Cell adhesion in Arabidopsis thaliana is mediated by ECTOPICALLY PARTING CELLS 1 – a...

Cell adhesion in Arabidopsis thaliana is mediated byECTOPICALLY PARTING CELLS 1 – a glycosyltransferase(GT64) related to the animal exostosins

Sunil Kumar Singh1, Cathlene Eland1, Jesper Harholt2, Henrik Vibe Scheller2 and Alan Marchant1,*

1Department of Forest Genetics and Plant Physiology, SLU, 901 83 Umea, Sweden, and2Department of Plant Biology, The Royal Veterinary and Agricultural University, Copenhagen, Denmark

Received 10 December 2004; revised 27 April 2005; accepted 6 May 2005.*For correspondence (fax þ46 0 90 7868165; e-mail [email protected]).

Summary

Despite the fact that several hundred glycosyltransferases have been identified from sequencing of plant

genomes, the biological functions of only a handful have been established to date. A Poplar glycosyltransf-

erase 64 (GT64) family member that is differentially expressed during the cell division and elongation phases of

cambial growth was identified from previously generated transcript profiling of cambium tissues. The

predicted Poplar GT64 protein has a closely related Arabidopsis homolog ECTOPICALLY PARTING CELLS

(EPC1). Mutation of the EPC1 gene, one of three Arabidopsis GT64 family members, results in plants with a

dramatically reduced growth habit, defects in vascular formation and reduced cell–cell adhesion properties in

hypocotyl and cotyledon tissues. Secondary growth is enhanced in epc1 hypocotyl tissues and it is proposed

that this results from the abnormal cell–cell adhesion within the cortical parenchyma cell layers. Loss of cell–

cell contacts within cotyledon and leaf tissues is also proposed to account for vascular patterning defects and

the fragile nature of epc1 tissues. The EPC1 protein thus plays a critical role during plant development in

maintaining the integrity of organs via cell–cell adhesion, thereby providing mechanical strength and

facilitating the movement of metabolites throughout the plant.

Keywords: glycosyltransferase, cell adhesion, Arabidopsis, cell walls.

Introduction

Glycosyltransferases (GTs) are a diverse class of enzymes

that typically catalyze the transfer of a sugar moiety from a

nucleoside diphospho-sugar to an acceptor molecule

forming a glycosidic bond. GTs function in a number of

different processes including the biosynthesis of glycoli-

pids (Jarvis et al., 2000; Jorasch et al., 2000) and polysac-

charides (Myers et al., 2000), the transfer of sugars to

small molecules such as indole-3-acetic acid (Szerszen

et al., 1994), salicylic acid (Lee and Raskin, 1999) and

flavonoids (Hirotani et al., 2000) as well as playing a cen-

tral role in the synthesis of the plant cell wall. The cell wall

is a vital and complex structure which regulates cell shape

and adhesion, has a role in signaling as well as providing

mechanical strength to the plant. Plant cell walls are

composed of the polysaccharides cellulose, hemicelluloses

and pectins as well as structural proteins. There are at

least 14 monosaccharides making up the various wall

polysaccharides and these are linked in a variety of com-

binations via glycosidic linkages. Pectin alone has been

estimated to contain over 50 different sugar–sugar linkages

and it has been proposed that a similar number of glyco-

syltransferases are required for its biosynthesis (Mohnen,

1999). Despite the undoubted importance of the cell wall,

relatively little is currently understood about the biosyn-

thesis of its structural components, although GTs are

thought to play a major role. GTs from a wide range of

organisms have been classified into 73 families based on

their predicted protein sequence (Campbell et al., 1997;

Coutinho et al., 2003; http://afmb.cnrs-mrs.fr/CAZY/).

Although over 400 GTs have been identified in Arabidop-

sis, the biochemical function or developmental role of only

a few are known. It is difficult to purify GTs in an active

form and furthermore lack of appropriate substrates has

hampered the determination of their biochemical function.

384 ª 2005 Blackwell Publishing Ltd

The Plant Journal (2005) 43, 384–397 doi: 10.1111/j.1365-313X.2005.02455.x

One of themajor functions of the cell wall is inmodulating

intercellular adhesion. In contrast to animals, in most cases

plants establish adhesion between cells during cytokinesis

and normally this contact is maintained during subsequent

development. The maintenance and coordination of the

growth of adjacent adhering cells is important not only to

preserve the integrity of an organ but also to maintain

connectivity via plasmodesmata. If these connections are

broken then cell–cell communication, transport of signaling

molecules and movement of metabolites will be affected.

Transcription factors such as KNOTTED and SHORTROOT

which are important in establishing organ patterning, move

between different cells via the plasmodesmata (Lucas et al.,

1995; Nakajima et al., 2001; Zambryski and Crawford, 2000).

Thus themaintenance and regulation of cellular connectivity

is of fundamental importance during plant development.

Intercellular adhesion between plant cells occurs at the

middle lamella which is rich in pectic polysaccharides and

proteins (Carpita and Gibeaut, 1993; Jarvis, 1984). In pea and

other angiosperms partially methyl esterified homogalac-

turonan (HGA) is found to localize to the ‘reinforcing zones’

between cells placing HGA in a central role in mediating

adhesion between cells. Pectin has been further implicated

in maintaining cell–cell adhesion via the identification of

mutants in Arabidopsis and tobacco. The Arabidopsis

quasimodo1 (qua1) is mutated in a GT8 family protein and

shows reduced cell adhesion properties. Analysis of the cell

wall sugars reveals a 25% reduction in the galacturonic acid

content leading to the suggestion that themutant phenotype

results from an alteration in the pectin composition (Bouton

et al., 2002). The tobacco nolac-H18mutant also has reduced

cell–cell adhesion properties and RGII glucuronic acid con-

tent is dramatically reduced. The phenotype results from

mutation of a gene encoding a GT47 protein related to the

animal exostosins (EXTs) (Iwai et al., 2002). The EXTs are

bimodular enzymes consisting of an N-terminal domain

belonging to the GT47 family and a GT64 family C-terminal

domain. The human EXT family consists of five members

(EXT1, EXT2 and EXT-LIKE 1–3), at least three of which are

involved in heparin sulfate synthesis (Lind et al., 1998;

McCormick et al., 1998). Mutations in EXT1 or EXT2 result

in the human hereditary multiple exostoses (HME) disorder

which manifests as benign outgrowths on the ends of long

bones (Solomon, 1963). The GT47 domain of the human

EXT1 and EXT2 proteins add b-1,4-GlcA residues and the C-

terminal GT64 domain adds a-1,4-GlcNAc residues (Kita-

gawa et al., 1999). The human and murine EXT-L2 proteins

are composed of just the GT64 domain and exhibit GalNAc/

GlcNAc transferase activity which is thought to function in

the initiation of heparin sulfate biosynthesis (Kitagawa et al.,

1999).

Here we describe the identification and characterization of

the Arabidopsis ectopically parting cells 1 (epc1) mutant

which is affected in its cell–cell adhesion properties within

hypocotyl and cotyledon tissues. The severity of the epc1

mutant phenotype demonstrates that the protein plays a

fundamental role during plant development and provides

further insight into the function of cell adhesion in plants.

Results

Identification and characterization of an Arabidopsis GT64

glycosyltransferase

Due to its relatively large physical size, the Poplar cambium

represents an ideal tissue in which to identify develop-

mentally important GTs. A Poplar EST library consisting of

2995 unique sequences has been previously generated

allowing transcript levels to be determined for specific

developmental zones of the cambium (Hertzberg et al.,

2001). This data set was searched for GT-encoding genes

upregulated in the A, B and C zones which correlate with

cell division and elongation processes to identify enzymes

potentially involved in primary cell wall biosynthesis. This

approach identified an EST (AI162408) encoding a GT rela-

ted to the animal EXTs from family GT64 (Figure 1a) (http://

afmb.cnrs-mrs.fr/CAZY/). Subsequent detailed analysis of

the region surrounding the cambial meristem has revealed

strongest expression on the phloem side of the presump-

tive stem cells (Schrader et al., 2004). The GT64 family of

glycosyltransferases includes members from a wide range

of species including humans, Drosophila and Caenorhab-

ditis as well as Arabidopsis, poplar and rice. Comparison of

the Poplar AI162408 EST against the Arabidopsis database

revealed highest homology to At3g55830 which was named

ECTOPICALLY PARTING CELLS (EPC1) based on subse-

quent mutant analysis. The EPC1 gene consists of five

exons (Figure 1b) and encodes a predicted protein of 334

amino acids with a single predicted transmembrane span

between amino acids 27 and 49 (http://www.cbs.dtu.dk/

services/TMHMM) (Krogh et al., 2001). EPC1 is predicted to

be Golgi localized (Yuan and Teasdale, 2002) consistent

with a putative role at the site of cell wall polysaccharide

synthesis. The predicted EPC1 protein contains a DXD motif

(residues 166–168) which is important in a number of GTs

for interacting with an Mnþ2 metal cofactor and thus sta-

bilizing the binding of the diphosphate moiety of the UDP-

sugar (Unligil and Rini, 2000). In Arabidopsis there are two

additional GT64 family members encoded by At1g80290

and At5g04500. The predicted At1g80290 protein (329 ami-

no acids) is similar in length to EPC1 whereas the

At5g04500 protein is predicted to be larger (764 amino

acids) showing homology to EPC1 in the carboxy-terminal

320 amino acids. Comparison of the three Arabidopsis GT64

family sequences reveals around 27% identity and 42%

similarity (Figure 1c). This work describes characterization

of the closest Arabidopsis homolog (EPC1 At3g55830) to the

Poplar cambium-expressed GT64 gene.

Glycosyltransferase mediates plant cell adhesion 385

ª Blackwell Publishing Ltd, The Plant Journal, (2005), 43, 384–397

Characterization of the epc1 mutant

To determine the developmental role of EPC1, four inde-

pendent T-DNA insertion lines (Syngenta SAIL 68A11, Salk

046781, Salk 014479 and Wisc DsLox 233-236invM1) were

identified (Figure 1b). PCR analysis revealed that each of the

lines contains an insertion within the EPC1 gene (data not

shown). All four insertion lines showed segregation of a

mutant exhibiting a reduced growth phenotype. Segrega-

ting seed from the SAIL 68A11 line produced 150 wild type

and 38 mutant seedlings (expect 141:47 for 3:1 segregation).

All seedlings exhibiting the mutant phenotype were con-

firmed to be homozygous for the T-DNA insertion in EPC1

using PCR and from 74 of the wild-type appearing plants, 53

were heterozygous for the T-DNA insertion in EPC1 and 21

wild type (expect 49:25 for 2:1 segregation) confirming that

themutation segregates as a recessive allele. All subsequent

analysis was performed using the SAIL 68A11 T-DNA

insertion allele.

Prior to further characterization of the mutant, genomic

complementation was performed to confirm that the epc1

mutant phenotype was as a result of the T-DNA insertion in

the At3g55830 gene sequence. A genomic copy of the

At3g55830 gene including 450 bp of upstream sequence

encompassing the promoter region up to the stop codon of

the next upstream gene was PCR amplified from wild-type

genomic DNA and cloned into the pMOG402 vector to create

pMOG:EPC1. Direct Agrobacterium-mediated transforma-

tion of the epc1mutant was not possible due to the very low

seed set and general poor growth habit of the mutant.

Therefore, pMOG:EPC1 was introduced into an epc1/EPC1

background and transformants selected on kanamycin for

the presence of the transgene. These kanamycin-resistant T1

plants were subsequently selected for heterozygosity of the

Figure 1. A Poplar GT64 family member is differentially expressed across the cambium.

(a) Quantitation of the AI162408 transcript within the Poplar cambium (data from Hertzberg et al., 2001).

(b) Schematic diagram of the EPC1 (At3g55830) gene showing positions of the SAIL, Salk and Wisc Ds Lox T-DNA insertions. Filled boxes indicate exons and open

boxes introns.

(c) Clustal-X alignment between the Poplar EPC1 protein PtEPC1, AtEPC1, At1g80290 and the C-terminal 323 amino acids of At5g04500. Shaded regions indicate

conserved residues. The position of the conserved DXD motif is indicated by ***.

386 Sunil Kumar Singh et al.


endogenous copy of the EPC1 gene by PCR. T2 seed from

epc1/EPC1 plants resistant to kanamycin were grown on MS

agar plates either in the presence or absence of kanamycin

selection. All kanamycin-resistant T2 seedlings from two

independent transformants were wild type in appearance

(n ¼ 153). In the absence of kanamycin selection 16 epc1

mutant and 208 wild-type seedlings (cumulative numbers

from two independent transformants) were observed con-

firming that the epc1 mutant is complemented by the

At3g55830 gene.

epc1 exhibits defects in root and shoot development

When germinated on MS agar plates, epc1 seedlings were

virtually indistinguishable from the wild type up to 4 days

after germination (dag) apart from a reduction in primary

root elongation (Figure 2a). Subsequently, epc1 cotyledon

and leaf expansion and primary root elongation were re-

duced compared with the wild type. After 9 days growth

wild-type primary roots were 54.3 � 0.8 mm (mean � SE,

n ¼ 30) in length compared with 18.3 � 0.6 mm for epc1

roots (mean � SE, n ¼ 30). Cotyledons of epc1 were curled

at the edges, thicker andmore fragile than the wild type. The

lateral root density of 10 dag epc1 seedlings (0.478 � 0.028

lateral roots/mm primary root) (mean � SE, n ¼ 30) was

more than double that of the wild type (0.228 � 0.016)

(mean � SE, n ¼ 30) (Figure 2b). In 6 dag dark-grown

seedlings, epc1 hypocotyl elongation (16.3 � 0.4 mm)

(mean � SE, n ¼ 30) was reduced compared with wild type

(21.2 � 0.6 mm) (mean � SE, n ¼ 30) (Figure 2c) and epi-

dermal cells of hypocotyls (Figure 2e) and roots (data not

shown) exhibited a left-handed spiraled organization which

was not present in the wild type (Figure 2d). Similar spira-

ling phenotypes but with a right-handed spiral have been

observed previously in the spr1 and spr2 mutants. It has

been proposed that SPR1 and SPR2 act antagonistically with

a microtubule-dependent process to control directional

elongation of cells (Furutani et al., 2000). The lefty1 and

lefty2 suppressors of spr1 cause a left-handed spiraling

arrangement (Thitamadee et al., 2002) and this is proposed

(f) (g) (h)

0

20

40

60

1 2 3 4 5 6 7 8 9

Wild-type

epc1

Days after germintaion

Roo

t len

gth

(a)

0

10

20

wt epc1H

ypoc

otyl

leng

th(m

m)

(c)

0

0.1

0.2

0.3

0.4

0.5

0.6

Late

ral r

oots

/m

m p

rimar

y ro

ots

wt epc1

(b)

(d)

(e)

Figure 2. Phenotype of the epc1mutant. Primary root elongation of wild type and epc1 seedlings up to 9 dag (a). Lateral root density of 10 dag wild type and epc1

seedlings (b). Hypocotyl elongation of 6 dag wild type and epc1 dark-grown seedlings. (c) Wild type (d) and epc1 (e) hypocotyl stained with toluidine blue showing

the spiraling of epc1 epidermal cells. Four-week-old wild type (f) and epc1 (g) grown on soil. Eight-week-old in vitro-grown epc1 (h). Bar (d) and (e) ¼ 100 lm; (f), (g)

and (h) ¼ 5 mm.



to result from reduced microtubule stability. The microtu-

bule organization in epc1 is currently being investigated

further.

Mutant epc1 seedlings transferred from MS agar to soil

15 dag either died before forming an inflorescence or

formed a single unbranched inflorescence which produced

few or no seed (Figure 2g). The rosette leaves remained

small compared with the wild type (Figure 2f) and became

pale or bleached after 1–2 weeks on soil. epc1 seedlings

could be maintained on MS agar plates for at least 8 weeks

and exhibited a bushy phenotype compared with soil-grown

plants forming multiple inflorescence stems. However, the

root system remained short and highly branched and the

leaves did not expand normally (Figure 2h).

Vascular patterning is disrupted in the epc1 mutant

The Arabidopsis EPC1 gene was initially identified by

homology to a Poplar homolog expressed in the region of

early vascular development. In order to establish whether

expression of the Arabidopsis EPC1 gene is vascular asso-

ciated, an EPC1::uidA reporter construct was used to mon-

itor expression in wild-type seedlings. A total of six

independent homozygous transformants were selected in

the T2 generation and detailed analysis showed that all

lines exhibited the same expression pattern throughout the

seedling. Expression in 8 dag seedlings was found

throughout cotyledons, leaves and hypocotyl tissues

(Figure 3a). Expression was also detected within columella

and lateral root cap cells of the primary root tip but not in the

differentiation or elongation zones (Figure 3b). Expression

was detectable again within mature root tissues. Expression

within the cotyledon was not only strongest within the

vascular bundle but was also evident throughout the rest of

the tissue (Figure 3c). Sections of hypocotyls of EPC1::uidA

seedlings showed detectable GUS expression in all cells

apart from the central xylem and some endodermal cells

(Figure 3d).

Vascular patterning is affected in the epc1 mutant

The expression profile of the Poplar cambium-expressed

GT64 gene indicates a possible role during early stages of

vascular development. Although the expression pattern of

EPC1 does not indicate a vascular-specific function, this

aspect of development was studied further. The cotyledon

vascular organization is relatively simple and so repre-

sents an ideal tissue for studying patterning alterations.

Wild-type cotyledons typically comprise three or four

closed loops linked to a central midvein (Figure 4a). In

7 dag epc1 cotyledons there was a reduction in the

average number of closed loops compared with the wild

type, although the number of loops varied between two

and five (Table 1, Figure 4b). The possibility that these

effects may be an indirect result of the cotyledon size

being reduced in epc1 rather than a direct effect of the

mutation cannot be discounted. Comparison of epc1 and

wild-type leaf tissues revealed a number of open-ended

vascular elements located at the margins of the mutant

leaf (Figure 4d) which were rarely found in the wild type

(Figure 4c). Additionally, the amount of tertiary and qua-

ternary order vasculature was reduced in epc1, although

this might reflect the smaller size of epc1 leaves com-

pared with the wild type.

Examination of cleared epc1 cotyledon and leaf tissues

allows visualization of the xylem network but does not

confirm whether phloem development is equally affected.

In order to address this question, the APL1::uidA marker

was crossed into the epc1 mutant background. APL1

functions to promote phloem formation while repressing

xylem formation, and expression is limited to the phloem

tissues (Bonke et al., 2003). Examination of 10 dag wild-

type APL1::uidA-expressing cotyledons and leaves shows a

continuous phloem network staining for GUS activity

(Figure 4e,g). In contrast, epc1 cotyledons and

leaves showed some discontinuous phloem elements

(Figure 4f,h). The pattern of APL1::uidA expression in

epc1 tissues mirrored that of the xylem indicating that

phloem and xylem development were both affected. The

APL1::uidA expression pattern in epc1 vasculature was

more intense compared with the wild type and also

showed a low expression level in the cells surrounding

the vascular strands (Figure 4f,h).

Figure 3. Expression analysis of EPC1 analyzed using promoter::GUS

fusions. EPC1::uidA expression within the aerial tissues of 8 dag seedlings

(a). EPC1::uidA expression is detected within the columella and lateral root

cap cells of the primary root with the elongation and differentiation

zones showing no apparent expression (b). Transverse section of 8 dag

cotyledon showing EPC1::uidA expression (c). Radial section of 8 dag

hypocotyl showing EPC1::uidA expression (d). Bar ¼ 1 mm (a) or 50 lm (b–d).



epc1 tissues display reduced cellular contacts and

hypocotyls undergo premature secondary growth

Alterations in the patterning of the cotyledon and leaf vas-

culature in epc1 prompted amore detailed investigation into

the cellular organization of epc1 tissues. Sections were

made of cotyledons and leaves from 12 dag wild type and

epc1 seedlings. It was noticeable that in comparisonwith the

wild type, mesophyll cells of epc1 cotyledons were much

more loosely arranged with large spaces between cells

(Figure 5a,b). In contrast, epidermal cells of epc1 cotyledons

remained attached indicating that the cell adhesion defect is

restricted to the inner cells.

The hypocotyls of epc1 were larger in diameter than the

wild type suggesting altered patterning or development in

the mutant tissues. Wild-type hypocotyls 12 dag exhibited

a similar degree of secondary growth (Figure 5c) to the

published findings for 15 dag wild-type plants grown

under short-day conditions (Chaffey et al., 2002). Examina-

tion of 12 dag epc1 hypocotyls grown under the same

conditions as the wild type showed more extensive

secondary growth with an average fivefold increase in

the radial area of the stele tissues compared with wild type

(Figure 5d). The increase in secondary growth results from

more cells rather than an increased cell size and largely

accounts for the enlarged diameter of the epc1 hypocotyls.

In addition to the increased secondary growth in epc1 it

was also found that the inner layer of cortical parenchyma

cells was disrupted with large gaps between individual

cells (Figure 5d). Intercellular spaces were seldom

observed in sections of the wild-type hypocotyls of an

Figure 4. Vascular patterning and development is abnormal in epc1. Wild

type (a) and epc1 7 dag cotyledon (b) cleared using chloral hydrate (Koizumi

et al., 2000). Wild type (c) and epc1 10 dag leaf (d). Triangles indicate open

vascular ends at the leaf margin which are much less frequently observed in

the wild type. APL1::uidA expression in 10 dag wild type (e) and epc1 (f)

cotyledons. APL1::uidA expression in 10 dag wild type (g) and epc1 (h) leaf

tissue. Bar ¼ 0.5 mm.

Table 1 epc1 cotyledons show a reduced vascular complexity

Number of closed loops 0 1 2 3 4 5 Mean

Wild type 0 0 6 28 28 0 3.38epc1 0 1 28 22 11 2 2.77

The numbers of closed vascular loops were counted in 10 dag wildtype and epc1 cotyledons.

Figure 5. epc1 tissues exhibit reduced cell–cell adhesion. Transverse section

of 12 dag wild type (a) and epc1 (b) cotyledon tissue. Transverse section of

12 dag wild type (c) and epc1 (d) hypocotyl tissue (bar ¼ 50 lM).



equivalent age. The outer layer of hypocotyl cortical

parenchyma cells was increased in size compared with

the wild type reflecting the overall increased diameter of

the epc1 hypocotyl. Sections were made of 20 dag wild-

type hypocotyls in order to determine whether the cortical

parenchyma cell separation is a consequence of the degree

of secondary growth. The amount of secondary growth

exhibited by the 20 dag wild-type seedlings was only

slightly increased compared with 12 dag samples and was

still considerably less than the epc1 12 dag samples. The

20 dag wild type hypocotyls exhibited no evidence of

cortical parenchyma cell separation (Figure S1).

The increased secondary growth observed in epc1 hy-

pocotyl tissues could have arisen from a larger number of

cells established in the stele during development. To

address this possibility sections were made of 6 dag wild

type and epc1 hypocotyls. There were no discernable

differences in the radial organization or size of the stele

tissues indicating that this cannot account for the effects on

secondary growth in epc1 (data not shown). There was also

no apparent evidence of cell separation within the cortical

parenchyma cells of 6dag epc1 hypocotyls. In order to

determine whether EPC1 is expressed within tissues under-

going secondary growth, hand sections were made of

EPC1:uidA-expressing hypocotyls grown under short-day

conditions for 50 days. GUS expression was found to be

strongest within the phloem tissues but was also evident

within the non-lignified cells of the central xylem (Fig-

ure S2). This expression of EPC1 with the secondary thick-

ened Arabidopsis hypocotyl contrasts with the cambium

transcript profile of the Poplar GT64 homolog (Figure 1a)

suggesting that the proteins may have differing functional

roles.

It was noticed that epc1 tissues were more fragile in

nature, than the wild type and this was investigated further

by making tissue squashes of cotyledon and leaf material

from 12 dag seedlings. Wild-type cotyledons and leaves

remained intact with little visible damage caused by the

applied force. In contrast, epc1 tissues readily disintegrated

and in particular, isolated cells were released in addition to

cell contents (data not shown).

Callose is ectopically deposited in epc1

The vascular development defects observed for the epc1

tissues prompted an investigation into callose deposition in

the vasculature. Callose is a b 1-3-linked glucan component

of the phloem found lining the pores at the ends of phloem

sieve plates. Callose in sieve plates is thought to function to

seal damaged sieve elements. Callose is also transiently

associated with the developing cell plate during cytokinesis

(Northcote et al., 1989), in pollen tubes and forms a struc-

tural component of the plasmodesmata. Besides these

developmental roles, callose is deposited in response to

different biotic and abiotic stresses (Aist, 1976; Jacobs et al.,

2003; Schreiner et al., 1994). To determine whether callose

distribution in the phloem was altered in epc1, 12 dag

seedlings were stained with aniline blue (Carland et al.,

1999). Wild-type cotyledon and leaf tissues showed a low

level of fluorescent signal associated primarily with the

vasculature (Figure 6a). Callose deposition in the vascula-

ture of epc1 cotyledons was reduced (Figure 6b) compared

Figure 6. Callose is ectopically deposited in epc1 tissues. Aniline staining of

callose in wild type (a) and epc1 (b) 10 dag cotyledons. Arrow heads indicate

the path of the vascular strand. Wild type (c) and epc1 (d) cotyledon showing

punctate distribution of callose. Wild type (e) and epc1 (f) roots stained for

callose deposition. Callose deposition in epc1 cotyledon associated with

fluorescent signal from mesophyll cells (g). Higher magnification of high-

lighted area in g (h). Bar ¼ 50 lM (a, b, e–h) and 0.5 mm (c, d).



with the wild type and the majority of 12 dag seedlings

showed no vascular-specific deposition. However, the

majority of epc1 seedlings showed extensive punctate dis-

tribution of fluorescent signal throughout the cotyledons,

leaves, hypocotyl and root tissues (Figure 6d,f) which was

absent in the wild type (Figure 6c,e). Cotyledons showing

this punctuate callose distribution also displayed a fluores-

cent signal from groups of mesophyll cells (Figure 6g). The

callose deposits are associated with the cell periphery rather

than being localized exclusively within the cell wall (Fig-

ure 6g,h). The origin of the background fluorescence linked

with the punctate callose deposition in epc1 is unclear.

However, wounding of wild-type cotyledons induces a

similar fluorescent signal within 24 h to that seen in a

number of epc1 cotyledons (Figure S3). This suggests that

epc1 cotyledons undergo a wound response that correlates

with the formation of ectopic callose deposition. The pattern

of distribution of callose is similar to that seen in Arabid-

opsis leaves challenged with powdery mildew fungus

(Jacobs et al., 2003) further suggesting that the ectopic

deposition could be due to a wounding-like response path-

way.

epc1 has reduced efficiency of phloem loading

Reduced cell–cell adhesion in the epc1 aerial tissues is likely

to have a significant impact on the symplastic transport of

photoassimilates as well as other metabolites such as auxin.

Coupled with this is the disruption of the vascular network in

epc1 which is also likely to negatively affect phloem-based

transport capacity. To test the phloem-based transport

capacity of epc1, carboxyfluorescein diacetate (CFDA) was

applied to the cut tips of 6 dag cotyledons (Duckett et al.,

1994). CFDA was only able to enter the cotyledon via the cut

surface as no signal was detectable after incubation of intact

seedlings (data not shown). Distribution of the fluorescent

marker was examined by fluorescence microscopy between

60 and 120 min after application. A similar distribution and

intensity of fluorescent signal was detectable within root

tissues of both wild type and epc1 seedlings indicating that

phloem-based transport was functional (data not shown).

However, the intensity of the fluorescent signal remaining in

the majority of epc1 cotyledons was higher than in the wild

type indicating a reduced efficiency of phloem loading or

transport in the mutant. This possibility was studied further

by applying a 30-min pulse of CFDA to the cut ends of cot-

yledons and then examining the distribution of the fluores-

cent signal remaining in the cotyledons after a further

60 min incubation in the absence of applied CFDA.Wild-type

cotyledons showed a distinctive signal following the route of

the vasculature within the basal half of the cut cotyledon

tissue (Figure 7a). epc1 cotyledons also exhibited a fluores-

cent signal within the vascular elements and had a more

diffuse signal within the blade of the cotyledon which was

not observed in the wild type (Figure 7b). This suggests that

phloem loading is less efficient in epc1 compared with the

wild type. However, the possibility that CFDA is able to

penetrate epc1 cotyledon tissue via a non-symplastic route

due to altered cell–cell adhesion properties cannot be dis-

counted.

Quantitation of epc1 cell wall monosaccharides

A number of plant glycosyltransferases including QUA1

(Bouton et al., 2002), MUR2 (Vanzin et al., 2002), MUR3

(Madson et al., 2003) and NtGUT1 (Iwai et al., 2002) have

been directly implicated in the biosynthesis of cell wall

components. Homology between EPC1 and the animal EXT

homologs suggests a possible role in metabolism of a UDP-

sugar which could be incorporated into a cell wall compo-

nent. Monosaccharide composition was determined for wild

type and epc1 5 dag dark-grown seedlings using HPAEC-

PAD of hydrolyzed material. The level of glucose was in-

creased by 32% in epc1 while the level of galactose was

decreased by 12%. If the contribution of glucose to the

overall molar percentagemeasurements is disregarded then

the level of galactose is only decreased by 9% in epc1. No

significant differences between wild type and epc1 were

found for any of the other assayed monosaccharides (L-Fuc,

L-Rha, L-Ara, D-Gal, D-Xyl, D-Man, D-GalUA and D-GlcA)

(Figure 8).

Discussion

This study has utilized detailed transcript profiling data

derived from the Poplar cambium (Hertzberg et al., 2001) as

a starting point to identify potential developmentally

important glycosyltransferases. The Poplar cambium has

some advantages over Arabidopsis in this approach due to

the physically larger size of the tissues. Distinct develop-

mental stages of cambial formation can be accurately dis-

sected allowing transcripts potentially involved in different

processes to be identified. However, analysis of gene

Figure 7. Movement of CFDA within epc1 cotyledon tissues is reduced.

Fluorescent detection of CFDA in wild type (a) and epc1 (b) 6 dag cotyledons.

Bar ¼ 0.5 mm.



function in Poplar is challenging due to its long generation

time and physical size prompting a number of research

groups to use Arabidopsis as a genetic model for wood

formation (Chaffey et al., 2002; Lev-Yadun, 1994; Oh et al.,

2003; Zhao et al., 2000). Adopting this approach has al-

lowed the identification of a Poplar GT64 family member

which shows highest expression on the phloem side of the

presumptive cambial stem cells (Schrader et al., 2004) and

in the early stages of cambial development. Comparison of

the Poplar GT64 sequence and the Arabidopsis genome

sequence has allowed the identification of a closely related

homolog. Although it cannot yet be stated that the Ara-

bidopsis EPC1 and related Poplar proteins perform the

same function, preliminary data show that the gene struc-

ture is conserved between the two species (E. Edvardsson

and AM, unpublished data) and complementation studies

are currently in progress. The finding that insertion muta-

tions in the EPC1 gene lead to severe developmental de-

fects demonstrates the importance of the protein in plant

growth and indicates that neither of the other two GT64

family proteins can fully compensate for the lack of a

functional EPC1 protein. The level of identity between the

three Arabidopsis GT64 family members is only around

27% and so it not unexpected that they may have different

biochemical functions. Additionally, analysis of ATH1

Affymetrix data (http://www.cbs.umn.edu/arabidopsis/) for

At1g80290 and At5g04500 shows that the expression level

is lower and less extensive than EPC1.

Cell–cell adhesion defects impact many aspects of

development

Numerous observations demonstrate that cell–cell contacts

are altered in epc1 compared with the wild type and this is

likely to be a major factor contributing to the phenotypes

exhibited by the mutant plant. First, sections of epc1 hy-

pocotyl and cotyledon tissues show large spaces between

cells which are not apparent in the wild type. These spaces

are not evident in newly formed tissues, suggesting that

their appearance may be linked to expansion of the organ

forcing loosely attached cells to separate. Secondly, epc1

tissues are fragile and readily disintegrate when a crushing

pressure is applied again indicating that the strength of cell–

cell connections is weak in epc1. Thirdly, reduced CFDA

movement from the epc1 cotyledon lamina into the vascular

system is likely, at least in part, to be caused by reduced

symplastic connections resulting from loss of cell–cell

adhesion. The most striking evidence of cell separation is in

the cortical parenchyma layers of the hypocotyl. In wild-type

hypocotyls undergoing secondary growth, the endodermal

cells become crushed against the surrounding cortex layer

(Chaffey et al., 2002) suggesting several important facts.

First, the expanding secondary growth must exert a con-

siderable compression force on the surrounding endoder-

mis. Secondly, the cortex layer of cells must be able to resist

this compression force such that the endodermis is trapped

between a non-compressible cell on the outer face and an

expanding layer on the inner face. The cortex and possibly

endodermis cell–cell adhesion properties of epc1 hypocotyls

are altered such that the expanding cambium forces the cells

to separate. We propose a model where the cortical paren-

chyma cell layers act like a belt repressing cambial expan-

sion during early seedling growth. In epc1, however, the

reduced strength of cell–cell adhesion results in the cortical

parenchyma cells being forced apart, thereby reducing or

removing the repression of secondary growth. Alternatively,

increased secondary growth may be initiated in epc1 in or-

der to strengthen the weak hypocotyl structure resulting

from altered cell–cell adhesion. This explanation is less likely

as hypocotyl tissues in this study were obtained from

seedlings grown on vertically oriented plates whereby the

agar surface provides a physical support to the hypocotyl

tissues.

A further consequence of cell separation in epc1 is the

formation of callose papillae-like structures which may seal

lesions or severed plasmodesmata connections. As this

callose deposition is seen in seedlings grown entirely in vitro

under sterile conditions the response cannot be the result of

pathogen attack or mechanical damage but rather an

inherent property of the mutant tissues. It is possible that

the cell–cell adhesion properties of epc1 are altered as a

result of changes in cell wall properties, suggesting that

cell expansion causes induction of a wounding response.

0

5

10

15

20

25

Fuc Ara Rham Gal Gluc Xyl Man GalA GlucA

Wild-type

epc1

M %

Monosaccharide

Figure 8. Monosaccharide composition of epc1 cell walls. Monosaccharide

levels of fucose (Fuc), arabinose (Ara), rhamnose (Rham), galactose (Gal),

glucose (Gluc), xylose (Xyl), mannose (Man), galacturonic acid (GalA) and

glucuronic acid (GlucA) measured in cell wall preparations of 5-day-old dark-

grown wild type and epc1 seedlings.



Alternatively, callose deposition may be a consequence of a

weak cell wall structure. The cyt1 mutant which has reduced

levels of celluloseaccumulates callosewithin theprimary cell

wall and it has been suggested that this is due to increased

mechanical stresses (Kang and Dengler, 2004). However, the

primary cell wall localization of callose in cyt1 is different

from the punctate distribution observed in epc1. Similar

analysis of the callose deposition in the procuste mutant

which is also affected in cellulose biosynthesis and exhibits

abnormally radially expanded epidermal root cells did not

reveal any ectopic callose deposition (S.K. Singh and

A.Marchant, unpublisheddata). This argues that aweakened

cell wall structure or reduced cellulose content is unlikely to

fully account for the ectopic callose deposition in epc1.

Abnormal cell–cell contacts affect vascular patterning in

epc1

The canalization of signal flow hypothesis implicates auxin

as a key factor in controlling vascular development in plants

(Sachs, 1991). Several Arabidopsis vascular patterning mu-

tants including scarface (Deyholos et al., 2000), monopteros

(Berleth and Jurgens, 1993; Przemeck et al., 1996), lopped 1

(Carland and McHale, 1996), axr6 (Hobbie et al., 2000) and

the van series of mutants (Koizumi et al., 2000) have been

identified and studies on these have highlighted auxin

transport as playing a key role in vascular patterning. How-

ever, the cvp1 and cvp2 mutants also show discontinuous

cotyledon vasculature but do not have defects in auxin

transport or perception indicating that auxin alone cannot

account for vascular pattern formation (Carland et al., 1999).

The altered cell–cell adhesion property of epc1 is likely to

affect vascular development in two ways. First, altered cell–

cell contacts in aerial tissues of epc1 will reduce the sym-

plastic movement of metabolites including auxin from

source to sink tissues. Reduced symplastic connectivity

within cotyledon tissues is demonstrated by the reduced

flux of the CFDA fluorescent label in epc1 (Figure 7).

According to the signal flow hypothesis, disruption of the

ability of a tissue to canalize the auxin signal will affect

vascular development. Additionally, reduced flux of auxin

from aerial tissues will affect development of the root sys-

tem. This is supported by the finding that epc1 primary root

elongation is partially rescued by the addition of low levels

of exogenous auxin (S.K. Singh and A. Marchant, unpub-

lished data). Secondly, loss of symplastic connections be-

tween mesophyll cells will affect the recruitment of cells to

become provasculature. This will result in premature strand

termination as observed in epc1 cotyledons and leaves

(Figure 4). Recent studies have proposed that polar secre-

tion of the proteoglycan xylogen by premature Zinnia tra-

cheary elements functions promote entry of neighboring

cells into the vascular differentiation pathway (Motose et al.,

2004). If a xylogen-mediated induction is involved in Ara-

bidopsis vascular development, then it would be expected

that cell separationwould affect the ability of the polar signal

to promote vascular formation in a directional manner. The

Drosophila EXT homologs tout-velu, BOTV and SOTV have

been shown to have roles in modulating movement of

morphogenic molecules such as Hedgehog, Wingless and

Decapentaplegic which are required to establish patterning

information (Bellaiche et al., 1998; Han et al., 2004). This role

is somewhat similar in concept to the function of EPC1 in

modulating the movement of auxin and establishing pat-

terning information.

What is the biochemical function of EPC1?

Although elucidating the biochemical function of EPC1 is

challenging, some tentative clues can be gained from stud-

ies on the animal EXTs. Glycosyltransferases can be classi-

fied either as retaining enzymes resulting in an a glycosidic

linkage or inverting forming a b linkage. Based on the known

activities of the human GT64 C-terminal domain it is pre-

dicted that EPC1 is a retaining GT. In animal systems, the

majority of EXT proteins are bimodular containing both

GT47 and GT64 domains. This contrasts with the situation in

Arabidopsis where the GT47 andGT64 domains are found as

separate proteins. It is interesting to note that the GT47

family in Arabidopsis contains 39 members and is much

larger than the GT64 family. The human and murine EXT-L2

proteins which contain just the GT64 domain represent the

simplest starting point to make comparisons with the Ara-

bidopsis EPC1 enzyme. Themurine EXT-L2 protein has been

crystallized and shown to bind both UDP-N-acetylglucosa-

mine (UDP-GlcNAc) and UDP-N-acetylgalactosamine (UDP-

GalNAc), although the UDP-GlcNAc transferase activity was

more than 10-fold higher than that of UDP-GalNAc (Peder-

sen et al., 2003). This suggests that EPC1 may catalyze the

formation of a glycosidic bond involving the transfer of ei-

ther a GlcNAc or GalNAc sugar. The predicted Golgi local-

ization of EPC1 along with the cell adhesion defect is

consistent with a possible role in pectin biosynthesis. How-

ever, some caution needs to be exercised in making hypo-

theses about the biochemical function of EPC1 based on

findings in the murine and human systems due to the rel-

atively low level of homology exhibited between EPC1 and

the animal EXTs. This is highlighted by analysis of the Ara-

bidopsis MUR3 gene which encodes a GT47 family member

that catalyzes the transfer of a b-1,2-galactose residue to the

third xylose within the XXXG core structure of xyloglucan

(Madson et al., 2003). This activity however differs from the

mammalian GT47s which transfer b-1,4-GlcA residues. A

second GT47 tobacco mutant (nolac-H18) has been charac-

terized and found to have a reduced cell wall glucuronic acid

content associated with an altered RGII structure (Iwai et al.,

2002). Further problems in predicting the biochemical

function of GT proteins based solely on homology have



been highlighted by the FUT family in Arabidopsis. AtFUT1

catalyzes the transfer of a terminal fucose residue to xylo-

glucan (Perrin et al., 1999). AtFUT1 is part of a family of 10

Arabidopsis proteins but analysis of heterologously ex-

pressed protein of a further three family members revealed

they do not catalyze the fucosylation of xyloglucan (Sarria

et al., 2001).

Analysis of the monosaccharide content of epc1 cell wall

material does not provide any strong evidence for the

biochemical function of EPC1. The major significant differ-

ence observed was a 32% increase in glucose levels in epc1

tissues. As 5 dag dark-grown seedlings do not produce

significant levels of starch as evidenced by iodine staining

(data not shown), it is unlikely that this polysaccharide can

account for the observed differences in glucose levels. A

more likely explanation is that the elevated glucose level

arises from the higher level of callose deposition observed in

the epc1 mutant (Figure 6). Although no significant differ-

ences were found for the other monosaccharides, it is

possible that epc1 is altered in a monosaccharide which was

not measured in this study. For example, the RGII molecule

consists of 12 different glycosyl residues joined by 20

different linkages. These 12 residues include the less com-

monly occurring sugars D-apiose, L-aceric acid, 2-O-methyl

L-fucose, 2-O-methyl D-xylose, L-galactose, 2-keto-3-deoxy-

D-lyxo-heptulosaric acid, and 2-keto-3-deoxy-D-manno-oct-

ulosonic acid (O’Neill et al., 2004) which were not measured

in this study. RGII has been implicated in maintenance of

cell–cell adhesion from studies of the nolacH18 tobacco

mutant which lacks a terminal a-L-Galp-(1 fi 2)-b-D-GlcpA

linkage. This adversely affects the ability of RGII to form

borate ester crosslinks between apiosyl residues resulting in

reduced cell adhesion in callus tissue. The nolac-H14

tobacco mutant also displays reduced cell adhesion proper-

ties and in this case the cell walls were depleted in pectic

polysaccharides. Additionally, the proportion of arabinose

was significantly reduced in nolac-H14 leading the authors

to conclude that arabinose-rich pectins may function during

intercellular attachment (Iwai et al., 2001). Further evidence

for pectin playing a role in cell adhesion comes from

analysis of the qua1 Arabidopsis mutant. Analysis of qua1

cell walls shows a reduction in galacturonic acid and

reduction in JIM5- and JIM7-reactive epitopes (Bouton et al.,

2002). These findings point to HGA in addition to RGII

functioning in cell adhesion. Further detailed analysis of the

sugar composition of epc1 cell walls coupled to heterolo-

gous expression and biochemical characterization will be

required to determine the biochemical function of EPC1.

This study presents the analysis of a member of the GT64

family in plants and clearly shows that EPC1 plays an

important role during plant development by modulating cell

adhesion. It remains to be seen whether EPC1 homologs in

other species such as Poplar play a similarly significant role

during development. However, further work is still required

in Arabidopsis to fully understand the function of EPC1. For

example, identification of the specific donor substrate and

acceptor molecules as well as the product of the EPC1-

catalyzed reaction has yet to be determined. Work is

currently ongoing to heterologously express EPC1 and to

establish biochemical assays to address these questions.

Experimental procedures

Growth of Arabidopsis

Wild type (Columbia 0 ecotype) and epc1 seedwas surface-sterilizedaccording to the method of Forsthoefel et al. (1992). Seed was sownin vitro on MS agar [4.3 g MS salts (Duchefa, Haarlem, the Nether-lands); 1% sucrose, pH 5.8, with 0.5 M KOH; 1% agar] and vernalizedat 4�C for 48 h prior to germination under a 16-h light period at 23�C.Soil-grown plants were maintained at 23�C under a 16-h light per-iod. Kanamycin-resistant seedlings were selected on MS agar con-taining 25 lg ml)1 antibiotic.

Analysis of T-DNA and transposon insertions

The presence of T-DNA insertions within the EPC1 gene was con-firmed using the following EPC1 gene- and insert-specific primercombinations – EPCgen (5¢-CACAGTTGACTTCTGAGACG-3¢),EPCR3 (5¢-ATCTACCGTGTGACAAGG-3¢) and LB1 (5¢-TCA-GAAATGGATAAATAGCC-3¢) for SAIL68A11; EPCR3, EPCgen, andSalkLBR1 (5¢-TCACGTAGTGGGCCATCG-3¢) for Salk046781; EPCRT5(5¢-CTCGAGATGGGCATGAGG-3¢), EPCRT3 (5¢-TTGCGGCTATC-CACGGC-3¢) and SalkLBR1 for Salk014479; EPCRT5, EPCRT3 andP745 (5¢-AACGTCCGCAATGTGTTATTAAGTTGTC-3¢) for WiscDsLox293-296invM1.

Plasmid constructs

Genomic complementation construct. The entire genomiccoding region of EPC1 including 450 bp of sequence upstream ofthe presumptive start codon was PCR amplified using EPCXba(5¢-GCTCTAGACGAAGATGATGATCC-3¢) and EPCBam (5¢-AG-TTCTGGATCCAATTGTGATGC-3¢) and cloned into the XbaI andBamHI sites of the pMOG402 binary vector (Sijmonds et al., 1990) tocreate pMOG:EPC1. Binary vectors were transformed into Agro-bacterium (C58 GV3850) (Wen-jun and Forde, 1989) which was thenused to transform plants using the floral dip procedure (Clough andBent, 1998).

GUS expression construct. To construct the EPC1 promo-ter–GUS fusions, the 2 kb genomic region upstream ofthe start codon was PCR amplified using forward (5¢-GGGG-ACAAGTTTGTACAAAAAAGCAGGCTGCCTCTATCGATGACATCAA-CGC-3¢) and reverse (5¢-GGGGACCACTTTGTACAAGAAAGCTGGG-TCATCTAAAATCACGAATTCAAG-3¢) primers and cloned into thepDONR201 Gateway vector (Invitrogen, Abingdon, UK). The pro-moter was subsequently transferred to the pGWB3 Gateway vector(provided by Tsuyoshi Nakagawa Shimane University) placing theuidA gene under the control of the EPC1 promoter. The expressionof uidA constructs was monitored according to the method of Wil-lemsen et al. (1998) and tissues cleared using the method of Mala-my and Ryan (2001). Cleared tissues were mounted in 50% glyceroland viewed using a Leica MZ95 microscope (Leica, Sollentuna,Sweden) Images were captured using a Leica DC300 camera.



Carboxyfluorescein diacetate transport

Amodified version of the procedure described byDuckett et al., 1994was followed. CFDA (Sigma, Stockholm, Sweden) was prepared as a6 mg ml)1 stock in acetone and diluted to 30 lg ml)1 in sterile dis-tilled water. Approximately 1 mmwas excised from the tips of 6 dagcotyledons using a double-sided thin razor blade. The cut tips wereplaced in contact with CFDA on parafilm for 30 min after which timethey were briefly rinsed in water and then placed on wetted filterpaper for a further 60 min. Fluorescence was visualized using anAxioplan 2 microscope under UV excitation and images capturedusing an Axiocam camera (Zeiss, Hallbergmoos, Germany).

Cell wall preparation and sugar quantitation

Seedlings for cell wall analysis were grown for 1 day in the lightfollowed by 4 days in the dark. Mutant epc1 seedlings wereselected from a segregating population based on hypocotyl lengthand spiraling of the epidermal cells of hypocotyl and root tissues.Alcohol-insoluble residue was prepared as described in Fry (1988)with adaptations. The tissue of interest was boiled in 96% ethanolfor 30 min. The supernatant was removed after centrifugation at10 000 g for 5 min. The pellet was washed with 70% ethanol withsubsequent centrifugation until it appeared free of chlorophyll. Afinal wash with 100% acetone was performed and the pellet wasevaporated under vacuum. Samples were hydrolyzed in 2 M tri-flouroacetic acid (Sigma) for 1 h at 120 �C. Triflouroacetic acid wasremoved by evaporation under vacuum. Monosaccharide com-position was determined by high-performance anion exchangechromatography with pulsed amperiometric detection (HPAEC–PAD) of hydrolyzed material using a PA10 column (Dionex,Sunnydale, CA, USA) (ØBro et al., 2004). The sugars were elutedwith a flow of 1 ml min)1 and a gradient consisting of water for40 min, which eluted the neutral sugars, followed by 500 mM ofNaOH for 20 min, which eluted GalUA and GlcUA. Post-columnaddition of 1 M NaOH at 0.05 ml min)1 was used to ensure PADresponse. Monosaccharide standards were obtained from SigmaChemical Co. and included L-Fuc, L-Rha, L-Ara, D-Gal, D-Glc, D-Xyl,D-Man, D-GalUA and D-GlcUA. A standard mixture run was per-formed before analysis of a batch of samples for verification ofthe response factors.

Tissue sections

Material was prepared and sectioned as previously described(Marchant et al., 1999). Tissue sections were stained either withruthenium red (0.05%), toluidine blue (0.05%) and viewed using anAxioplan 2 microscope (Zeiss). Images were captured using anAxiocam camera (Zeiss).

Acknowledgements

Funding was provided by the Swedish Foundation for StrategicResearch (SSF) (AM and CE) and Formas grant 22.0/2003-1246(SKS). We would like to thank Yka Heliarutta for the APL1::uidAseed. The pGWB3 binary vector was kindly donated by TsuyoshiNakagawa (Shimane University, Japan). Morag Whitworth (Not-tingham University UK) provided valuable support in growing epc1mutant plants. We would like to thank Malcolm Bennett for supportduring the early phase of the project as well as Florence Goubet forcritical reading of the manuscript and for helpful discussion. TMRI,NASC and the SALK institute are acknowledged (Alonso et al., 2003)for supplying epc1 knockout lines.

Supplementary Material

The following supplementary material is available for this articleonline:Figure S1. Wild-type hypocotyls 20 dag do not exhibit corticalparenchyma cell separation.Figure S2. EPC1:uidA is expressed within secondary-thickenedhypocotyl tissues.Figure S3. A fluorescent signal that can be mimicked by woundingis associated with the presence of punctate callose deposition inepc1 cotyledons.

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