the fused leaves1/adherent1 regulatory module is …2011; oshima et al., 2013; lee and suh, 2015)....

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THE FUSED LEAVES1/ADHERENT1 REGULATORY MODULE IS REQUIRED

FOR MAIZE CUTICLE DEVELOPMENT AND ORGAN SEPARATION

Xue Liu1, Richard Bourgault2, Josh Strable3, Mary Galli1, Zongliang Chen1,

Jiaqiang Dong1, Isabel Molina2, Andrea Gallavotti1,4

1 Waksman Institute of Microbiology, Rutgers University, Piscataway, NJ 08854-

8020, USA 2 Department of Biology, Algoma University, Sault Ste. Marie, ON P62A 2G4,

Canada 3 School of Integrative Plant Science, Plant Biology Section, Cornell University,

Ithaca, NY 14853, USA 4 Department of Plant Biology, Rutgers University, New Brunswick, NJ 08901, USA

Corresponding author: Andrea Gallavotti, [email protected]

ORCID IDs: 0000-0002-9928-7114 (X.Liu); 0000-0002-0260-8285 (J. Strable); 0000-0002-1901-2971 (A. Gallavotti)

Running title: maize cuticle formation

One sentence summary: The classic maize mutant adherent1, first isolated a

century ago, is affected in an enzyme responsible for cuticle formation that is

regulated by the MYB transcription factor FUSED LEAVES1.

ABSTRACT

In land plants all aerial epidermal cells are covered by the cuticle, an extracellular

hydrophobic layer. The cuticle represents a primary barrier between cells and the

.CC-BY-NC-ND 4.0 International license(which was not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprintthis version posted February 12, 2020. . https://doi.org/10.1101/2020.02.11.943787doi: bioRxiv preprint

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external environment, provides protection against abiotic and biotic stresses, and

prevents organ fusion during development. Here we report the cloning and

characterization of a classic mutant of maize called adherent1 (ad1), first described

a century ago, and we show that AD1 encodes a 3-KETOACYL-CoA SYNTHASE

involved in the deposition of cuticular wax on the epidermis of leaves and

inflorescences. ad1 mutants show decreased amounts of various wax components

as well as a range of organ fusion defects during vegetative and reproductive

development. Accordingly, we find that AD1 is strongly expressed in the epidermis

of various developing organs where it is directly regulated by the MYB transcription

factor FUSED LEAVES1 (FDL1), which in turn controls a series of additional genes

involved in cuticle formation. Altogether, our results identify a major pathway of

cuticle biosynthesis essential for the development of maize plants, and a key

regulatory module that is conserved across monocot and eudicot species.


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INTRODUCTION The plant cuticle covers the aerial epidermis of all land plants, providing protection

against external environmental stresses, including drought, extreme temperatures,

UV light, pests and pathogens, and represents one of the most important

adaptations for the terrestrial life of land plants (Yeats and Rose, 2013). It is

synthesized by the plant epidermis, a single sheet of cells derived from the L1 layer

of meristems that provides a necessary barrier for separating individual developing

organs (Javelle et al., 2011). The cuticle structure, composition and properties

influence cuticle permeability, which has been hypothesized to be critical in

preventing organ fusion in early organ development (Yephremov et al., 1999; Pruitt

et al., 2000; Ingram and Nawrath, 2017). Much of our understanding of postgenital

fusion, defined as fusions occurring at the margins of fully formed organs (Specht

and Howarth, 2015), of the epidermis comes from studies in several species

including Catharanthus roseus, maize and Arabidopsis (Walker, 1975; Li et al.,

2016; Vialette-Guiraud et al., 2016). Organ fusion has been observed in several

cuticular mutants of Arabidopsis, such as wax1, wax2, fiddlehead/kcs10 (fdh),

lacerata, hothead, bodyguard, desperado and long-chain acyl-CoA synthetases1

long-chain acyl-CoA synthetase2 (lacs1 and lacs2) (Jenks et al., 1996; Yephremov

et al., 1999; Pruitt et al., 2000; Wellesen et al., 2001; Chen et al., 2003; Kurdyukov

et al., 2006b; Kurdyukov et al., 2006a; Panikashvili et al., 2007; Weng et al., 2010).

In maize, mutations in CRINKLY4 (CR4), a receptor kinase, affect epidermal cells

and lead to adhesion events taking place between organs (Becraft et al., 1996;

Becraft et al., 2001). Most of these mutations cause organ fusion defects both in

juvenile vegetative and adult reproductive organs, and display cuticle defects due

to mutations in genes directly involved in cutin or cuticular wax formation, the main

components of the cuticle.


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Cutin is a polyester matrix composed mainly of glycerol and long-chain (C16

and C18) hydroxy fatty acid monomers (Kolattukudy, 1980; Graca et al., 2002).

Cuticular waxes are instead composed of a mixture of aliphatic and alicyclic

compounds. Wax aliphatics are derived from very-long-chain fatty acids (VLCFAs;

C20–C34) (Buschhaus and Jetter, 2011). VLCFA biosynthesis begins with de novo

C16 or C18 fatty acid biosynthesis in the plastid of epidermal cells. Exported to the

cytosol as acyl-CoAs, further elongation up to C34 is achieved by the FATTY ACID

ELONGASE (FAE) complex at the endoplasmic reticulum (ER). The FAE complex

catalyzes the two-carbon condensation from malonyl-CoA to an acyl-CoA, and

consists of four enzymes, 3-KETOACYL-CoA SYNTHASE (KCS), 3-KETOACYL-

CoA REDUCTASE (KCR), 3-HYDROXYACYL-CoA DEHYDRATASE (HCD) and

ENOYL-CoA REDUCTASE (ECR). During the elongation process, the first step is

the condensation of C2 units to acyl-CoA by KCS, the enzyme that confers

substrate specificity to the complex. VLCFAs are subsequently transformed into

primary alcohols and wax esters, or into aldehydes, alkanes, secondary alcohols

and ketones (Kunst and Samuels, 2003; Samuels et al., 2008), and are exported

to the cuticle by transporters at the plasma membrane (Pighin et al., 2004;

Panikashvili et al., 2007).

Cuticular wax biosynthesis is regulated at the transcriptional level mainly by

members of the MYB and AP2/ERF families of transcription factors (TFs). In

Arabidopsis, WAX INDUCER1(WIN1)/SHINE1(SHN1), SHN2, SHN3 and

WRINKLED4 (WRI4) are AP2/ERF TFs that directly activate genes in the wax

synthesis pathway (Aharoni et al., 2004; Broun et al., 2004; Park et al., 2016),

while DECREASE WAX BIOSYNTHESIS (DEWAX) and DEWAX2, negatively

regulate wax production (Go et al., 2014; Kim et al., 2018). The MYB TF family

also includes important regulators of cuticular wax biosynthesis, such as AtMYB16,


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AtMYB30, AtMYB94, AtMYB96, and AtMYB106 (Raffaele et al., 2008; Seo et al.,

2011; Oshima et al., 2013; Lee and Suh, 2015). AtMYB16 and AtMYB106 were

shown to regulate cuticle biosynthesis through direct activation of WIN1/SHN1 and

cuticle biosynthetic genes in standard conditions (Oshima et al., 2013), while

AtMYB30, AtMYB94 and AtMYB96 were found to activate wax biosynthetic genes

under biotic or abiotic stress (Raffaele et al., 2008; Seo et al., 2011; Lee and Suh,

2015). Recently, in maize, two MYB family TFs, FUSED LEAVES1/MYB94 (FDL1)

and GLOSSY3/MYB97, were identified as positive regulators of leaf cuticular wax

accumulation. While glossy3 mutants did not show any fusion defects, fdl1 mutants

showed organ fusion during the juvenile phase (Liu et al., 2012; La Rocca et al.,

2015).

In this study, we characterized the adherent1 (ad1) mutant of maize and show

that AD1 encodes a 3-KETOACYL-COA SYNTHASE required for cuticular wax

biosynthesis. Furthermore, using genome-wide DNA binding analysis we show

that AD1 and a series of other enzymes involved in cuticle formation are directly

activated by FDL1/MYB94. These findings establish a major pathway for cuticle

formation in maize, and highlight a common module for the regulation of cuticle

biosynthesis, involving MYB TFs and KCS genes, that is conserved between

eudicot and monocot species.

RESULTS

The adherent1 mutant shows abnormal fusions between organs and cells

In search of modifiers of the ramosa1 enhancer locus2 (rel2) mutant, we performed

an ethyl methanesulfonate (EMS) enhancer/suppressor screen of the pleiotropic

rel2-ref mutant in the A619 genetic background (Liu et al., 2019). Among the M2

families segregating developmental defects we noticed a strong upright tassel

branch phenotype segregating in one family (M2-92-224) as a single recessive


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locus (Supplemental Figure 1). After generating an F2 population for positional

cloning purposes, we determined that this phenotype was segregating

independently of the rel2-ref mutation and strongly resembled the phenotype of a

classic maize mutant, originally isolated a century ago, called adherent1 (ad1)

(Kempton, 1920; Sinha and Lynch, 1998). F1 plants obtained from crosses

between the mutant found in the M2-92-224 family and previously isolated alleles

(ad1-109D and ad1-110E) failed to complement the upright tassel branch

phenotype, indicating that the mutant identified in M2-92-224 was a new allele of

ad1, and we therefore renamed it ad1-224.

Subsequently, we carefully analyzed the mutant phenotype of the ad1-224

allele. The most notable phenotypes of ad1 plants were the adherence of leaves

in the juvenile stages of seedling development and the fusion of tassel branches

in mature plants. In particular, the first, second, and occasionally third leaf blades

upon germination were fused with themselves or with each other, giving rise to a

characteristic phenotype in seedlings (Figure 1A). Normal maize tassels are

characterized by long branches borne at the base of a long axis, the central spike

(Figure 1B). Both long branches and the central spike are covered in spikelets,

specific structures, enclosed by two glumes, that contain two florets. In mature ad1

plants, all tassel branches were completely attached to each other and the central

spike (Figure 1B), while spikelets appeared fused together and shriveled, with

partially emerging anthers (Figure 1C,D, Supplemental Figure 1). Within each

floret, anthers frequently showed fusion as well (Figure 1E).

Longitudinal and transverse sections of germinating shoots, tassels and

spikelets of wild type and ad1 mutants revealed widespread organ fusion defects.

For example, the coleoptile and the first leaf, the first and second leaves were

fused in ad1 germinating shoots (Figure 1H-K), with both the adaxial and abaxial


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sides of the blade participating in fusion events (Figure 1I,K), while ad1 tassel

branches and glumes adhered to each other and to the central spike (Figure 1F,G).

Unlike tassels, mature ears of ad1 mutants did not show a visible phenotype

(Supplemental Figure 1).

To examine seedling and tassel fusion events in greater detail, we used

scanning electron microscopy (SEM). ad1 mutants displayed several types of

fusion events i.e. a seedling leaf curled up and fused to itself (Figure 1M), the

second leaf fused to the surface of first leaf along the margin and the macrohair

from one surface fused to epidermal cells on the other surface (Supplemental

Figure 1). Areas of fusions between juxtaposed epidermal surfaces were quite

extensive and often appeared seamless, making it difficult to identify the boundary

between the partners of the fusion event. Similar extensive and seamless fusion

events of distinct epidermal cell layers were observed in ad1 tassel glumes, which

were reduced in size, and anthers (Figure 1N-Q; Supplemental Figure 1). We also

checked developing ear spikelets using SEM and observed fusion events in

adjacent glumes (Supplemental Figure 1). The seemingly normal appearance of

mature ears therefore may be simply due to the reduced outgrowth of glumes in

ear spikelets when compared to tassel spikelets. Taken together these

observations indicate that the organ adherence of ad1 mutants were caused by

epidermal fusions among and between different tissues and organs in both juvenile

and reproductive stages, and suggest that AD1 plays an important role in

establishing epidermal properties required to maintain proper organ separation

throughout maize development.

AD1 encodes a 3-KETOACYL-CoA SYNTHASE involved in cuticular wax

biosynthesis


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Using an F2 segregating population and a positional cloning approach, the ad1-

224 allele was mapped to a 10.8 Mb window on chromosome 1 between SSR

markers umc1147 and umc2080 (Figure 2A). We subsequently performed Bulked

Segregant Whole Genome Sequence analysis to identify point mutations in coding

genes within the mapping window (Dong et al., 2019). A G to A transversion was

found in the ad1-224 mutant bulk sample within the coding region of

GRMZM2G167438/Zm00001d032728 (B73v3/v4), which introduced a premature

stop-codon (W210>STOP). GRMZM2G167438/Zm00001d032728 encodes a 3-

KETOACYL-CoA SYNTHASE (KCS), a key enzyme in cuticular wax biosynthesis

(Yeats and Rose, 2013). To confirm that GRMZM2G167438/Zm00001d032728

corresponded to AD1, we sequenced the candidate gene in three additional alleles

(ad1-109D, ad1-110E and ad1-09-2121). Sequencing results showed that ad1-

109D and ad1-110E contained an identical and unique 1067 bp insertion in the

second exon, leading to a frame shift (hereafter ad1-109D and ad1-110E are

referred to as ad1-ref; Supplemental Figure 2), while ad1-09-2121 carried a C to T

transversion within the second exon that introduced a premature stop-codon

(Figure 2A,B; Q327>STOP). Mutations in all three independent alleles were

predicted to produce truncated AD1 proteins (Figure 2B). These results confirmed

that knockout of GRMZM2G167438/Zm00001d032728 caused the adherence

phenotype of ad1 mutants.

AD1 is an enzyme of 466 amino acids containing a conserved domain that

includes substrate binding, active and product binding sites (Figure 2B). Based on

qRT-PCR and available RNA-seq data, and consistent with the severe phenotype

observed in seedlings and tassels, AD1 showed high levels of expression in

tassels, ears and seedling leaves, moderate levels in silks and embryos, and weak

levels in endosperm, mature leaves and roots (Supplemental Figure 2). To


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characterize the expression pattern of AD1 in more detail, we carried out RNA in

situ hybridizations in germinating shoots, 20 DAP embryos and immature tassels.

In all tissues tested, AD1 showed strongest expression in the epidermal L1 layer

of young leaves, embryos and tassels (Figure 2C-F). To determine its subcellular

localization, we performed confocal co-imaging of a YFP-AD1 fusion protein and

the endoplasmic reticulum (ER) marker mCHERRY-CNX1 (Gao et al., 2012) in N.

benthamiana leaf epidermal cells. Strong co-localization of the YFP-AD1 and

mCHERRY-CNX1 signals was observed (Figure 2G). Altogether, the expression of

AD1 in epidermal cells and the subcellular localization of AD1 in the ER are

consistent with the role of AD1 in cuticular wax biosynthesis (Yeats and Rose,

2013).

The KCS family is plant specific and shows extensive genetic redundancy

(Yeats and Rose, 2013). The Arabidopsis genome contains 21 KCS genes

(Costaglioli et al., 2005; Joubes et al., 2008), and several KCS genes have been

previously described for their role in the synthesis of VLCFAs (James et al., 1995;

Todd et al., 1999; Yephremov et al., 1999; Fiebig et al., 2000; Gray et al., 2000;

Pruitt et al., 2000; Franke et al., 2009; Lee et al., 2009; Kim et al., 2013). A

homology-based search showed that the maize B73v3 reference genome contains

28 maize KCS genes. Neighbor-joining analysis classified the 21 Arabidopsis KCS

proteins and 28 maize KCS proteins into several different clades, and placed AD1

within the clade that includes Arabidopsis KCS3, KCS12 and KCS19. FDH/KCS10,

whose mutants also showed organ fusion phenotypes in Arabidopsis (Yephremov

et al., 1999; Pruitt et al., 2000), belongs to a separate distant clade (Supplemental

Figure 3). To understand why ad1 single mutants showed a severe phenotype in

maize, we analyzed the expression level of the 28 KCS genes in different tissues

based on publicly available RNA-seq datasets (Stelpflug et al., 2016). In general,


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KCS genes were broadly expressed in all tissues throughout maize development,

although some differences could be observed based on expression levels in aerial

vs. root tissue (Supplemental Figure 2). Taken together, these results suggest that

loss of AD1 function cannot be completely compensated by related family

members despite showing largely overlapping expression patterns with them (i.e.

GRMZM2G162508/ZmKCS15).

Cuticular wax biosynthesis and deposition are defective in ad1 mutants

To investigate whether loss of AD1 function influenced cuticular wax biosynthesis

and deposition, we tested the “lotus effect”, a self-cleaning mechanism of many

plant leaves in which water tends to roll to the ground as droplets, collecting and

washing particles and debris from the leaf surface in the process. The efficiency of

this self-cleaning mechanism has been correlated with the abundance of

epicuticular wax crystals (Barthlott and Neinhuis, 1997). Wild type and ad1 mutant

seedlings were misted with water, and water droplets accumulated only on the

surface of ad1 mutant leaves (Figure 3A), suggesting epicuticular wax crystal

defects. We therefore analyzed the deposition of epicuticular wax crystals on the

leaf surface by SEM. Strikingly, the number of epicuticular wax crystals was

considerably reduced and wax crystals appeared smaller in ad1 mutant leaves

than wild type (Figure 3B). We also quantified the accumulation of toluidine blue

stain in germinating coleoptiles, an assay used to measure cuticle permeability,

and detected a higher accumulation of the dye in ad1 mutants (Figure 3C,D),

suggesting defects in cuticle formation during embryogenesis (Tanaka et al., 2004;

Doll et al., 2020). In addition, we also examined cuticular wax accumulation by

measuring cuticular transpiration and chlorophyll leaching (Figure 3E,F), which are

frequently used to examine cuticular defects in leaves (Chen et al., 2003). This

analysis showed that both cuticular transpiration, measured as loss of leaf weight,


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and chlorophyll extraction occurred more rapidly in ad1 mutant alleles relative to

wild type, further confirming the observed defects in cuticular wax accumulation of

ad1 mutants.

The reduced amount of epicuticular wax crystals on the surface of ad1 leaves

together with the water loss and chlorophyll leaching assays suggested that AD1

is involved in cuticular wax biosynthesis. Therefore, we evaluated the amount and

composition of cuticular waxes from the third leaves of wild type and ad1 plants

using gas chromatography-mass spectrometry (GC-MS) and gas chromatography

coupled to a flame ionization detector (GC–FID) (Figure 4 and Supplemental

Figure 4). The amount of total wax was significantly lower in ad1-224 (∼18%) and

ad1-ref (∼23%) than in wild type leaves (Figure 4A, Supplemental Figure 4). Maize

leaf cuticular waxes are composed of a mixture of compounds including VLCFAs,

alkanes, alcohols, aldehydes, ketones, wax esters and alicyclic compounds

(Bourgault et al., 2020). All components were significantly decreased in ad1-224

compared with wild type, with the exception of alkanes (Figure 4A). Similar results

were obtained with the ad1-ref mutant, although primary alcohols and fatty acids

were also not significantly different from wild type levels (Supplemental Figure 4).

The primary alcohol fraction was the most abundant component class in juvenile

leaf waxes (Figure 4A, Supplemental Figure 4), with the C32:0 primary alcohol being

the dominant homolog, as expected (Bianchi et al., 1978). In fact, this single

component (C32:0 primary alcohol) constituted over 60% of the overall wax load in

the samples studied. The level of C32:0 primary alcohol in ad1-224 and ad1-ref

mutants was decreased by approximately 11% and 13% relative to the wild type,

respectively (Figure 4B, Supplemental Figure 4). Another abundant component

was C32:0 aldehyde, which constituted about 25% of the total wax load. In ad1-224

and ad1-ref mutants, the C32:0 aldehyde load was reduced by approximately 30%


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and 48%, respectively, compared with values in wild type samples (Figure 4D,

Supplemental Figure 4). Less abundant components, including C30:0-C34:0 fatty

acids, C30:0 and C34:0 aldehydes, C33:0 alkane and all four identified alicyclic

compounds, showed lower concentrations in ad1-224 mutants than in control

samples (Figure 4C,E,G). In ad1-ref mutants, leaf waxes showed reduced

amounts of C34:0 fatty acid, C28:0-C34:0 aldehydes, C33:0 and C39:0 alkanes,

tocopherolin and campesterol (Supplemental Figure 4). Interestingly, all

components of the wax ester fraction, which corresponds to less than 5% of the

overall wax load, were equally affected in both alleles of ad1 (Figure 4F,

Supplemental Figure 4). Altogether, ad1 mutants had reduced amounts of cuticular

wax components with acyl chains longer than 31 carbons (with the exception of

the primary alcohol fraction), suggesting a key role for AD1 in the formation of wax

compounds with more than 31 carbons.

The MYB transcription factor FUSED LEAVES1 positively regulates AD1

expression

The previously characterized maize mutant fused leaves1 (fdl1) has a phenotype

similar to ad1, in particular regarding seedling leaf blade fusion and cuticular wax

accumulation defects. FDL1 (GRMZM2G056407/Zm00001d022227) encodes

MYB94, a MYB transcription factor (La Rocca et al., 2015) whose Arabidopsis

homologs are known to be involved in the regulation of cuticular wax biosynthesis

(Raffaele et al., 2008; Seo et al., 2011; La Rocca et al., 2015). To understand

whether AD1 was regulated by FDL1/MYB94, we obtained a transposon insertion

(mu1092890) in the FDL1 gene, which disrupts its second exon (Figure 5A).

Homozygous fdl1-Mu mutant plants showed organ fusion defects at the seedling

stage (Supplemental Figure 5). Similar to ad1 mutants, a reduced amount of

epicuticular wax crystals, increased loss of epidermal water and faster chlorophyll


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leaching were also observed in fdl1-Mu plants (Supplemental Figure 5) mirroring

what was previously reported for another fdl1 allele (La Rocca et al., 2015). Using

RNA in situ hybridizations, we determined that FDL1 showed strong expression in

the epidermal layer of young leaves and tassels, in a pattern remarkably similar to

AD1 (Figure 5B, Supplemental Figure 5). We next generated ad1;fdl1 double

mutants by crossing ad1-ref with fdl1-Mu plants. At the seedling stage, both single

mutants and double mutants showed the characteristic leaf fusion phenotype

(Figure 5C). SEMs of the leaf surface showed that the epicuticular wax crystal

deposition defects of ad1-ref were enhanced by the fdl1 mutation (Figure 5D).

Accordingly, water loss by cuticular transpiration was slightly faster in ad1-ref;fdl1-

Mu double mutants than in ad1-ref, fdl1-Mu and wild type samples, and chlorophyll

leaching occurred more rapidly in ad1-ref;fdl1-Mu double mutants compared with

each single mutant and wild type (Figure 5E,F). The accumulation of toluidine blue

stain in germinating coleoptiles was also higher in ad1;fdl1 double mutants relative

to single mutants (Figure 5G,H), and in double mutant mature plants we also

noticed a higher frequency of fusion events in leaves of adult plants (Supplemental

Figure 6).

To investigate the hypothesis that FDL1/MYB94 could directly regulate the

expression of AD1 and other KCS genes, we performed DAP-seq, an in vitro DNA-

TF binding assay that captures genomic DNA binding events in their native

sequence context (O'Malley et al., 2016; Galli et al., 2018). In vitro expressed

HALO-FDL1 proteins were incubated with maize genomic DNA libraries and FDL1-

bound DNA fragments were identified using next-generation sequencing. In total,

10,028 FDL1/MYB94 binding events were detected of which 2,737 (27%) were

located at distances up to 10Kb upstream of the transcription start site (TSS),

within the gene or 3Kb downstream of the transcription termination site (TTS)


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(Figure 6A), putatively targeting 2,533 unique genes. FDL1 preferentially bound to

sequences containing the core CCAACC motif (Figure 6B). This consensus motif

was highly similar to the motif observed previously for the distantly related

P1/MYB3 TF in maize (Grotewold et al., 1994; Morohashi et al., 2012) as well as

the motif identified in DAP-seq experiments for AtMYB94 and AtMYB96 (O'Malley

et al., 2016). It also resembled that reported in the promoters of several cuticle-

related target genes of AtMYB94 and AtMYB96, although several nucleotides

outside of the core AAC motif were divergent in these genes (Seo et al., 2011; Lee

and Suh, 2015).

A major functional category of genes directly bound by FDL1 included those

encoding a subset of wax biosynthetic enzymes, such as homologs of Arabidopsis

KCR1, CER1 and CER3, as well as many KCS family genes (Supplemental Table

1)(Yeats and Rose, 2013). Overall, of the 28 KCS genes in maize, we observed

strong peaks in putative regulatory regions of five KCS genes including AD1

(Figure 6C; Supplemental Figure 7, Supplemental Table 1), suggesting that FDL1

may regulate cuticular wax biosynthesis in maize via transcriptional control of

multiple KCS genes. An additional seven KCS genes also showed weak binding

(adjusted p-value cutoff 0.01). Maize homologs of genes encoding the putative wax

transporter AtLTPG1 and regulators of cuticular wax biosynthesis AtMYB30,

AtMYB106 were also directly bound by FDL1, suggesting that cuticular wax

biosynthesis and transport were broadly regulated by FDL1 (Supplemental Table

1). Many additional genes for which no direct role in wax formation has yet been

described, were also found to be strongly bound by FDL1. These included

ZmLTP2/GRMZM2G010868, encoding a lipid transfer protein whose closest

Arabidopsis homolog is expressed in the L1 layer; ZmPSS1 (phosphatidylserine

synthase1/GRMZM2G039385), a gene involved in lipid metabolism that affects


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Arabidopsis pollen and meristem development (Liu et al., 2013); and

ZmEXPA1/GRMZM2G339122, encoding alpha expansin1, a cell wall modifying

enzyme (Ramakrishna et al., 2019).

As mentioned above, AD1 was among the list of KCS genes directly bound by

FDL1. A strong peak was located in the 5’UTR region along with a minor peak in

the first intron (Figure 6C). We subsequently checked the sequence of the proximal

AD1 promoter and found that three core CCAACC motifs were present in the FDL1

binding peak region (Figure 6C, Supplemental Figure 7). Electrophoretic mobility

shift assays (EMSAs) using a glutathione S-transferase (GST)-FDL1 fusion protein

produced in E. coli were performed to test FDL1 binding to the AD1 promoter. A

208 bp AD1 promoter sequence containing the three core CCAACC motifs was

used as the target probe with a mutated core motif sequence containing TTTTTT

as a negative control. As shown in Figure 6D, GST-FDL1 bound the probe

containing the CCAACC motifs resulting in a visible shift of the protein-DNA

complex. This shift was not observed when GST-FDL1 was incubated with the

negative control probe containing the mutated core motif. Furthermore, addition of

excess amounts of unlabeled wild type probe outcompeted labeled wild type probe,

indicating a specific interaction. These results indicated specific binding of FDL1

to the AD1 promoter sequence in vitro. To investigate whether FDL1 binding to the

AD1 promoter could affect its transcription, we used a transient dual-luciferase

(LUC) expression assay in maize protoplasts. An FDL1 effector construct and a

luciferase reporter construct driven by a 1.6-kb wild type or mutated core CCAACC

motif promoters of AD1 were transiently co-expressed in protoplasts, and a 2-fold

increase in the luminescence intensity compared with the control was observed

(Figure 6E,F). Considerably reduced expression was observed in the reporter line

driven by the mutated promoter (Figure 6F). These results indicate that FDL1


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recognized the AD1 proximal promoter region and activated its expression in

maize protoplasts. In agreement with these findings, the expression level of AD1

and other KCS genes (i.e. ZmKCS13, ZmKCS15 and ZmKCS24), assessed by

qRT-PCR in fdl1 mutant embryos, were significantly reduced compared to wild type

(Figure 6G), confirming that FDL1 regulates AD1 and KCS transcription. These

results suggest that FDL1 directly activates AD1 expression in vivo, most likely by

binding to the CCAACC motifs in its 5’UTR region. Collectively, these observations

suggest that FDL1 functions as a positive regulator of AD1 as well as other

enzymes involved in cuticular wax biosynthesis in maize.

DISCUSSION

Maize ad1 mutants showed fusions between cells and organs, and fusion events

were evident from embryo through tassel and floret development. These results

highlight the importance of proper cuticle formation for maize shoot development,

and show that AD1, a 3-KETOACYL-CoA SYNTHASE involved in cuticular wax

biosynthesis, is necessary for keeping organs separated and prevent postgenital

fusion events. Previously reported maize glossy mutants have wax accumulation

defects, but unlike ad1, they do not display organ fusions (Bianchi et al., 1978;

Moose and Sisco, 1996; Xu et al., 1997; Sturaro et al., 2005; Li et al., 2014). A

similar situation is seen in Arabidopsis, where the fdh mutant, defective for a KCS

enzyme distinct from AD1, shows severe organ fusions (Yephremov et al., 1999;

Pruitt et al., 2000), while other mutations in KCS genes do not display similar

defects (James et al., 1995; Todd et al., 1999; Yephremov et al., 1999; Fiebig et

al., 2000; Gray et al., 2000; Pruitt et al., 2000; Franke et al., 2009; Lee et al., 2009;

Kim et al., 2013). While redundancy in the KCS family is thought to be

advantageous among higher terrestrial plant species, it is likely that AD1 and FDH,

which belong to distinct clades (Supplemental Figure 3), have evolved specialized


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and predominant roles that cannot be complemented by other closely related

family members. In addition, other gene families involved in cuticular wax as well

as cutin biosynthesis when mutated also exhibit organ fusion (i.e. wax1, wax2, lcr,

hth and lacs1 lacs2) indicating that cuticle formation involves a complex array of

components, each critical for plant development (Jenks et al., 1996; Wellesen et

al., 2001; Chen et al., 2003; Kurdyukov et al., 2006b; Kurdyukov et al., 2006a; Bird

et al., 2007; Panikashvili et al., 2007; Weng et al., 2010). While the mechanism of

how wax and cutin biosynthetic pathway mutants affect cuticle structure,

composition and properties is not well understood, it is clear that proper cuticle

formation which starts from L1 cells during embryo development, plays an

essential role in maintaining organ separation and preventing postgenital fusions

(Yephremov et al., 1999; Pruitt et al., 2000; Ingram and Nawrath, 2017). Detailed

characterization of various organ fusion mutants in early stages of development

should improve understanding of the molecular and cellular mechanics of how the

cuticle suppresses epidermal fusion in plants. For example, recent work in

Arabidopsis revealed mechanistic details of the early steps of cuticle deposition by

the action of an endosperm localized subtilase and peptide/receptor complexes

derived from the embryo (Doll et al., 2020).

Genome-wide DAP-seq analysis indicated that FDL1, a previously described

MYB TF (La Rocca et al., 2015), directly binds to many genes involved in wax

biosynthesis, including those encoding enzymes, transporters and upstream

transcriptional regulators. A major category of targets bound by FDL1 and

validated by qRT-PCR belongs to the KCS gene family, including AD1 itself. These

data suggest that FDL1 may regulate cuticular wax biosynthesis through

transcriptional control of a suite of KCS genes. In agreement with these findings,

genetic analysis showed that ad1;fdl1 double mutants enhanced the cuticle defects


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of single ad1 mutants, suggesting that other genes (i.e ZmKCS13,15 and 24) were

the targets of FDL1 activity during juvenile development. Accordingly, both AD1

and FDL1 genes were predominantly expressed in epidermal cells of germinating

seedlings. These data indicate that FDL1 is likely a key regulator of cuticular wax

biosynthesis, and AD1 and FDL1 function is essential to form a proper barrier to

prevent epidermal fusions in the early stages of maize development. Many

additional genes whose role in cuticle development has yet to be described were

also directly bound by FDL1, providing new avenues for the discovery of new

players involved in this process.

In Arabidopsis, several MYB TFs are known to play a role in cuticle formation

i.e. AtMYB16, AtMYB30, AtMYB94, AtMYB96, and AtMYB106 (Raffaele et al.,

2008; Seo et al., 2011; Oshima et al., 2013; Lee and Suh, 2015). Of these,

AtMYB94 and AtMYB96, the MYBs most-closely related to FDL1/ZmMYB94, have

been shown by microarray, EMSA, and ChIP-PCR to directly regulate a handful of

distinct cuticle biosynthetic genes (Seo et al., 2011; Oshima et al., 2013; Lee and

Suh, 2015). The maize orthologs of several of these genes were also directly

bound by FDL1 in our DAP-seq dataset (Supplemental Table 1). To the best of our

knowledge, no regulatory information has been reported for any Arabidopsis KCS

genes belonging to the AD1 clade, suggesting possible regulatory variation among

these two species. Therefore, while a core regulatory module composed of MYB

TFs and KCS genes appears conserved between Arabidopsis and maize, many

species-specific features are likely to exist highlighting the importance of obtaining

empirical data from different species.

Similar to ad1 mutants, fdl1 mutant plants show cuticular wax deposition

defects and fusions in leaves but, unlike ad1, no fusion events are observed during

inflorescence development. We therefore hypothesize that other closely related


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maize MYB family members may function redundantly to FDL1 during reproductive

development. It is important to note that the Arabidopsis MYBs most closely related

to FDL1 (AtMYB30/94/96), are induced by stress i.e. drought or pathogens, and

positively regulate cuticular wax biosynthesis (Raffaele et al., 2008; Seo et al.,

2011; Lee and Suh, 2015). These similarities suggest that the maize FDL1/AD1

regulatory module may also enhance stress resistance by activating cuticular wax

biosynthesis. Overall, these findings discovered an essential pathway for cuticle

and organ development in maize, an important step to devise strategies for

improving environmental adaptability and stress tolerance in variable

environments for maize and other crops.

METHODS

Plant materials and phenotyping

The ad1-224 allele, originally identified in an EMS mutagenesis screen for rel2

enhancers/suppressors in the A619 background, was back-crossed to A619 before

phenotypic measurements. The ad1-ref allele was obtained from the Maize

Genetics Cooperation Stock Center (stock ad1-109D and ad1-110E), while the

ad1-9.2121 allele was originally obtained in an EMS enhancer screen of a weak

ramosa1 mutant. The FDL1 insertion line mu1092890 (stock UFMu-13110) was

obtained from the Maize Genetics Cooperation Stock Center. The phenotype of

single and double mutant plants was analyzed at Rutgers University, NJ, in field

and greenhouse grown plants. Student’s t-test was used to determine statistical

significance for all measurements.

Positional cloning

For map-based cloning of the AD1 locus, we constructed a fine-mapping F2


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population by crossing ad1-224 mutant from the original M2 genetic background

to the B73 inbred line. Using the PCR-based molecular markers, we mapped ad1

to a 10.8 Mb window on chromosome 1 between markers UMC1147 and

UMC2080. For Bulked Segregant Whole Genome Sequence analysis, a single

bulk genomic DNA sample was obtained from 10 ad1 plants. Library preparation

and sequencing of the sample was performed by Macrogen. 150bp pair end

sequencing was done on a NovaSeq 6000 Illumina instrument. The resulting fastq

files were used for analysis by a previously published pipeline (Dong et al., 2019),

using B73v3 as a reference genome. Two index plots were produced by the

ggplot2 package in the R programming language, one for all SNPs and one for all

INDELs identified by the pipelines (Dong et al., 2019). Then checked SNPs within

the 10.8 Mb ad1 mapping window using the Integrative Genomics Viewer (IGV;

http://www.broadinstitute.org/igv/). The sequence of AD1 was deposited in

GenBank (accession no. MN872839) and corresponds to

GRMZM2G167438_T01/Zm00001d032728_T001 (B73v3/v4).

Microscopy

For light microscopy, thin sections (8 µm thick), were stained with Safranin

O/Alcian Blue and images were acquired using a Leica DM5500B microscope

equipped with a DFC450 C digital camera. For the analysis of organ fusion defects

and epicuticular wax crystals, fresh tissue was imaged using a JMC-6000PLUS

Scanning Electron Microscope.

For confocal microscopy, YFP-AD1 and mCHERRY-CNX1 were transiently

expressed in N. benthamiana using Agrobacterium-mediated leaf injections.

Images were obtained on a Leica SP5 confocal microscope. The YFP signal was

imaged using 514 excitation and 520–575 emission, while mCHERRY was imaged


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using 594 excitation and 625-655 emission settings. Acquired images were

analyzed using FIJI.

Expression analysis

For quantitative real-time PCR (qRT-PCR), total RNA was extracted from different

tissues using the RNeasy Plant Mini Kit (Qiagen). Retrotrascription was performed

using the qScript cDNA Synthesis kit. cDNA was amplified with PerfeCTa®

SYBR® Green FastMix® (Quanta Biosciences) on an Illumina Eco Real-Time PCR

System. Relative expression levels were calculated using 2−ΔΔCt method with

UBIQUITIN as control. The primers used for qRT-PCR are listed in SI Appendix

Table S2.

For in situ hybridizations, 0.2 cm B73 germinating seedlings and tassels were

dissected and fixed using paraformaldehyde acetic acid (PFA). Samples were

dehydrated and embedded in paraplast. Hybridizations were performed at 59°C,

overnight. After several washes, samples were treated with anti-digoxygenin (DIG)

antibody (Roche) and signals were detected using NBT/BCIP (Promega). The AD1

and FDL1 antisense probes were synthesized using T7 RNA polymerase

(Promega) of AD1 3’UTR and FDL1 3’UTR cloned into pENTR223.1-Sfi and

digested with EcoRI.

Phylogenetic analysis

Full length amino acid sequences of the KCS family were obtained from

Phytozome 12.1. Sequences of maize and Arabidopsis were aligned with Clustal

Omega. The alignment file was then used to generate a neighbor-joining rooted

tree with MEGA 5.0, applying the Bootstrap method and 1000 bootstrap

replications (Simmons and Freudenstein, 2011).


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Water loss and chlorophyll leaching assays

For water loss assays, 3-week-old plants grown in greenhouse potting media were

used. Whole shoots of dark-acclimated seedlings were excised and leaves soaked

in water for 60 min in the dark. Subsequently, excess water was removed from the

leaves, and 3cm pieces of leaves were weighed at the indicated time points using

a precision balance. Three measurements were averaged per each time point in

each genotype.

Chlorophyll leaching assays were performed on leaves of 3-week-old plants.

Two grams of each leaf sample was incubated on ice for 30 min and immersed in

30 mL of 80% ethanol in 50 mL conical tubes at room temperature. Aliquots of 100

µL were removed from the solution at every 15 min after initial immersion. The

amount of extracted chlorophylls was quantified by measuring absorbance at 647

and 664 nm using a spectrophotometer (Thermo Scientific). Three measurements

were averaged for each time point in each genotype.

Toluidine blue permeability test

For assessing cuticle integrity, toluidine blue tests were performed, as previously

described (Tanaka et al., 2004; Doll et al., 2020). Two-day old etiolated

coleoptiles were stained for 5 minutes in a toluidine blue solution (0.05% w/v)

with Tween 20 (0.1% v/v) and washed in tap water. Pictures were acquired using

a Leica DM5500B microscope equipped with a DFC450 C digital camera. For

quantification, 5 coleoptiles were excised, placed in tubes containing 1 mL of

80% ethanol, and incubated for 4 hours in the dark until all dye and chlorophyll

had been extracted. Toluidine blue absorbance of the resulting solution was


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analyzed using a spectrophotometer. Per each treatment, 5 repeats were

performed.

Cuticular wax analysis

Waxes were extracted from an expanded portion of juvenile leaves (3rd leaf). A

small section of each leaf used for the analysis was preliminary scanned by SEM

to verify loss of crystal waxes in the mutants. Wax components were identified by

GC-MS and quantified by gas chromatograph coupled to a flame ionization

detector (GC–FID) following the protocol described in Bourgault et al (2020)

(Bourgault et al., 2020).

DAP-seq, read mapping, filtering, and peak calling

HALO-FDL1 and HALO-GST (negative control) DAP-seq samples were performed

using protein expressed in the TNT rabbit reticulocyte expression system

(Promega) as previously described (Bartlett et al., 2017) with the exception that 1

microgram of genomic maize B73 DNA library was added to the protein bound

beads. Protein bound beads and gDNA were rotated for 1h at RT. Beads were

washed four times in 1X PBS+NP40, followed by two washes with 1X PBS. Beads

were transferred to a new tube and DNA was recovered by resuspending in 25μl

EB and boiling for 10min at 98°C. Eluted samples were enriched and tagged with

dual indexed multiplexing barcodes by performing 20 cycles of PCR in a 50μl

reaction. Samples were pooled and sequenced on a NExtSeq500 with 75bp single

end reads. A total of 10–30 million reads were obtained for each sample.

Reads were mapped to the B73v3 genome as described in Galli et al., 2018 (Galli

et al., 2018). In brief, fastq files were trimmed using trimmomatic with the following

parameters ILLUMINACLIP:TruSeq3-SE:2:30:10 LEADING:3 TRAILING:3


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SLIDINGWINDOW:4:15 MINLEN:50. Trimmed reads were mapped to the B73v3

reference genome (nuclear chromosomes only) using bowtie2 v2.2.8. Mapped

reads were filtered for reads containing >MAPQ30 using samtools (samtools view

–b –q 30) in order to restrict the number of reads mapping to multiple locations in

the genome. MAPQ filtered reads were used for all subsequent analysis. Peaks

were called using GEM v2.5 using the GST-HALO negative control sample for

background subtraction and an FDR of 0.00001 (--q 5). Peak calling was

performed with the following parameters: --d Read_Distribution_default.txt --k_min

6 --k_max 20. For visualization in the Integrative Genome Browser (IGV), bam files

containing mapped reads were converted to bigwig files using deep Tools v2.5.3

bam Coverage with 10bp bin size and FPKM normalization. Peaks were

associated with their closest putative target genes using ChIPseeker (Yu et al.,

2015). The FDL1 DAP-seq dataset was deposited in the Gene Expression

Omnibus (GEO) database (accession no. GSE142847).

Electrophoretic Mobility Shift Assays

For EMSAs, a selected region -411 to -233 relative to AD1 start codon (+1) within

the AD1 promoter that was enriched for core CCAACC elements was PCR

amplified, gel purified, and biotinylated using the Biotin 3′ End DNA Labeling Kit

(Thermo Scientific) according to the manufacturer’s recommendations. DNA

binding assays were performed using the Lightshift Chemiluminescent EMSA kit

(Thermo Scientific) as follows: binding reactions containing 1× Binding Buffer, 50

ng/μL polydI/dC, 2.5% glycerol, 2 μL biotinylated probe, and 1 μL purified GST-

FDL1 protein were incubated at room temperature for 20 min and loaded on a 6%

DNA retardation gel (Life Technologies) before transfer to a nylon membrane.

Subsequent detection was carried out according to the manufacturer’s


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recommendations. Competition with unlabeled probe was carried out using 50-fold

excess of unlabeled probe. Mutated probe was generated by annealing two

complementary oligos in which the core CCAACC elements were changed to

TTTTTT. Primers used are listed in Supplemental Table 2.

Transient Transcription Dual-LUC Assay

The isolation of B73 maize mesophyll protoplasts, PEG-calcium transfection of

plasmid DNA, and protoplast culture were performed as described previously

(Sheen, 2001). The effector vector pRI101 was used for the expression of the

FDL1 gene driven by a 35S promoter. The reporter vector pGreenII 0800-LUC was

used for detecting the transactivation activity of the AD1 promoter. The ratio of

LUC/REN activity was measured using the DLR assay system (Promega). Primers

are listed in SI Appendix Table S2.

AUTHOR CONTRIBUTIONS

X.L., J.S. and A.G. conceived the study; X.L., J.S., R.B., Z.C., M.G., J.D. and A.G.

performed all experiments; X.L., R.B., M.G., J.D., I.M. and A.G. analyzed data;

X.L. and A.G. wrote the manuscript with inputs from all authors.

DATA DEPOSITION

The sequence of AD1 was deposited in GenBank (accession no. MN872839) and

corresponds to GRMZM2G167438_T01/Zm00001d032728_T001 (B73v3/v4). The

FDL1 DAP-seq dataset was deposited in the Gene Expression Omnibus (GEO)

database (accession no. GSE142847).

ACKNOWLEDGMENTS


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The authors wish to thank the Maize Genetics Cooperation Stock Center for seeds;

Mariusz Roszkowski and Jonathan Kunkel-Jure for help with positional cloning;

Erik Vollbrecht for providing the ad1-9.2121 allele. This work was supported by the

National Science Foundation (IOS#1456950) to A.G., by a National Science

Foundation Postdoctoral Research Fellowship in Biology grant (IOS#1710973) to

J.S., and by funding from the Canada Research Chairs program and the Natural

Sciences and Engineering Research Council of Canada (RGPIN-2019-05923) to

I.M.


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Figure 1. The adherent1 mutant shows organ fusion defects.

(A) The seedling phenotype of wild type and ad1-224 plants. The second leaf is

rolled and fused to the adaxial surface of first leaf (upper image). The second leaf

and first leaf show fusion at the leaf tip (lower image). (B) Mature tassel phenotype.

Tassel branches are fused together in ad1-224. (C and D) In ad1 tassel spikelets

adhere to each other, and the glumes of adjacent spikelets are fused together.

Scale bars, 0.1cm. (E) Anthers in the same floret show fusion in ad1-224. Scale

bars, 0.1cm. (F) Cross sections of wild type and ad1-224 mature tassels. Scale

bars, 0.1cm. (G) Saffranin-O Alcian Blue staining of transverse sections of wild

type and ad1-224 mature tassel. Scale bars, 0.2mm. (H-K) Longitudinal and


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transverse sections of wild type (H and J) and ad1-224 (I and K) germinating

seedlings. Higher magnification (lower image) of the area framed in upper image.

Scale bars, 0.2 mm. (L and M) SEMs of wild type leaves (L) show no fusion defects,

while a seedling leaf blade curled up and fused to itself in ad1-224 mutants (M).

Scale bars, 100 μm. Arrows point to regions of fusion. (N and O) Spikelet

phenotype of wild type and ad1-224 mutants. Note that the glumes of adjacent

spikelets are separated in wild type but fused in ad1-224 mutants. Higher

magnification (right image) of the area framed in left image. (P and Q) Anthers of

same floret are separated in wild type tassels, but fused in ad1-224 mutants.

Higher magnification (right image) of the area framed in left image. Scale bars in

N-Q, 500 μm.


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Figure 2. AD1 encodes a 3-KETOACYL-CoA-SYNTHASE.

(A) Positional cloning of AD1. Schematic representation of the AD1 gene and the

position of the mutant alleles. Exons are depicted as black rectangles and UTRs

are depicted as grey rectangles. (B) Schematic representation of the AD1 protein.

ACP_syn_III_C, 3-Oxoacyl-[acyl-carrier-protein (ACP)] synthase III C terminal

(Pfam). (C to F) RNA in situ hybridizations with AD1 antisense probes. Expression

pattern in wild type germinating seedlings (C), higher magnification of the area

framed in C (D), shoot region of 20 DAP embryos (E), and immature tassel (F).

Scale bars, 0.2 mm. (G) Confocal images of YFP-AD1 shows co-localization with


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the ER marker mCHERRY-CNX1 in N. benthamiana leaf epidermal cells. Scale

bars, 50 μm.


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Figure 3. Epicuticular wax crystals and transpiration analysis.

(A) Leaves of wild type and ad1-224 seedlings misted with water. (B) SEM images

of epicuticular wax crystals on the third leaf sheath (top) and the third leaf lamina

(bottom) in wild type and ad1-224 mutants. Scale bars, 5 μm. (C) Toluidine blue

tests on etiolated coleoptiles. Scale bars, 1 mm. (D) Quantification of toluidine blue

uptake by the coleoptile of young seedlings, normalized to chlorophyll content. n=5

(5 seedlings per repetition; * Student-test P < 0.001). Error bars represent SD. (E)

Chlorophyll leaching assays. Extracted chlorophyll contents at individual time

points were expressed as percentages of that at 24 h after initial immersion. Error

bars show SD, n=3. (F) Water loss assays. Error bars show SD, n=3.


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Figure 4. ad1 cuticular wax analysis.

(A) Total wax coverage and amount of each wax class in ad1-224 mutants and

wild type leaves. The inset shows the less abundant wax classes at a different

scale to more clearly visualize significant differences. (B to G) Concentration of

individual components in each wax class: primary alcohol (B), fatty acid (C),

aldehyde (D), alkane (E), wax ester (F) and alicyclic (G). Means of 4 replicates and

SD are reported. The asterisks represent significant difference determined by the

Student’s two-tail t test; *, P < 0.05; **, P < 0.01.


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Figure 5. fdl1-Mu enhances the cuticle defects of ad1 mutants.

(A) Schematic representation of the FDL1 gene showing the transposon insertion

in the second exon (black and grey rectangles, exons and UTR regions,

respectively). (B) RNA in situ hybridization with an FDL1 antisense probe in

germinating seedlings. (C) The seedling phenotype of wild type, ad1-ref, fdl1-Mu

and ad1-ref;fdl1-Mu. (D) SEM images of epicuticular wax crystals on the third

leaves in wild type, ad1-ref, fdl1-Mu and ad1-ref;fdl1-Mu. Scale bars, 5 μm. (E)

Water loss assays of dark-acclimated plants. Error bars show SD, n=3. (F)

Chlorophyll leaching assays. Extracted chlorophyll contents at individual time

points were expressed as percentages of that at 24 h after initial immersion. Error

bars show SD, n=3. (G) Toluidine blue assay on etiolated coleoptiles. Scale bars,

1 mm. (H) Quantification of toluidine blue uptake by the aerial parts of young


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seedlings, normalized to chlorophyll content (n=5, 5 seedlings per replicate; *

Student-test, P < 0.001). Error bars represent SD. UT, untreated samples.


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Figure 6. FDL1 directly regulates AD1 expression.

(A) Distribution of FDL1 peaks (qvalue e-5 Q30) within gene features. (B) Top

FDL1 binding motif identified by DAP-seq. (C) FDL1 directly binds to AD1 5’UTR

in DAP-seq. Screenshot of aligned reads. In green are significant peaks. Gene

models show location of EMSA probe and the promoter used for the Luciferase

assay (boxed) and the core binding motif predicted by DAP-seq marked in red lines

that were mutated to generate mprobe (in EMSAs) and mPromoter (mpro, in

transcriptional activation assay). (D) EMSAs show that GST-FDL1 directly bind to

the promoter of AD1. (E) Schematic diagram of effector and reporter constructs

used for transcriptional activation assay. (F) AD1 transcriptional activation by

FDL1. Error bars show SD, n=4. (G) Comparison of AD1 and selected KCS genes

expression levels between wild type and fdl1-Mu. The maize UBIQUITIN gene was

used as reference. Values are means ± SD (n=3). Asterisks represent significant

difference determined by the Student’s two-tail t test at P < 0.001.


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