FACT, CYP86A1, CYP86B1, FAR1, FAR4,and FAR5 (Table I; Supplemental Data Set S1). In con-trast, the transcript abundance of cutin biosyntheticgenes, such as CYP86A2, CYP86A8, CYP77A6, DCF,GPAT6, and GPAT8, showed no significant changes(Table I; Supplemental Data Set S1).

In general, qRT-PCR analyses of two independentbiological repeats using RNAs extracted from themixed siliques (repeats 1 and 2 in Table I) validated thetranscriptional changes of those suberin biosyntheticgenes found in the RNA-seq data sets (Table I). LACS2and GPAT4 that encode enzymes known for cutinsynthesis also appeared to be down-regulated inmyb107 (Table I), which agrees with their proposedadditional roles in suberin biosynthesis (Li-Beissonet al., 2013). The expression of KCS2 and KCS20, how-ever, was not affected in myb107. KCS2 and KCS20 arethe fatty acid elongases functionally redundant in thetwo-carbon elongation to very-long-chain fatty acidsthat is required for the biosynthesis of cuticular waxesand root suberin (Franke et al., 2009; Lee et al., 2009).

Figure 3. Aromatic and aliphatic monomers of seed coat polyesters by HPLC and gas chromatography-mass spectrometry (GC-MS). A, Portion of UV light-HPLC profiles of the phenolics from seed coat suberin of the wild type (WT) and myb107. Numbersrepresent the identified phenolics: 1, ferulate; 2, caffeate; 3, sinapate; 4, coumarate. B, Contents of aromatic compounds in seedcoat suberin quantified byHPLC. C, Contents of aliphatic monomers, including fatty alcohols, diols,v-hydroxy fatty acids (v-OHFAs), dicarboxylic acids (DCA), and fatty acids (FAs), in cell wall residues of seeds. The data represent means 6 SD from threebiological replicates. Asterisks indicate statistical differences compared with the wild type (Student’s t test, ***P, 0.001 [B] and*P , 0.05 [C]). CW, Cell wall; DW, dry weight.







The recently identified suberin ATP-binding cassettetransporter genesABCG2,ABCG6, andABCG20 (Yadavet al., 2014) did not experience significant down-regulation in myb107 (Table I), suggesting thatMYB107 does not affect the expression of genes in-volved in the process of polyester monomer transport/deposition. In addition, genes involved in seed coatpigmentation, namely the TRANSPARENT TESTA (TT)genes (Debeaujon et al., 2000; Appelhagen et al., 2014),also did not show significant changes (SupplementalTable S3; Supplemental Data Set S1).

Conversely, when transcripts of suberin biosyntheticgenes, represented by FAR1 and FACT, were examinedin complementation lines of myb107, we found thattheir defective expression in siliques could be restoredto levels even slightly higher than those of the wild type(Supplemental Fig. S11A), confirming that MYB107 issufficient for activating seed suberin biosynthetic geneexpression.

Coexpression of MYB107 with Seed Suberin BiosyntheticGenes in a Temporal Manner

In the above RNA-seq and qRT-PCR experimentsusing RNAs from mixed siliques, the detected changesin expression levels of suberin biosynthetic genes inmyb107 were somehow less substantial. We suspectedthat MY107 may temporally regulate suberin biosyn-thetic gene expression at a specific seed developmentalstage and that the real differences might have been di-luted when RNAs of the mixed siliques were used.Therefore, we dissectedMYB107 expression patterns atdifferent seed developmental stages and examined itspotential correlation with other suberin biosyntheticgenes. Total RNAs were extracted from seeds at fivedefined developmental stages (Supplemental Fig. S12):stages I (1–5 d after flowering [DAF]), II (6–10 DAF), III(11–15 DAF), IV (16–20 DAF), and V (mature seeds).qRT-PCR analyses revealed that the relative expression

Figure 4. TEM observation of suberinultrastructures in seed coats of the wildtype (WT) and myb107. Mature seedswere ultrathin sectioned, and the sec-tions (90–100 nm) were treated with10% H2O2 and then stained with 1%aqueous uranyl acetate and Sato lead.Enlarged images from the squared areasin A are shown in B, and enlarged imagesfrom the squared areas in B are shownin C. bpl, Brown pigment layer; oi, outerintegument. Arrows point to suberin la-mellae in C. Bars = 2 mm (A), 500 nm (B),and 100 nm (C).


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level of MYB107 was extremely low at stages I and II,surged to its maximum at stage III, declined at stage IV,and was nearly absent at stage V (Fig. 5A). Coinci-dently, nearly all the known suberin biosynthetic genes,including HHT, FACT, GPAT5, FAR1, FAR4, FAR5,CYP86A1, and CYP86B1, showed an essentially iden-tical expression pattern to that of MYB107 (Fig. 5). Incontrast, although cutin-like polyesters were proposedto occur in the seed coat of Arabidopsis (Molina et al.,2008), the expression of cutin biosynthetic genes, in-cluding DCF, GPAT4, GPAT6, GPAT8, CYP86A2,CYP86A8, and CYP77A6, exhibited no obvious ex-pression pattern like that ofMYB107 (Fig. 5). These dataindicate that MYB107 coexpresses temporally with su-berin biosynthetic genes but not cutin biosyntheticgenes during seed development and that stage III (11–15 DAF) of developing seeds is a crucial period for theonset of suberin biosynthetic gene expression.Subsequently, we undertook a third repeat of the

qRT-PCR analysis using RNAs from the seeds at stage

III of both the wild type andmyb107. As expected, mostof the known suberin biosynthetic genes were foundmore significantly down-regulated in myb107 (Fig. 6;repeat 3 in Table I) compared with the data from twoprevious repeats. Among them, the reductions of FARs,FACT, and HHT transcripts were most profound,showing more than 80% decline (Fig. 6; Table I). Incontrast, expression of the genes involved in cutin bi-osynthesis, such as DCF, CYP86A2, and GPAT6, wasnot changed substantially (Fig. 6; Table I). Taken to-gether, these data offer evidence thatMYB107 regulatessuberin biosynthetic gene expression in Arabidopsisdeveloping seeds in a temporal manner.

Nuclear Localization and Interaction of MYB107 withSuberin Biosynthetic Gene Promoters

In agreement with its proposed function as a TF, wefound that MYB107 localized in the nucleus when thep35S::MYB107:GFP construct was transiently expressed

Table I. Comparisons of the expression levels of suberin and cutin biosynthetic and deposition-related genes in the wild type (Col-0) and myb107,showing the fold change of gene expression in myb107 versus the wild type quantified by RNA-seq and qRT-PCR

For repeat 1 and repeat 2 of the RNA-seq and qRT-PCR experiments, RNAs from mixed siliques of different growth stages were used. For repeat 3 ofthe qRT-PCR experiment, RNAs from stage III developing seeds were used. Fold change values less than 0.5 are highlighted in boldface. ND, Notdetermined.

Gene Name Arabidopsis Genome Initiative No. Pathways

RNA-Seq (myb107/Wild

Type) qRT-PCR (myb107/Wild Type)

Repeat 1 Repeat 2 Repeat 1 Repeat 2 Repeat 3

BAHD acyltransferaseHHT/ASFT AT5G41040 Suberin 0.59 0.48 0.48 0.63 0.15FACT AT5G63560 Suberin 0.10 0.11 0.39 0.39 0.06DCF AT3G48720 Cutin 0.95 0.59 1.18 1.74 0.73DCR AT5G23940 Cutin 1.15 1.17 0.99 0.80 1.02

Cytochrome P450 monooxygenaseCYP86A1 AT5G58860 Suberin 0.49 0.17 0.49 0.57 0.59CYP86B1 AT5G23190 Suberin 0.38 0.46 0.39 0.32 0.25CYP86A2 AT4G00360 Cutin 0.89 0.87 0.76 0.76 0.63CYP86A8 AT2G45970 Cutin 0.79 0.71 0.85 0.60 0.65CYP77A6 AT3G10570 Cutin 1.16 0.92 1.22 0.60 1.23

Long-chain acyl-CoA synthetaseLACS1 AT2G47240 Cutin 1.00 0.92 ND ND NDLACS2 AT1G49430 Cutin/suberin 0.57 0.62 0.40 0.45 0.49

Glycerol-3-phosphate acyltransferaseGPAT5 AT3G11430 Suberin 0.84 0.60 0.68 1.12 0.36GPAT4 AT1G01610 Cutin/suberin 0.95 0.49 0.71 0.43 0.45GPAT6 AT2G38110 Cutin 0.84 1.02 0.83 0.63 1.02GPAT8 AT4G00400 Cutin 0.91 1.64 0.98 0.83 ND

Alcohol-forming fatty acyl-CoA reductaseFAR1 AT5G22500 Suberin 0.33 0.53 0.20 0.37 0.08FAR4 AT3G44540 Suberin 0.24 0.23 0.28 0.36 0.15FAR5 AT3G44550 Suberin 0.57 0.59 0.36 0.67 0.28

3-Ketoacyl-CoA synthaseKCS2 AT1G04220 Wax/suberin 1.00 0.85 0.78 0.73 1.06KCS20 AT5G43760 Wax/suberin 0.82 0.79 0.81 0.70 0.58

ABC transporterABCG2 AT2G37360 Suberin 0.53 0.72 ND ND NDABCG6 AT5G13580 Suberin 1.55 1.21 0.93 0.65 0.86ABCG20 AT3G53510 Suberin 0.86 0.80 0.56 0.58 0.84ABCG11 AT1G17840 Cutin 0.92 1.02 1.43 0.68 0.64





in the leaf cells of Nicotiana tabacum (Fig. 7A). To detectwhether MYB107 indeed activates suberin biosyntheticgene expression, we examined the interactionof MYB107 and suberin biosynthetic gene promotersvia yeast one-hybrid (Y1H) assay and chromatin im-munoprecipitation (ChIP) coupled with quantitativePCR (qPCR) analysis. For the Y1H analysis, the full-length promoters of the selected representative suberin

biosynthetic genes FAR1, GPAT5, CYP86A1, HHT, andFACTwere placed upstream of the LacZ reporter gene tocreate the promoter::reporter constructs. Both the full-length MYB107 and its DNA-binding domain(MYB107BD) were hybridized with the yeast GAL4 ac-tivation domain (AD) and then transferred into the yeastcells harboring the promoter constructs (Fig. 7B). Yeastharboring an N-terminal fragment of the mutator-like

Figure 5. Expression patterns of MYB107 and suberin/cutin biosynthetic genes during seed development. Developing siliquesand mature seeds were grouped into five different developmental stages. The relative expression levels of MYB107 and threeBAHD family genes (A), fourGPAT genes (B), three FAR genes (C), and five CYP genes (D) at each developmental stage (I–V) areshown.UBIQUITIN10 (UBQ10) was used as the reference gene for the normalization. The expression level of each gene in stage Iof developing seeds was set as 1. All data represent means 6 SD of three experimental repeats. Asterisks indicate statistical dif-ferences of each stage compared with that at stage I (Student’s t test, ***P , 0.001).

Figure 6. Expression of suberin/cutin biosynthetic genes in seed developmental stage III of the wild type (WT) andmyb107. Therelative expression levels of three BAHD family genes (A), threeGPAT genes (B), three FAR genes (C), and three CYP genes (D) atseed developmental stage III are shown. UBQ10 was used as the reference gene for the normalization. The expression level ofeach gene in the wild type was set as 1. All data represent means6 SD of three experimental repeats. Asterisks indicate statisticaldifferences of each stage compared with that at stage I (Student’s t test, ***P , 0.001).


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transposase FHY3 (FHY3N) and its targeting promoterof FHL (pFHL; Lin et al., 2007) was used as the positivecontrol. We found that yeast harboring AD:MYB107 (orAD:MYB107BD) and pFAR1::LacZ or pGPAT5::LacZyielded obvious positive color reaction in b-Gal assays;the yeast cells containing AD:MYB107 (or AD:MYB107BD) and the promoters of HHT and FACT

produced a relatively weak coloration; while yeastcontaining AD:MYB107 (or AD:MYB107BD) andpCYP86A1::LacZ or pFHL::LacZ constructs, or harboringthe AD alone with any of promoter::reporter constructs,did not yield any coloration (Fig. 7C). These data suggestthe potential interactions of MYB107 with the promotersof FAR1, GPAT5, HHT, and FACT. To validate thein vitro Y1H results, we performed ChIP coupled withqPCR using Arabidopsis seedlings transiently express-ing MYB107:GFP and free GFP (as the control) againstanti-GFP antibody. The resulting DNAs were amplifiedagainst the primers complementary to the sequences ofpromoters and/or coding regions of FAR1, GPAT5,HHT, FACT, CYP86A1, and DCF genes (Fig. 8). TheqPCR data indicated that the promoter regions of FAR1,GPAT5, HHT, and FACT were significantly enriched inthe MYB107:GFP-mediated immunoprecipitates relativeto the immunoprecipitates of the free GFP control (Fig.8). No significant enrichment was detected for the pro-moter regions of CYP86A1 and DCF (Fig. 8). These re-sults are consistent with Y1H assays and indicate thatMYB107 does bind specifically to the promoters of par-ticular suberin biosynthetic genes in vivo.

DISCUSSION

Suberin is an essential physical barrier contributingto seed coat impermeability (Pollard et al., 2008;Nawrath et al., 2013). A defect of suberin synthesischanges the seed coat sealing property and lowers seedviability and tolerance to biotic and abiotic stresses(Beisson et al., 2007; Gou et al., 2009; Molina et al., 2009;Vishwanath et al., 2013). While the knowledge on su-berin synthesis and deposition is relatively advanced,the regulatory mechanisms underlying seed coatsuberin formation remain elusive. In this study, weidentified and defined a TF, MYB107, a predicatedR2R3-MYB family member of subgroup 10 (Duboset al., 2010), as a positive regulator controlling suberinsynthesis in the Arabidopsis seed coat. MYB107 coex-presses with a set of suberin biosynthetic genes in atemporal manner during seed development (Fig. 5).Down-regulation ofMYB107 suppresses the expressionof most known suberin biosynthetic genes (Fig. 6; TableI). Consequently, the suberization of the myb107 seedcoat is impaired, which is demonstrated by the drasticreduction of suberin aliphatic and aromatic monomersand the altered lamellation of the suberin layer in outerintegument of the seed coat (Figs. 3 and 4). Thesechemical and structural changes ultimately result in theenhanced seed coat permeability and increased abioticstress susceptibility of the myb107 seed (Figs. 1 and 2;Supplemental Fig. S3). Conversely, complementation ofMYB107 expression in myb107 restores suberin bio-synthetic gene expression, defective suberin synthesis,seed coat impermeability, and abiotic stress tolerance(Figs. 1 and 2; Supplemental Fig. S6). Moreover, bothin vitro and in vivo studies show that MYB107 interactsdirectly with the promoters of at least four suberin

Figure 7. Subcellular localization of MYB107 and its physical inter-actions with the suberin biosynthetic gene promoters. A, Subcellularlocalization of theMYB107:GFP fusion protein. Confocal images of freeGFP (top row) and MYB107:GFP (bottom row) in tobacco leaf cells aredisplayed. BF, Bright field; Chl, chlorophyll autofluorescence; GFP, GFPfluorescence; Combined, themerged images. Bars = 25mm. B, Diagramof the expression constructs of MYB107 and the promoters of suberinbiosynthetic genes used in the Y1H assay. The full-length MYB107 andMYB107 DNA-binding domains (MYB107BD) were fused with theyeast GAL4 activation domain (AD). The suberin biosynthetic genepromoters were placed to drive a LacZ reporter gene. C, Color reactionof yeast cells harboring AD:MYB107 or AD:MYB107BD with the pro-moter::LacZ construct. The images were captured after growing theyeast for 8 d on an SC(2Trp,2Ura) plate containing 5-bromo-4-chloro-3-indolyl b-D-galactopyranoside (X-Gal). CK+, AD:FHY3N+pFHL::LacZ; CK-(1), AD:MYB107+pFHL::LacZ; CK-(2), AD:MYB107BD+pFHL::LacZ.







biosynthetic genes, GPAT5, FAR1, HHT, and FACT,although the promoters of the latter two genes exhibi-ted relatively weak interactions with MYB107 in theY1H assay (Fig. 7). GPAT5 is critical for the propersynthesis of suberin and associated waxes via theformation of sn-2 monoacylglycerol intermediates(Beisson et al., 2007); FAR1 is the fatty acyl-CoA re-ductase responsible for producing 22:0 primary fattyalcohol in suberized tissues; while both HHT and FACTare aromatic acyltransferases responsible for the for-mation of feruloyl and caffeoyl esters, respectively, insuberin or some of the suberin-associated waxes (Gouet al., 2009; Molina et al., 2009; Domergue et al., 2010;Kosma et al., 2012; Vishwanath et al., 2013). All thesedata suggest that MYB107 functions as a positive reg-ulator dominating the expression of suberin biosyn-thetic genes and, thus, the synthesis and assembly ofboth suberin aliphatics and aromatics in the Arabi-dopsis seed coat (Supplemental Fig. S13).

The determined regulatory role of MYB107 in the seedcoat appears specific for suberin biosynthetic genes.MYB107 coexpresses dominantly with genes involved insuberin biosynthesis but not those for cutin synthesis or forthe formation of seed coat pigmentation (SupplementalTable S2). The down-regulation of MYB107 essentially didnot affect the expression of specific cutin biosynthetic genesor TT genes (Table I; Fig. 6; Supplemental Table S3). Fur-thermore, the myb107 mutant showed no obvious mor-phological andultrastructural alterationsassociatedwith thecuticles (Supplemental Figs. S9 and S10). Its seeds also haveno tt phenotype (Supplemental Fig. S4C), as seen in other tt

mutants (Debeaujon et al., 2000; Appelhagen et al., 2014).These data suggest thatMYB107 controls seed coat perme-ability by specifically regulating suberinbiosynthesis butnotother elements contributing to the seed-sealing property.They also are consistentwith the previousdiscovery that thesynthesis of suberin and cutin, the two analogous polyestersderived from fatty acids, is under distinct transcriptionalcontrols (Molina et al., 2008). Such regulatory distinctionmight be the key determinant in establishing the uniquetissue- and/or organ-specific distribution of the two chem-ically analogous lipid-based polymers.

Arabidopsis seed coat and root peridermal or endo-dermal cells are the major sites of suberization. Whileseveral lines of evidence suggest that MYB107 is aregulator that controls the onset of programmed ex-pression of suberin biosynthetic genes during seed de-velopment, knocking down MYB107 has little effect onsuberin gene expression in roots (Supplemental Fig.S11B). The synthesis of suberin aliphatics and aromaticsin the roots of myb107was not impaired (SupplementalFig. S8). These data indicate that MYB107 does notdominate suberin synthesis in roots. The exact reasonsfor MYB107 exhibiting no function on root suberinsynthesis remain unclear. This may indicate the exis-tence of additional redundant TF(s) in roots.

The down-regulation of MYB107 affects the expres-sion of a set of suberin biosynthetic genes; moreover,MYB107 directly targets the promoters of a few suberinbiosynthetic genes (Figs. 7 and 8). This result impliesthat MYB107 regulates suberin biosynthetic genes bothdirectly and indirectly. The mode of MYB107 action is

Figure 8. ChIP-qPCR assays of MYB107-DNA complexes. The scheme of the primer design for each gene is shown at top, inwhich black lines represent the promoter region and black boxes represent the gene-coding region. Red lines beneath the genestructures with numbers mark the locations of amplicons amplified by ChIP-qPCR.UBQ10 (UBQ) was used as a negative control.Two biological replicates were performed; the data presented represent means 6 SD of three technical repeats from one repre-sentative biological replicate. Asterisks indicate statistical differences compared with the free GFP control (Student’s t test, *P ,0.05, **P , 0.01, and ***P , 0.001). IP, Immunoprecipitate.


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reminiscent of the TFs involved in cutin synthesis (YeatsandRose, 2013) and in secondary cellwall formation (Zhaoand Dixon, 2011). The TFs regulating cuticle formationappear to form a regulatory cascade. WIN1/SHN1 regu-lates the expression of a set of cutin and/or wax biosyn-thetic genes accompanied by the effect of wax load andcutin synthesis (Broun et al., 2004; Kannangara et al., 2007);meanwhile, WIN1/SHN1 and its homologous memberscan directly activate promoters of several cutin biosyn-thetic genes (Kannangara et al., 2007; Shi et al., 2011). Inaddition, two MYB TFs, MYB106 and MYB16, that wereimplicated in cuticle developmentwere demonstrated toact upstream of WIN1/SHN1 (Oshima et al., 2013). Sim-ilarly, in a hierarchical regulatory network for secondarycell wall formation, the NAC-type TFs VND6/7 andNST1-3 act as the top master switches controlling thebottom transcriptional activators or repressors and,meanwhile, directly targeting some of the specific ligninor cellulose biosynthetic genes (Zhao and Dixon, 2011).The temporal expression pattern of MYB107 in Arabi-dopsis developing seeds indicates that MYB107 mayposition in an unidentified regulatory cascade or net-work and itself is developmentally controlled by up-stream regulators; meanwhile, it also may control and/or coordinate the actions of additional TFs to regulatesuberin biosynthetic gene expression (Supplemental Fig.S13). Indeed, a few other TF genes, such as the WRKY,bZIP, andMYB familymembers, were found to be eithercoexpressed with MYB107 or cosuppressed in myb107(Supplemental Table S2; Supplemental Data Set S1).These include MYB41, a TF that was defined recentlyas the suberin regulator likely controlling the stress-induced suberin synthesis in roots (Kosma et al., 2014).

CONCLUSION

Several lines of evidence from bioinformatics, molecu-lar genetics, biochemistry, microscopic, and physiologicalstudies suggest that Arabidopsis MYB107 positively reg-ulates the expression of a set of suberin (but not cutin) bi-osynthetic genes in developing seeds, by which it controlsthe synthesis and assembly of seed coat suberin, thus af-fecting the sealingproperty of the seed coat.MYB107 exertsits regulatory role directly and indirectly on suberin bio-synthetic genes, suggesting that it positions in an unde-fined regulatory network or cascade. Identifying MYB107as apositive regulator for suberin synthesis in seeds offers abasis for further discovering the potential transcriptionalnetwork behind one of the most abundant lipid-basedpolymers in nature and for further elucidating its interplayand coordination with other transcriptional regulators inpolyester synthesis, deposition, and assembly.

MATERIALS AND METHODS

Arabidopsis Plant Growth, Mutant Genotyping,and Staining

Arabidopsis (Arabidopsis thaliana) seeds were surface sterilized with 20%(v/v) bleach for 5 min. Seeds were stratified at 4°C for 2 to 3 d and germinated

on a one-half-strength MS medium plate (Murashige and Skoog, 1962) con-taining 0.7% agar and 1% Suc. Seven-day-old seedlings were transferred to soil(Metro-Mix 200; SunGro) or to a hydroponic device (Tocquin et al., 2003) andgrown in growth chambers under photoperiodic cycles of 16 h of light and 8 h ofdark at 22°C. The T-DNA insertion mutant lines myb107-1 (SAIL_242_B04) andmyb107-2 (SALK_203615) were obtained from the Arabidopsis Biological Re-source Center and genotyped with the primers listed (Supplemental Table S4).The Tetrazolium Red staining of seeds and the Sudan Black B staining of rootlipid polyesters were conducted for 12 and 1 h, respectively, following de-scribed methods (Beisson et al., 2007). To characterize leaf cuticles, 10-d-oldseedlings or 35-d-old plants were stained with 0.05% Toluidine Blue solutionfor 15 min or 2 h, respectively, as described (Li et al., 2007).

RNA Extraction and qRT-PCR Analysis

Plant tissues, including 10-d-old plate-grown seedlings and 8-week-oldhydroponic-grown roots, stems, leaves, flowers, and siliques, were collectedfor RNA extraction. Siliques at different growth stages of the soil-grown plantsalso were collected and dissected to obtain young seeds for RNA extraction,except that the siliques in stage I (instead of young seeds) were used directly forRNA extraction (Supplemental Fig. S12).

Total RNAs were extracted from collected plant materials using the Trizolreagent (Invitrogen) as instructed by the manufacturer, except that a Trizol-based two-step method was specifically used for extracting RNAs from thesiliques and seeds (Meng and Feldman, 2010). M-MuLV reverse transcriptase(New England Biolabs) was used to synthesize cDNA from the mRNAs. qRT-PCR was performed by following the defined standards and guidelines(Udvardi et al., 2008) and using the primers listed (Supplemental Table S4).SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) was used for the re-action. The cycle threshold value was calculated by the CFXManager Softwareversion 3.0 (Bio-Rad). Relative gene expression levels were calculated byfollowing the documented standard comparative cycle threshold method (i.e.22DDCt method; Livak and Schmittgen, 2001). Briefly, either the housekeepinggeneUBQ10 or TUBULIN2was used as the reference gene. The expression levelfor a given gene was first normalized to that of either UBQ10 or TUBULIN2(only for Fig. 1D) and then was normalized to the expression level of the samegene in the defined control sample. The data were presented as the fold change.

Complementation of the myb107 Mutant

A 5,705-bp genomic fragment of the MYB107 gene, including the 3,839-bpnative promoter sequence upstream of the start codon ATG and the 176-bp 39untranslated region sequence downstream of the stop codon TAG, was am-plified using the primers PCR8LB-PMYB107-1-F and PCR8RB-MYB107-ORF-3UTR-R (Supplemental Table S4) and was cloned into the pCR8/GW/TOPOentry vector by the ligation-independent cloning method (Jeong et al., 2012).The genomic piece was then subcloned into the binary pGWB501 (Nakagawaet al., 2007) by LR reaction (Invitrogen) to generate the native promoter-drivenMYB107 expression construct. Full-length cDNA of MYB107 (with or withoutthe stop codon) was amplified with the primers ATTB1-MYB107-F and ATTB1-MYB107-R2 or ATTB1-MYB107-R1 (Supplemental Table S2) and cloned into theGateway entry vector pDONR207 by the BP reaction (Invitrogen). They werethen subcloned into pMDC32 and pGWB405 (Nakagawa et al., 2007) vectors,respectively, to generate the 35S promoter-driven MYB107 or MYB107:GFPfusion construct. The gene expression constructs were transformed into themyb107 mutant or the wild type by the floral dip method. T2 mature seeds ofindividual transgenic lines were stained with Tetrazolium Red as describedabove to check the seed coat permeability. Two-week-old seedlings, 3-week-oldroots, and mixed siliques of different developmental stages of the individual T2transgenic lines were used for RNA extraction and qRT-PCR analysis ofMYB107 expression levels. Mature seeds of two individual lines were used tomeasure the suberin monomers.

Analyses of Suberin Monomers

For compositional determination and measurement of suberin, 500 mg ofmature (dry) seedswasused toprepare cellwall residues following thedescribedprotocol (Li-Beisson et al., 2013). For depolymerization, we followed the pre-viously described method of Franke et al. (2005) with some modifications.Briefly, 10 mg of cell wall residues was mixed with 2 mL of boron trichloride/methanol (12%, w/w; Sigma) in a screw-cap Teflon-sealed tube and incubatedat 80°C for 2 h. At this step, 100 nmol of chrysin was added as the internal














standard for HPLC analysis, and 5 mg heptadecanoic acid (C17) and 10 mg of1-tricosanol (C23) were added as the internal standards for GC-MS. Two mil-liliters of water was added to terminate the reaction, and 3 mL of chloroformwas used to extract the hydrolysates twice. The extracts were pooled and di-vided into two portions and then dried under a stream of N2 gas. One portion ofresidue was dissolved in 250 mL of 80% methanol, and 30 mL was injected forHPLC analysis. Another portion was dissolved in 50 mL of pyridine, furtherderivatized with 50 mL of N-methyl-N-(trimethylsilyl)trifluoroacetamide(Sigma) at 70°C for 1 h, and then dried under a stream of N2 gas. The driedresidues were dissolved in 100 mL of heptane:toluene (1:1) for GC-MS analysis.

HPLCanalysiswasperformedwithagradientof solventB (0.2%acetic acid inacetonitrile) in solventA(0.2%aceticacid inwater) as follows: 2min, 15%;35min,100%; 38 min, 100%; and 40 min, 15%, at a linear flow rate of 0.8 mL min21 in areverse-phase C18 column [Luna C18 (2), 5 mm; Phenomenex]. To quantify thecontent of suberin phenolics, the UV light-absorptive area of a particular peakfrom each sample was first normalized to that of the internal standard, thencalibratedwith the standard curve of ferulate, caffeate, p-coumarate, or sinapateestablished in the same HPLC, running using a series of concentrations of au-thentic chemicals after the same boron trichloride/methanol treatment andextraction.

For GC-MS, we followed the reported protocol (Franke et al., 2005). Briefly,4-mL samples were injected via splitless mode and resolved on a DB-5MScapillary column (30 m 3 0.32 mm, 0.1 mm; J&W) on an Agilent 7890A gaschromatograph equipped with an Agilent 5975C mass detector. The tempera-ture settings were as follows: inlet, 300°C; the oven temperature program wasset to 50°C for 2 min and then increased to 150°C at a rate of 10°C min21; aftermaintaining it for 1 min, the temperature was increased to 310°C at the rate of3°C min21 and maintained for 10 min. The helium flow rate was 1.5 mL min21.The contents of fatty acids and dicarboxylic acids were quantified using hep-tadecanoic acid as the standard, whilev-hydroxy fatty acids, alcohols, and diolswere quantified using 1-tricosanol as the standard.

SEM and TEM Analyses

For SEM, plant tissues, including leaf, stem, flower, and silique from 5-week-old plants as well as fully matured seeds, were chemically fixed in 2.5% glu-taraldehyde and 2% paraformaldehyde diluted with 0.1 M cacodylate bufferand then processed for SEM analysis. The samples were washed with the samebuffer, dehydrated in a graded ethanol series, and dried with a Tousimiscritical-point drying apparatus (Tousimis Research). All dried samples weremounted onto aluminum stubs, sputter coated with gold, and imaged with aQuanta 200 environmental scanning electron microscope (FEI).

For TEM, mature and dried seeds were imbibed in distilled water for 24 hand then carefully pricked with a microneedle. Plant tissues, including thepricked seeds and leaves from 5-week-old plants, were fixed in 2.5% glutaral-dehyde and 2% paraformaldehyde diluted with 0.1 M cacodylate buffer andpostfixed in 1% osmium tetroxide diluted with the same buffer. Samples thenwere dehydrated through a graded acetone series and embedded in Spurr’sepoxy resin (ElectronMicroscopy Science). All the samples were sectioned withan Ultracut E microtome (Reichert-Jung, Cambridge Instruments). Thin sec-tions (90–100 nm) were mounted onto 200-mesh copper grids, treated with 10%H2O2 for 10 min, and then stained with 1% aqueous uranyl acetate and Satolead. Three to five sections for each sample were examined and imaged at120 kV with a JEM-1400 transmission electron microscope (JEOL). Represen-tative images are presented.

Abiotic Stress Treatment

Surface-sterilized wild-type (Col-0) and myb107 seeds were stratified andgerminated on a one-half-strength MS plate supplemented with 150 mM NaCl,300 mM mannitol, and 5mM H2O2. After 7 d of growth under light, the numbersof seedlings with two fully established cotyledons were counted, and the es-tablishment rates were calculated. Three plates were set for each treatment (asthree biological replicates), and approximately 80 seeds were plated in eachrepeat.

RNA-Seq

The mixed siliques of 8-week-old myb107 and wild-type (Col-0) plants werecollected. RNAs were extracted using the method described above. The as-sessment of purity and concentration of each RNA sample, and the strand-specific RNA-seq library preparation, were conducted using the Polar

Genomics service following a protocol described by Zhong et al. (2011). Se-quencing was done using the Illumina HiSeq2500 platform via 100-bp single-end reads ofmultiplexed RNA samples. RNA-seq reads first were aligned to therRNA database (Quast et al., 2013) using Bowtie (Langmead et al., 2009), andthose mapped were excluded for downstream analysis. The resulting cleanedreads were aligned to the Arabidopsis genome sequence (TAIR10) usingTopHat (Trapnell et al., 2009) allowing one mismatch. Following these align-ments, raw counts for each Arabidopsis gene were derived and normalized toreads per kilobase of exon model per million mapped reads. The differentiallyexpressed genes were identified with the integrated Cuffdiff program(Langmead et al., 2009; Trapnell et al., 2010) based on a false discovery rate-adjusted P value (i.e. q value) at the cutoff of 0.1 and fold change greater than orless than 2.

Confocal Microscopy of MYB107-GFP

Both pGWB405:MYB107 (MYB107:GFP) and the empty vector pCAM-BIA1302 (free GFP) were transformed into Agrobacterium tumefaciens strainGV3101. Six-week-old Nicotiana tabacum plants were infiltrated with an A.tumefaciens strain harboring each of the constructs. Fluorescence images werecaptured via a Leica TCS SP5 Laser Scan ConfocalMicroscopewith excitation at488 nm; an emission signal between 493 and 560 nm was collected for GFPfluorescence, and excitation at 496 nm and an emission signal of 630 to 720 nmwere collected for chlorophyll autofluorescence.

Y1H Assay

Full-length cDNA of MYB107 (with the stop codon) was amplified usingprimers ATTB1-MYB107-F andATTB1-MYB107-R2 (Supplemental Table S2). AcDNA fragment of MYB107 encoding the DNA-binding domain (MYB107BD)was amplified using primers ATTB1-MYB107-BD-F and ATTB2-MYB107-BD-R(Supplemental Table S2). Both cDNAs first were cloned into pDONR207 by theBP reaction (Invitrogen) and then subcloned into the pDESTAD-2m vector bythe LR reaction (Invitrogen) to generate the Y1H-TF prey constructs (Reece-Hoyes et al., 2011). The promoters of suberin biosynthetic genes were ampli-fied using the primers listed (Supplemental Table S4), first cloned into pDONRP4P1R (Invitrogen) by the BP reaction, and then subcloned into the pMW#3vector to generate the pMW#3:DNA bait constructs. The Y1H experiment wasperformed following the methods described (Deplancke et al., 2006; Reece-Hoyes et al., 2011) with slight modifications. Briefly, pMW#3:DNA bait con-structs first were transformed and integrated into the genome of the yeast strainY1H-aS2. The positive colonies selected from SC(2Ura) medium were used forthe self-activation test by streaking them onto the SC(2Ura) plate spread with100 mL of 30 mg mL21 X-Gal (Sigma). The colonies showing no blue colorationafter streaking for 8 d were picked for culturing and retransformed with eitherthe TF prey constructs or pDESTAD-2m empty vector (as the control). Theresulting positive colonies selected from the SC(2Trp,2Ura) medium wererestreaked onto the SC(2Trp,2Ura) plate spread with 100 mL of 30 mg mL21

X-Gal and incubated for 8 d before then capturing the color changes. Yeastharboring a well-characterized N-terminal fragment of mutator-like trans-posase FHY3 (FHY3N) and its targeting promoter of FHL (pFHL; Lin et al.,2007) was used as the positive control. Meanwhile, yeast harboring MYB107or MYB107BD with the pFHL DNA construct was used as the negativecontrols.

ChIP-qPCR Analysis

The pCAMBIA1302 (p35S::GFP) and pGWB405:MYB107 (p35S::MYB107:GFP) constructs were transiently transformed into 4-d-old Arabidopsis wild-type (Col-0) seedlings byA. tumefaciens-mediated transformation using vacuuminfiltration (Marion et al., 2008). One gram of seedlings after transformation for3 d was collected and cross-linked using 40 mL of cross-linking buffer (50 mM

KH2PO4/K2HPO4, pH 5.8, and 1% formaldehyde) for 15 min under vacuum.The cross-linking was then quenched using quenching buffer (50 KH2PO4/K2HPO4, pH 5.8, and 0.3 M Gly) for 5 min by vacuum. The materials wereground into fine power. The chromatin isolation and nuclei lysis were per-formed as described previously (He et al., 2013). Chromatin was then sonicatedon ice eight times for 10 s each (30-s interval) with power setting at 2 on a FisherScientific 550 sonic dismembrator to shear DNA into 500- to 1,500-bp fragments(He et al., 2013). After dilution to 1.5 mL using ChIP dilution buffer, 3% (45 mL)of the sheared sample was used as the input fraction. The rest was preclearedwith 20 mL of coated Dynabeads Protein G beads (1003D; Thermo Fisher


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Scientific) for 3 h, and 10 mg of Living Colors monoclonal GFP antibody (JL-8;Clontech) was added and incubated overnight. The protein-DNA complexeswere captured by incubating with 30 mL of newly added coated DynabeadsProtein G beads for 2 h at 4°C. The beads were washed with low- and high-saltbuffer and LiCl wash buffer and eluted with 100 mL of elution buffer as de-scribed (He et al., 2013). The elution was then incubated overnight at 65°C toreverse cross-linking. Four microliters of RNase (10 mg mL21) was added andincubated for 1 h at 37°C, and 4mL of proteinase K (20mgmL21) was added andincubated for another 2 h at 45°C. The DNA was purified with the PureLinkPCR Purification Kit (K310001; Thermo Fisher Scientific). Eluted solutions wereused for qPCR. UBQ10 was used as a negative control. Three or four pairs ofprimers were synthesized for each tested gene with matching to their promoterand/or coding regions (Fig. 8). The primer sequences are listed in SupplementalTable S4. Three biological experiments were performed. Data are normalized bydividing qPCR signal from the ChIP samples with qPCR signal from the inputsamples (i.e. percentage of input) following the manufacturer’s instructions(https://www.thermofisher.com/us/en/home/life-science/epigenetics-non-coding-rna-research/chromatin-remodeling/chromatin-immunoprecipitation-chip/chip-analysis.html).

Accession Numbers

Sequence data of the gene investigated in this study can be found in TheArabidopsis Information Resource under accession number At3g02940(MYB107).

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. In silico expression patterns of HHT and DCF indifferent tissues.

Supplemental Figure S2. In silico expression patterns of MYB107 in dif-ferent tissues.

Supplemental Figure S3. Characterization of the myb107-2 mutant.

Supplemental Figure S4. Morphologies of wild-type and myb107 plants.

Supplemental Figure S5. Cosegregation of the T-DNA insertion of myb107with the enhanced tetrazolium staining phenotype.

Supplemental Figure S6. Complementation of the myb107 mutant bypMYB107::MYB107.

Supplemental Figure S7. GC-MS profile of the aliphatic monomers fromseed coat cell wall of the wild type and myb107.

Supplemental Figure S8. Aromatic and aliphatic monomers of suberin inroots.

Supplemental Figure S9. Microscopic observation of suberin ultrastruc-tures in seed coats of the wild type and myb107.

Supplemental Figure S10. Phenotypic characterization of wild-type andmyb107 plants.

Supplemental Figure S11. Expression of suberin biosynthetic genes inMYB107 complementation lines.

Supplemental Figure S12. Sample collection of Arabidopsis young seedsat the defined developmental stages.

Supplemental Figure S13. Proposed model of MYB107-mediated regula-tion of suberin biosynthesis.

Supplemental Table S1. Gene coexpression analysis using HHT/ASFT(At5g41040) as the bait.

Supplemental Table S2. Gene coexpression analysis using MYB107(At3g02940) as the bait.

Supplemental Table S3. Expression of TT genes in the wild type andmyb107 detected by RNA-seq.

Supplemental Table S4. PCR primer sequences used in this study.

Supplemental Data Set S1. Complete RNA-seq data set of the wild type(Col-0) and the myb107 mutant.

ACKNOWLEDGMENTS

We thank Drs. Lifang Zhang and Doreen Ware at Cold Spring HarborLaboratory for advice on the Y1H assay system and Jiapei Yan and ZhixueWang at Cornell University for advice on the ChIP experiment.

Received October 18, 2016; accepted December 10, 2016; published December13, 2016.

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