Haba New Phytologist

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Early gene expression programs accompanying

trans-differentiation of epidermal cells of Vicia faba
cotyledons into transfer cells
Blackwell
Oxford,
New
NPH
1469-8137
0028-646X
March
10.1111/j.1469-8137.2009.02822.x
2822
8
0
Original
877???
XX
63???
ThePhytologist
Authors
2009
UK
Article
Publishing
(2009).Ltd
Journal compilation New Phytologist (2009)
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Stephen J. Dibley, Yuchan Zhou, Felicity A. Andriunas, Mark J. Talbot, Christina E. Offler, John W. Patrick and
David W. McCurdy
School of Environmental and Life Sciences, The University of Newcastle, Callaghan, New South Wales 2308, Australia
Summary
Author for correspondence:
David W. McCurdy
Tel: +61 2 49 21 5879
Email: David.McCurdy@newcastle.edu.au
Received: 17 November 2008
Accepted: 6 February 2009
New Phytologist (2009) 182: 863877

doi: 10.1111/j.1469-8137.2009.02822.x
Key words: cDNA-AFLP, transdifferentiation, transfer cells, Vicia faba,

wall ingrowths.
Transfer cells (TCs) trans-differentiate from differentiated cells by developing

extensive wall ingrowths that enhance plasma membrane transport of nutrients.
Here, we investigated transcriptional changes accompanying induction of TC
development in adaxial epidermal cells of cultured Vicia faba cotyledons.
Global changes in gene expression revealed by cDNA-AFLP were compared
between adaxial epidermal cells during induction (3 h) and subsequent building
(24 h) of wall ingrowths, and in cells of adjoining storage parenchyma tissue, which
do not form wall ingrowths.
A total of 5795 transcript-derived fragments (TDFs) were detected; of these, 264
TDFs showed epidermal-specific changes in gene expression and a further 207 TDFs
were differentially expressed in both epidermal and storage parenchyma cells. Genes
involved in signalling (auxin/ethylene), metabolism (mitochondrial; storage product
hydrolysis), cell division, vesicle trafficking and cell wall biosynthesis were specifically
induced in epidermal TCs. Blockers of auxin action and vesicle trafficking inhibited
ingrowth formation and marked increases in cell division accompanied TC development.
Auxin and possibly ethylene signalling cascades induce epidermal cells of V. faba
cotyledons to trans-differentiate into TCs. Trans-differentiation is initiated by rapid
de-differentiation to a mitotic state accompanied by mitochondrial biogenesis driving
storage product hydrolysis to fuel wall ingrowth formation orchestrated by a modified
vesicle trafficking mechanism.
Introduction
Transfer cells (TCs) are characterized by wall ingrowths that
protrude into the cytoplasm forming a complex labyrinth
(Talbot et al., 2001) that acts as a scaffold for an amplified
plasma membrane enriched in nutrient transporters (Offler
et al., 2003). Transfer cells trans-differentiate from diverse cell
types in response to developmental cues, stress or other factors
(Offler et al., 2003). Despite their importance in nutrient
exchange in plants and, consequently, plant development
(Offler et al., 2003), little is known of the identity of genes
that orchestrate their induction and building of their wall
labyrinths.
Several genes nominated as TC-specific have been identified
in basal endosperm TCs of developing maize kernels. These
The Authors (2009)

include four defensin-like genes, BETL1-4 (Thompson et al.,

2001), a novel cell wall-related protein, MEG1 (Thompson
et al., 2001; Gutirrez-Marcos et al., 2004), and a transcriptional
activator, ZmMRP-1 (Gmez et al., 2002). The transcriptional
activator has been shown to activate BETL and MEG1 promoters
(Gutirrez-Marcos et al., 2004), and the ZmMRP-1 promoter
itself is active in regions of active transport between source
and sink tissues (Barrero et al., 2009). Furthermore, a putative
type-A response regulator gene, ZmTCRR-1, was shown to be
specifically expressed in the basal endosperm layer of maize
(Muiz et al., 2006). While these studies provide important
insights, our wider understanding of the molecular processes
underlying TC development remains poor.
Most TCs develop deep within complex tissues (e.g.
phloem parenchyma TCs in minor veins; Haritatos et al.,
New Phytologist (2009) 182: 863877 863

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864 Research
Fig. 1 The cDNA-amplified fragment length
polymorphism (AFLP) analysis of transfer cell
(TC) development using adaxial epidermal
peels of Vicia faba cotyledons. (a) Scanning
electron microscopy (SEM) image of an
isolated adaxial epidermal peel showing the
epidermal cell sheet (ECS) with a tag of
storage parenchyma tissue (SPT) that was
removed by dissection along the dotted line
indicated. (b,c) Higher magnification views of
epidermal peels revealing intact cellular
contents. Nuclei are clearly visible by (b) 4,6diamidino-2-phenylindole (DAPI) staining
(merged bright field/fluorescence image)
or visualization by SEM (c). The inner surface
of the epidermal layer is shown in (ac).
Bar, 1 mm (a), 20 m (b,c). (d) The cDNAAFLP banding patterns obtained from
independently prepared and amplified cDNA;
(e) cDNA-AFLP of amplified cDNA prepared
from epidermal peels of cotyledons cultured
for 0, 3 or 24 h. Induction (arrow head),
upregulation (asterisk) and down-regulation
(closed circle) of gene expression.
2000) and thus are not readily accessible for experimental

analysis. However, this issue is obviated by the readily accessible
epidermal cells of Vicia faba (Faba bean) cotyledons. During
in planta cotyledon development, abaxial epidermal cells transdifferentiate to form TCs but adaxial epidermal cells do not
(Offler et al., 1997). However, when cotyledons are cultured
with their adaxial surface in contact with nutrient agar, adaxial
epidermal cells form small papillate wall ingrowths within 3 h
(Wardini et al., 2007b), and functional TCs with a complex,
transporter-rich wall labyrinth by 48 h (Offler et al., 1997;
Farley et al., 2000; Wardini et al., 2007a). Thus, the V. faba
cotyledon culture system provides a large population of
developing TCs that share the same induction event, and
importantly, are morphologically and functionally equivalent
to TCs that form in planta.
To analyse transcriptional changes accompanying induction
and development of TCs in adaxial epidermal cells of V. faba
cotyledons, we developed an efficient and simplified cDNAamplified fragment length polymorphism (AFLP) procedure
incorporating nonsaturating PCR cDNA amplification to
profile transcripts derived from isolated epidermal cells. We
show that large-scale changes in gene expression occur within
3 h of TC induction, with many genes being induced,
upregulated or rapidly switched off specifically in the
adaxial epidermal cell layer undergoing trans-differentiation.
Of particular interest among genes exhibiting upregulated
and selective expression in trans-differentiating adaxial
epidermal cells were suites involved in auxin signalling,
cell division, vesicle trafficking associated with cell wall
biosynthesis, mitochondrial biogenesis and storage product
hydrolysis. Blocking auxin action or vesicle trafficking
inhibited wall ingrowth formation, thus confirming these

www.newphytologist.org
processes as key participants in TC development. Enhanced

numbers of mitotic figures present in 3-h cultured adaxial
epidermal cells demonstrated that these cells underwent
rapid de-differentiation on exposure to inductive conditions.
Collectively, these observations provide new insights into the
early gene expression events leading to the induction and
formation of TCs.
Materials and Methods

Plant material, cotyledon culture and tissue processing
Vicia faba L. (cv. Fiord) plants were grown in controlled glasshouse
and growth cabinet conditions (Talbot et al., 2001). At harvest,
cotyledons of 80120 mg FW were removed surgically
from their seed coats and either fixed immediately in ice-cold
ethanol and acetic acid (3 : 1, v : v) for 1 h at 4C or cultured
adaxial face down on modified Murashige and Skoog (MS)
media containing 50 mm glucose and 50 mm fructose (Farley
et al., 2000) for 3 h or 24 h and then fixed (see earlier). Fixed
tissue was processed rapidly by rinsing briefly in distilled
water before isolating sheets of adaxial epidermal cells as
epidermal peels. The adhering tag of parenchyma tissue
(Fig. 1a) was surgically removed and each epidermal peel
snap frozen in liquid nitrogen. Light and scanning electron
microscopy (SEM) observations revealed that most epidermal
cells in the peels were sheared along their anticlinal walls and
their cellular contents remained mostly intact (Fig. 1b,c).
Following peeling, 1-mm thick discs of storage parenchyma
tissue, free of epidermal cells, were collected from 3-h cultured
cotyledons using a 5-mm diameter cork borer and immediately
snap frozen.
The Authors (2009)

Research
RNA extraction and cDNA amplification

Epidermal peels, from a minimum of three cotyledons per
treatment, were pooled and total RNA extracted using an
RNeasy RNA isolation kit (Qiagen). Total RNA was extracted
from corresponding storage parenchyma discs using an
RNeasy RNA isolation kit following treatment with Trizol
reagent (Invitrogen). Extracted RNA was reverse transcribed with
Powerscript reverse transcriptase using 3-rapid amplification
of cDNA ends (RACE) CDS primer A and SMART II A
oligonucleotide (Clontech; see the Supporting Information,
Table S1) to generate fully transcribed first-strand cDNA
tagged with short sequences complementary to the SMART
II A oligonucleotide at both the 5 and 3 ends. This first
strand cDNA was purified with phenolchloroform and
ethanol precipitated using linear polyacrylamide as a carrier
(Ambion, Austin TX, USA) and then used as template for
full-length cDNA amplification following the Super SMART
PCR cDNA synthesis kit (Clontech) protocol. Amplification
parameters were optimized empirically by electrophoretic
analysis of small aliquots of PCR products from every two
cycles. The resulting double-stranded amplified cDNA were
purified and adjusted to equal total amounts by comparing
amplification of V. faba GAPDH1 and elongation factor 1-
(VfGAPDH1-FP/RP and VfEF1-FP/RP primer pairs,
respectively; see Table S1) by semiquantitative PCR.
RNA fingerprinting with cDNA-AFLP
The cDNA-AFLP fingerprinting reactions were carried out
using a protocol modified from Bachem et al. (1998). Briefly,
equal amounts of amplified cDNA from each experimental
sample were digested with MseI and ApoI (NEB, Ipswich,
MA, USA). The resulting digestion fragments were ligated to
enzyme-specific adaptors (Milioni et al., 2002, and Table S1)
using T4 DNA ligase (MBI Fermentas, Burlington, Canada).
Fragments ligated to the ApoI adaptor, biotinylated at the 5
terminus, were collected following binding to streptavidincoated paramagnetic Dynabeads (Dynal, Oslo, Norway). A
1/10 dilution of this ligation reaction was preamplified using
primers targeted to the adaptor sequences (Table S1). A 1/100
dilution of preamplification product was used for each selective
PCR determined by two specified bases at the 3 end of each
primer extending into the fragment sequence. In contrast to
Bachem et al. (1998), PCR primer concentration was 250 nm
to allow fragment visualization by silver staining. The PCR
reactions were incubated at 94C for 10 min followed by 13
cycles of 94C for 30 s, 65C for 30 s and 72C for 1 min,
with the annealing temperature dropping by 0.6C each
cycle. The reactions were completed by 23 cycles of 94C for
30 s, 56C for 30 s and 72C for 1 min. All 256 possible PCR
primer combinations were tested. Products from these
reactions were run on 16 cm-long 5% polyacrylamide gels for
4.5 h at 40 mA and stained using a rapid silver staining
The Authors (2009)

procedure (Qu et al., 2005). Fragments were visualized on a

Molecular Imager gel documentation XR system (Bio-Rad).
Relative band intensities were determined using quantity
one software (version 4.6.3; Bio-Rad), and a band was
classified as differentially expressed if its intensity showed
5-fold temporal change.
Transcript-derived fragment (TDF) extraction,
verification and sequencing
Each TDF of interest on silver-stained polyacrylamide gels
was stabbed with a sterile 200 l pipette tip and incubated in
15 l of 10 mm Tris-HCl (pH 8.0) for 30 min at room
temperature. Each fragment was reamplified using 5 l of the
fragment extract as template and subjected to the selective
PCR cycle program with an additional seven cycles at an
annealing temperature of 56C. Reamplified products were
separated on agarose gels, DNA bands were extracted using
the Wizard gel and PCR purification kit (Promega) and
cloned directly into the TA cloning vector pGEM-T Easy
(Promega). Clones were sequenced using T7 primer and BDT
sequencing chemistry (Invitrogen). Gene homology analysis
was performed using the blast program (Altschul et al.,
1997) at the NCBI website (http://www.ncbi.nlm.nih.gov/
BLAST/) and the TIGR database (http://www.jcvi.org/) with
default parameters. The TDF sequences were searched against
blastx and blastn of NCBI or blastx and blastn of TIGR.
Promoter sequences (a maximum of 2 kb up-stream of the
ATG start codon) of each Arabidopsis orthologue were screened
for regulatory cis-elements by Athena (OConner et al., 2005).
Expression of selected TDFs was validated by quantitative
real-time PCR, with Platinum Taq polymerase and SYTO9
dye (Invitrogen), on unamplified cDNA produced from
independently isolated RNA. Reactions were performed using
a Corbett RotorGene 6000 with fluorescence acquisition
through the green channel. Expression quantification utilized
the two standard curve method as described in the Corbett
Rotor-Gene 6000 software package (version 1.7), using V. faba
elongation factor 1- (VfEF1-) standard curve to normalise
expression.
Scanning electron microscopy of treated cotyledons
Cotyledon cultures were established as described earlier
except that sister cotyledons were divided between culture
media with or without the specified treatment (Wardini et al.,
2007b) or prepared under green light. After 15 h, adaxial
epidermal peels were prepared, washed in 2% (w : v) NaOCl
for 3 h and subsequently dehydrated at 4C through a 10%
step-graded ethanoldistilled H2O series, changed at 30-min
intervals. Peels were critical point-dried with liquid CO2 in a
critical-point drier (Balzers Union, Liechtenstein) and secured
outer face down onto sticky tabs to reveal the cytoplasmic face
of their outer periclinal cell walls. Samples were sputter-coated

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Table 1 Categories of verified gene
expression profiles identified in adaxial
epidermal cells of Vicia faba cotyledons
induced to form transfer cells (TCs)
Expression profilea, b
Epidermal peels
Hours in culture
24
SP
Number (%) of
TDFs
Epidermalspecific
Induced
Late-Induced
Early Transient-Induced
Up-Regulated
Rapidly Switched-Off
Gradually Switched-Off
69 (15)
22 (5)
21 (4)
30 (6)
102 (22)
20 (4)
Epidermal and
storage
parenchyma
Induced
Late-Induced
Early Transient-Induced
Up-Regulated
Rapidly Switched-Off
Gradually Switched-Off
116 (25)
13 (3)
19 (4)
19 (4)
26 (6)
14 (3)
Total number of TDFs
471 (100)
cDNA-AFLP analysis examined expression profiles from epidermal peels of cotyledons cultured
for 0, 3 or 24 h, or storage parenchyma tissue (SP) isolated from 3-h cultured cotyledons.
TDFs, transcript-derived fragments.
aOnly gene fragments with changes in expression levels 5-fold across the culture period are
included.
b
Black bars are schematic representations of PCR fragments visualized on silver stained
polyacrylamide gels and illustrate identified patterns of differential gene expression.
with gold to a thickness of 20 nm in a sputter-coating unit

(SPI Suppliers, West Chester, PA, USA), and viewed at 15 kV
with a Philips XL30 SEM.
Analysis of cell division
Cotyledons were cultured and fixed as described earlier. After
washing briefly in phosphate-buffered saline (PBS), adaxial
epidermal peels were collected and stained with 1 g ml1
4,6-diamidino-2-phenylindole (DAPI) for 5 min. Epidermal
peels were rinsed 2 5 min in PBS and mounted in Mowiol
(Calbiochem, San Diego, CA, USA) with 0.1% (w : v) pphenylenediamine. Tissue was viewed with a Zeiss Axiophot
epifluorescence microscope equipped with a 50 W short-arc
mercury lamp and a UV (365420 nm) filter (Osram). Mitotic
indices were estimated as percentages of cells containing
mitotic profiles from at least 100 cells scored per replicate.
Results
Isolation of RNA and amplification of cDNA from
epidermal peels
Recovery of total RNA obtained from either single or pooled
(maximum of five) epidermal peels was not sufficient to yield
reliable banding patterns using standard cDNA-AFLP protocols
(data not shown). These protocols typically use up to 100 g
of total RNA for starting material (Bachem et al., 1998)

compared with nanogram amounts retrieved from epidermal

peels. We therefore incorporated a nonsaturating PCR-based
cDNA amplification step based on procedures developed for
cDNA microarray analysis of small tissue samples (Hertzberg
et al., 2001; see the Materials and Methods section). Figure 1d
shows that transcript-derived fragments (TDFs), generated by
selective PCR of amplified cDNA, were consistent between
technical repeats and detected temporal changes in selective
gene expression (Fig. 1e). Using this modified procedure, we
were able to profile gene expression in adaxial epidermal cells
of freshly isolated cotyledons (no culture) or those cultured
for 3 h and 24 h, and to compare these profiles with those of
storage parenchyma cells from 3-h cultured cotyledons to
identify changes in gene expression occurring specifically in
adaxial epidermal cells and therefore likely to be related to
TC induction and development (Table 1). Expression profiles
deduced from our cDNA-AFLP approach (Table 1) were
verified using real-time PCR on unamplified cDNA (see Fig. S1
and associated text). This analysis demonstrated that conclusions
of cell-specific expression profiles could be drawn with
confidence but distinction between induced and upregulated
gene expression was less clear.
cDNA-AFLP analysis of transcriptional regulation
accompanying induction and development of TCs
Analysis of the 256 primer combinations containing two
basepair overhangs yielded a total of 5795 TDFs, ranging in
The Authors (2009)

Research
size from 50 bp to 500 bp, from adaxial epidermal cells and

storage parenchyma tissue. From this pool of TDFs, 756
demonstrated differential expression, defined here as a 5-fold
change (up or down) in band intensity detected on silver-stained
gels (see the Materials and Methods section). Of these, a total
of 471 fragments were verified as true cDNA-AFLP fragments
by extracting each band from the polyacrylamide gel and
reamplifying using the original selective primer pair. An
analysis of 234 V. faba cDNAs present in GenBank revealed
that 72% were cut at least once by both ApoI and MseI (data
not shown). Applying this percentage to the 471 differentially
expressed TDFs identified (Table 1), we estimate that TC
formation may involve differential expression of c. 650 different
genes. This estimate compares well with the numbers of
developmentally regulated genes detected during tracheary
element formation in cultured Zinnia mesophyll cells by
cDNA-AFLP (562; Milioni et al., 2002) or by microarray
(523; Demura et al., 2002) analyses. Furthermore, of the 471
differentially expressed TDFs, our approach identified 142
TDFs (Table 1 and hence an estimated 195 genes totally) that
were induced or upregulated specifically in epidermal cells
during TC formation. This number of genes displaying
epidermal-specific changes in expression is within the range of
preferentially expressed genes reported for epidermal cells of
maize coleoptiles (130; Nakazono et al., 2003) and Arabidopsis
stems at defined stages of development (180; Suh et al., 2005).
These comparisons support the conclusion that our cDNAAFLP study has successfully identified the majority of genes
being differentially regulated specifically in epidermal cells
during TC formation. Moreover, this conclusion is supported
by the finding that genes known to be expressed exclusively in
epidermal layers, such as fiddlehead (Pruitt et al., 2000) and
B1-type cyclin (Boudolf et al., 2004), were identified in the
epidermal-specific cohort of TDFs (Table 2).
Temporal patterns of expression were classified as Induced,
Late-Induced, Early Transient-Induced, Up-Regulated,
Rapidly Switched-Off and Gradually Switched-Off (Table 1).
Of those genes displaying epidermal-specific changes in
expression, approximately equal numbers were either induced/
upregulated or switched-off rapidly or gradually (Table 1).
Responses of differential gene expression were typically rapid,
with 85% of differential expression occurring within 3 h of
culture (Table 1).
Ontology-deduced functions of induced epidermalspecific genes relate to cell wall biosynthesis,
metabolism and protein synthesis/metabolism and are
potentially regulated by auxin and/or ethylene
Cotyledon culture induces the formation of TCs in adaxial
epidermal cells but not in cells of the underlying storage
parenchyma tissue (Farley et al., 2000; Talbot et al., 2007).
Consequently, attention was focused on identifying, by
homology searching (see the Materials and Methods section),
The Authors (2009)

the 112 TDFs showing epidermal-specific, induced expression

(Induced, Late-Induced, Early Transient-Induced; Table 1).
Genes showing this expression pattern are more likely to be
directly related to TC development, compared with those
associated with stress responses which are expected to be
expressed comparably in the adjacent storage parenchyma tissue.
Functional classifications were determined by searching blast
similarity matches (blast expectation values [E] of 103)
through the Gene Ontology (http://www.geneontology.org)
and KEGG BRITE (http://www.genome.jp/kegg/brite.html)
databases, with confirmation by reference to the literature.
This process enabled TDFs to be placed into one of nine
predicted functional groups (Table 2; groupings based on the
categories used by Milioni et al., 2002). Of the 112 TDFs, 44
(39%) returned no significant match to any database entry
(data not shown) and a further 15 (23%) matched database
entries for hypothetical or unknown proteins (Table 2; Fig. 2a).
The remaining 68 TDFs showed significant alignments and
were placed in functional groups. The major groups were
metabolism, energy and storage (12 TDFs; 18% of 68),
protein synthesis and metabolism (11 TDFs; 16%), cell
wall and vesicle trafficking (9 TDFs; 13%), and transcription
(6 TDFs; 9%) (Table 2; Fig. 2a).
The development of TCs in tomato roots is regulated
by auxin and ethylene (Schikora & Schmidt, 2001, 2002).
Accordingly, promoter regions of Arabidopsis orthologues
of identified V. faba TDFs (Table 2) were screened using
Athena (OConner et al., 2005) for the presence of auxin- and
ethylene-regulatory cis-element sequences. Of the 48 Arabidopsis orthologues identified, 24 (50%) contained at least one
repeat of the auxin-responsive element, AuxRe (TGTCTC;
Guilfoyle & Hagan, 2007) in its corresponding promoter
region, while nine (19%) contained at least one ET-responsive
element (GCC-box; Ohme-Takagi & Shinshi, 1995, and
see Table 2). These percentages are substantially higher than the
41% and 9% for AuxRe and the GCC-box elements, respectively, found by searching all promoter regions in the Arabidopsis genome using the Data Mining application of Athena.
Genes encoding hypothetical and unknown proteins
are abundant in those rapidly switched off within 3 h of
cotyledon culture
Of the 102 TDFs whose epidermal-specific expression was
rapidly switched-off (Table 1), 34 were selected for sequencing
based on their size (c. 150400 bp) and band intensity on the
silver-stained gels. Of this cohort only three returned no
significant hits, and of those exhibiting low E values (Table 3;
Fig. 2b), a substantial proportion (14 TDFs; 45%) matched
hypothetical and unknown proteins suggesting the possibility
of novel functions linked with trans-differentiation of TCs.
The proportion of genes distributed among the various
functional groupings was generally similar to that observed
for induced genes (compare Fig. 2b with 2a).

867
Functional group
Clone ID
Expression
profile
Size
(bp)
Transcription
V39A
V225E
V231A
V234A
V36A-2
V252A
V9A
V43A
V76A
V97C-1
V134C
V134F
V230B
V234B
I
I
I
I
ETI
ETI
I
I
I
I
I
I
I
I
201
362
137
436
325
311
428
337
224
158
349
161
160
144
V40D
V196B
V219B
V3A
V92B
V194C
V233B
V245B
V113F
V253C
V66G
V196C
ETI
ETI
ETI
I
I
I
I
I
LI
LI
ETI
ETI
150
249
412
309
170
392
429
139
244
134
124
50
V26A
V51F
V146A
V167A
V170C
I
I
I
I
I
330
201
355
226
138
V174A
V222F
V224B
V225F
V234F
V170B
V243B
I
I
I
I
I
LI
ETI
154
108
196
577
54
243
266
Protein synthesis
and metabolism
Cell wall and

vesicle trafficking
The Authors (2009)

Metabolism, energy
and storage
Gene product
Organisma
Accession
numbera
E valueb
Locus IDc
Promoter
motifd,e
Splicing factor Prp8

CONSTANs-like zinc finger protein
KH domain RNA binding protein
Mismatch repair protein
GH1 protein (AUX/IAA-like)
cAMP response element binding (CREB) protein
Ubiquitin-protein ligase
Aspartate aminotransferase
Flavonoid 3-O-galactosyl transferase
Polyubiquitin (UBQ12)
In2-1 (glutathione-S-transferase)
Serine carboxypeptidase S10 family protein
Peptidase M, neutral zinc metallopeptidase
Integrase, catalytic region; zinc finger,
CCHC-type; peptidase aspartic
Insulin-degrading enzyme
Putative aminopeptidase
Ribosomal protein S26
UDP-glucuronosyl/UDP-glucosyltransferase
ADP-ribosylation factor (ARF1)
YKT61 (similar to yeast SNARE YKT6 1)
Putative clathrin coat assembly protein
Glycoside hydrolase, family 17: X8 domain
Pectin methylesterase inhibitor
Hydroxyproline-rich glycoprotein-2 related cluster
Glycoside hydrolase, family 17; X8 domain
Putative ras-GTPase-activating protein,
SH3-domain-binding protein
Mitochondrial rpl5, rps14 and cob genes
Vicilin precursor
Aconitate hydratase
AMP-dependent synthetase and ligase
Oxidoreductase, short-chain dehydrogenase/
reductase family protein
Triacylglycerol/steryl ester lipase-like protein
Glyceraldehyde-3-phosphate dehydrogenase-like
Malic oxidoreductase
Mitochondrial NADH dehydrogenase subunit 7 (nad7)
Mitochondrial cytochrome oxidase subunit I (coxI)
Isoamylase isoform 1
Nitrilase/cyanide hydratase & apolipoprotein
N-acyltransferase
Oryza sativa
Pisum sativum
Medicago truncatula
Arabidopsis thaliana
Glycine max
Medicago truncatula
Medicago sativa
Vigna mungo
Glycine max
Medicago sativa
Medicago truncatula
Medicago truncatula
NP_001054734
AAX47173
ABE91933
AAD04176
AF016633
ABE93018
NP_201183
CAA43779
BAA36972
NP_564675
AAG34872
AAZ32845
ABE84181
ABE92232
3e-25
2e-18
1e-11
1e-56
4e-231
2e-29
2e-10
2e-48
2e-18
1e-08
2e-30
2e-04
2e-13
7e-13
At1g80070
At2g24790
At1g09660
At3g18524
Aux/Eth
Aux
Aux/Eth
Solanum esculentum
Pisum sativum
Medicago truncatula
Medicago sativa
Medicago truncatula
Medicago sativa
Medicago truncatula
Trifolium pratense
CAC67408
AAN72085
AAD47346
ABE82273
DQ455181
NP_200614
AAL38763
ABE89157
NP_196116
CO513118
ABN09816
AB236858
5e-05
8e-30
2e-28
5e-22
5e-421
4e-53
3e-53
2e-102
4e-05
1e-083
9e-07
3e-041
Pisum sativum
Vicia faba
Oryza sativa
Medicago truncatula
Oryza sativa
AJ132231.1
CAA68559
ABF93861
ABE86318
ABA98146
1e-411
2e-10
1e-40
3e-25
3e-06
Medicago truncatula
Medicago truncatula
Medicago truncatula
Beta vulgaris
Pisum sativum
Pisum sativum
Medicago truncatula
AAR29056
ABE82032
ABN05792
ABD36076
X14409
AAZ81835
ABE91350
2e-14
5e-07
5e-12
1e-06
3e-081
1e-37
2e-38
At3g12250
At5g63780
At5g11520
At5g17030
At1g55060
At5g02790
At1g28110
At5g10540
At4g27210
At2g41790
At1g63770
At3g56340
At2g36790
At5g58060
At3g50860
At2g01630
At5g04970
At1g15825
At1g64760
Aux
Aux
Aux/Eth
Aux
Aux
Aux
Aux
Aux
Aux
Eth
Eth
At2g05710
At1g51680
At5g50590
Aux
At5g14180
At1g13440
At4g00450
Aux
Aux
Eth
At2g07769
At2g39930
At4g08790
Aux
Aux
868 Research

Table 2 Functional classification and highest similarity match in NCBI or TIGR databases for transcript-derived fragments (TDFs) identified by cDNA-amplified fragment length polymorphism
(AFLP) to be induced specifically in adaxial epidermal cells in response to cotyledon culture
The Authors (2009)

Table 2 continued
Functional group
Clone ID
Expression
profile
Size
(bp)
Cell growth, division

and DNA synthesis
V25C
V120A
V245D
V24B
V104A
V112D
V89C
V165B
V190B
V227A
V187A
V178A
V200B
V129B
V36A-1
V36B
I
I
I
I
I
LI
I
I
I
I
ETI
I
I
LI
ETI
I
319
543
105
174
531
298
185
143
322
313
263
109
204
323
299
288
V37D
V38D
V42B
V71D
V110D
V110F-1
V113E
V140C
V159C
V202D
V230C
V216E
V256C
V165A
I
I
I
I
I
I
I
I
I
I
I
LI
LI
ETI
144
111
186
109
148
137
284
107
288
273
124
118
203
298
Transport facilitation
Signal transduction
Others
Hypothetical and
unknown proteins
Organisma
Accession
numbera
E valueb
Locus IDc
Promoter
motifd,e
AtMUS81; endonuclease/nucleic acid binding

Mitotic cyclin B1-type
Nucleosome/chromatin assembly factor C
P-type H+-ATPase
Ammonium transporter (AtAMT1;2)
Vacuolar (H+)-ATPase G subunit, prokaryotic type
Fiddlehead-like protein
Protein kinase; Adipokinetic hormone
GIL1 (gravitropic in the light)
Putative nodulin protein MtN21
Protein phosphatase type 2C
Epoxide hydrolase
Embryonic abundant protein USP87 precursor
Transferase-family protein
Importin-, N-terminal
Unknown protein (clone Ps-cos16 LTR
and Ogre retrotransposons)
Unknown protein (clone MTH2-172C6)
Unknown protein (clone MTH2-116F22)
Unknown protein
Conserved hypothetical protein
Unknown protein (clone MTH2-39B3)
Hypothetical protein T21J18_150
Hypothetical protein
Unknown protein (clone MTH2-3G18)
Unknown protein (clone MTH2-9B23)
Unknown protein
Unknown protein (clone MTH2-36J11)
Unknown protein F28N24.7
Protein of unknown function DUF649
Unknown protein (clone MTH2-32M22)
Glycine max
Zea mays
Zostera marina
Medicago truncatula
Gossypium hirsutum
Medicago truncatula
Oryza sativa
Medicago sativa
Medicago truncatula
Vicia faba
Medicago truncatula
Pisum sativum
NP_194816
BAA09467
AF384037
BAF03589
NP_176658
ABD32809
AAL67993
ABE77704
NP_851217
BAB92246
CAA72341
TA21728_3880
P21746
NP_181527
ABE90433
AY299398
6e-18
7e-41
1e-031
4e-16
1e-34
6e-25
1e-15
3e-13
2e-37
3e-14
1e-23
2e-042
7e-29
3e-12
8e-45
1e-081
At4g30870
At2g26760
At2g16780
At1g80660
At1g64780
At3g01390
At2g26250
At1g06840
At5g58960
At1g44800
At1g07160
Aux
Aux
Eth
Aux
Aux
Aux
Eth
At2g39980
At5g17020
Aux
Aux
Medicago truncatula
Medicago truncatula
Medicago truncatula
Medicago truncatula
Medicago truncatula
Medicago truncatula
Medicago truncatula
Oryza sativa
Medicago truncatula
Medicago truncatula
Medicago truncatula
Medicago truncatula
CT967315
AC174306
NP_683417
ABE89743
CT009540
TA25031_3880
AK229089
AC174350
AC139526
AAV44205
AC148482
TA9157_35883
ABE77839
AC122165
4e-101
2e-051
3e-09
0.001
2e-341
3e-082
9e-051
1e-201
2e-701
1e-21
2e-131
6e-042
2e-24
8e-131
At1g51355
Aux
Aux
Eth
At1g17210
At3g52760
Expression profiles of Induced (I), Late-Induced (LI) and Early Transient-Induced (ETI) are shown in Table 1.
a
Accession number and organism refer to the closest gene match returned from BLAST searching of NCBI database, unless noted () to correspond to a TIGR contig.
b
All values taken from BLASTx searching of NCBI, except where otherwise noted: 1, BLASTn of NCBI; 2, BLASTx of TIGR; 3, BLASTn of TIGR.
cLocus ID tag provided for closest Arabidopsis match when present as a significant BLAST match (E value of < 103).
d
Auxin and ethylene response motifs identified in promoter sequences (2000 to 0 bp upstream of ATG) in corresponding Arabidopsis genes. Aux: AuxRE (TGTCTC); Eth: GCC-Box (GCCGCC).
e
Promoter analysis only performed where significant Arabidopsis match (BLAST E value of < 103) was obtained from TDF sequence.
Research

Gene product
869
Functional group
Clone ID
Size (bp)
Gene product
Organism
Accession numbera
E valueb
Locus IDc
Promoter motifd,e
Transcription
V91B
V140A
V143A-46
V190E-61
V100B
V201B
V203A-73
V231D
V66C-14
V118C
V68A
V176B
325
435
354
232
305
242
147
184
201
271
152
103
Medicago truncatula
Medicago truncatula
Medicago truncatula
Spinacia oleracea
Medicago truncatula
Pisum sativum
Hyacinthus orientalis
Glycine max
Glycine max
Pisum sativum
TA70974_3847
ABE79807
ABE79544
S15348
ABE91897
X80854
EF468471
AAM93434
AK227260
AF516879
X06281
NM_128949
3e-083
6e-70
4e-27
7e-18
2e-14
4e-341
1e-161
1e-17
2e-05
5e-091
7e-081
3e-091
At3g24070
At1g21700
At1g27730
At5g50250
At4g13780
At2g07769
Aux/Eth
Aux
Eth
Eth
Aux
At2g17360
At3g55260
Aux/Eth
Aux
At2g33980
Aux
V41C
V222B
V234G
V40C
V216C
162
173
118
173
157
Medicago truncatula
Pyrgus communis
Medicago truncatula
ABE86426
AAR25995
TA30584_3880
NP_567059.1
ABO78428
1e-15
1e-19
1e-053
9e-13
7e-13
At5g57710
Aux
At3g23750
At3g57890
At1g65320
Aux
Aux
V46A
V66E
V91A
V97A
V143A-47
V163A-49
V163A-51
V184C
V190E-62
V191A
V203A-74
V213A
V244D
425
292
315
178
274
119
193
326
90
271
270
306
185
Zinc finger, CCHC-type

SWIRM
Zinc finger, C2H2-type
RNA-binding protein, 28K
Aminoacyl-tRNA synthetase, class Ia
Mitochondrial rps10, trnF and trnP
18S ribosomal RNA gene
40S ribosomal S4 protein
-N-acetylhexosaminidase-like protein
Expansin (EXP1)
Alcohol dehydrogenase
Nucleoside diphosphate-sugar
(NUDIX) hydrolase (AtNUDT22)
Heat-shock protein 101
Putative senescence-associated protein
Putative LRR receptor protein kinase
Tubulin-specific chaperone C-related
CBS (cystathionine--synthase)containing protein
Hypothetic protein (Os12g0630700)
Unknown protein (clone MTH2-17N24)
Hypothetical protein precursor
Unknown protein (clone GMW1-45c9)
Hypothetical protein (ACLA_028940)
Genomic sequence (contig VV78X023217.49)
Unknown protein (clone GMW1-105h23)
OSJNBa0027O01.6 protein
Oryza sativa
Medicago truncatula
Vitis vinifera
Medicago truncatula
Phillyrea latifolia
Glycine max
Medicago truncatula
Vitis vinifera
Medicago truncatula
Aspergillus clavatus
Vitis vinifera
Glycine max
Oryza sativa
NP_001067339
AC147712
CAN83392
AC138526
CAK18872
AC173960
ABO81572
CAN63098
AC147434
XP_001269594
AM430398
AC187294
TA121_57577
2e-10
4e-04
3e-07
1e-051
1e-22
5e-201
2e-11
5e-13
2e-101
2e-07
6e-701
3e-49
1e-052
Protein synthesis
and metabolism
Cell wall and

vesicle trafficking
Metabolism, energy
and storage
Signal transduction
Others
Hypothetical and
unknown proteins
The Authors (2009)

aAccession
At5g45170
At3g16895
At2g24660
At1g41880
Aux
number and organism refer to the closest gene match returned from BLAST searching of NCBI database, unless noted () to correspond to a TIGR contig.
All values taken from BLASTx searching of NCBI, except where otherwise noted: 1, BLASTn of NCBI; 2, BLASTx of TIGR; 3, BLASTn of TIGR.
c
Locus ID tag provided for closest Arabidopsis match when present as a significant BLAST match (E value of < 103).
dAuxin and ethylene response motifs identified in promoter sequences (2000 to 0 bp upstream of ATG) in corresponding Arabidopsis genes. Aux: AuxRE (TGTCTC); Eth: GCC-Box (GCCGCC).
e
Promoter analysis only performed where significant Arabidopsis match (BLAST E value of < 103) was obtained from TDF sequence.
b
870 Research

Table 3 Functional classification of transcript-derived fragments (TDFs) identified by cDNA-amplified fragment length polymorphism (AFLP) to be rapidly and specifically switched off in adaxial
epidermal cells of 3-h cultured cotyledons based on the highest match resulting from BLAST searching of NCBI and TIGR databases
Research
Fig. 2 Classification of predicted gene

functions derived from sequenced,
differentially-expressed transcript-derived
fragments (TDFs). Genes showing cultureinduced, epidermal-specific (a) expression
and (b) rapid down-regulation or switchingoff. Predicted functions were made on the
basis of top matches obtained from BLAST
searches of the NCBI and TIGR databases,
using Expect (E) values 103 to indicate
significant matches. Gene sequences
returning no significant match with database
entries were not included in calculating
percentage values.
Similar to the cohort of induced genes, promoter analysis

of the pool of Arabidopsis orthologues closely matching
V. faba TDFs, which were rapidly switched off, revealed an
increase in the frequency of AuxRe and GCC-box motifs
within the promoter regions of these identified genes (65%
and 24%, respectively; Table 3).
Testing key functional pathways predicted by cDNAAFLP gene discovery light, auxin, vesicle trafficking
and cell division
Predicted functions of V. faba genes deduced from ontology
searches of databases, which were rapidly and specifically
induced in adaxial epidermal cells, indicated possible light
(e.g. Gravitropic in the light (GIL1) and Constans-like 3
(COL-3)) and auxin-mediated (e.g. GH1 and AuxRe promoter
The Authors (2009)

motifs) signalling pathways leading to wall ingrowth induction

(Table 2). The operation of these predicted signalling pathways
were tested experimentally by culturing cotyledons under green
light or in the presence of the competitive auxin inhibitor,
p-chlorophenoxyisobutyric acid (PCIB; Oono et al., 2003).
Cotyledon culture in the absence of an early light signal had
no significant effect on wall ingrowth induction (Fig. 3e). By
contrast, PCIB reduced numbers of adaxial epidermal cells
forming wall ingrowths by 60% (Fig. 3e).
More than 10% of genes showing induced, epidermal-specific
expression encoded proteins predicted to be involved in
vesicle trafficking and cell wall synthesis (Fig. 2a), for example,
ADP-ribosylation factor 1 (ARF1), YKT61 and a pectin
methylesterase inhibitor (Table 2). To examine a requirement
for vesicle trafficking in wall ingrowth formation, cotyledons
were cultured in the presence of Brefeldin A, a potent inhibitor

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872 Research
Fig. 3 Effect of white light, auxin and vesicle

trafficking on wall ingrowth formation and
measures of mitosis during transfer cell (TC)
induction. (ad) Scanning electron
microscopy (SEM) images of cytoplasmic
faces of outer periclinal walls of adaxial
epidermal cells of Vicia faba cotyledons
cultured following exposure to (a) white
(control) or (b) green light, or on Murashige
and Skoog (MS) media containing (c) 100 M
p-chlorophenoxyisobutyric acid (PCIB) or
(d) 100 g ml1 Brefeldin A. Bar, 20 m.
Wall ingrowths (arrow) and starch grains
(arrowhead) are labelled in (a) and (b).
(e) Adaxial epidermal cell numbers containing
wall ingrowths (e.g. cells marked with * in (c))
in specified treatments expressed as
percentages of total cells scored (150200
cells counted per replicate). (f) Mitotic indices
of adaxial epidermal cells in control (0 h) and
3-h cultured cotyledons (100 cells scored per
replicate). All values are mean SE of six (e)
or four (f) independent replicates.
of vesicle formation (Ritzenthaler et al., 2002). Under

these conditions, wall ingrowth formation was abolished
(93% inhibition; Fig. 3e), demonstrating an absolute requirement for vesicle trafficking in wall ingrowth deposition.
Induction of a mitotic cyclin, an endonuclease and chromatin
assembly factor C specifically in epidermal cells (Table 2)
suggested activation of the cell cycle upon TC induction.
Comparisons of mitotic index in adaxial epidermal cells
showed a dramatic rise in mitotic rates following cotyledon
culture, rising from 0.5 to 7.4 in the first 3 h (Fig. 3f).
Discussion
We used experimental induction of adaxial epidermal TCs in
V. faba cotyledons to reveal transcriptional changes accompanying
trans-differentiation of epidermal cells into functional TCs
(Tables 2 and 3). Rapid (< 3 h) epidermal-specific induction
of genes (Table 1) is consistent with the finding of Wardini
et al. (2007b) that all biosynthetic machinery required to form
wall ingrowths is transcribed within 1 h following exposure of

cotyledons to inductive signal(s). Concurrently there is an

equal number of genes rapidly switched off (122 TDFs;
Table 1) upon exposure to culture, reflecting a major change
in the epidermal transcriptome associated with transdifferentiation of epidermal TCs. Generic responses, including
those to abiotic stress, may be distinguished from those peculiar
to trans-differentiation of epidermal TCs by analysing genes
specifically induced in these cells (Epidermal-specific; Table 1).
This assumption is supported by the absence of gene functions
associated with generic stress responses from this cohort of
genes specifically induced in adaxial epidermal cells upon
cotyledon culture (Table 2). The relative distribution of these
genes among functional categories (Fig. 2a) matches those
reported for tracheary element formation (Milioni et al., 2002)
except for expression of transporter genes and those linked
with cell division. Expression of transporter genes (e.g.
ammonium transporter, P-type and vacuolar H+-ATPases;
Table 2) is consistent with TC function (Offler et al., 2003)
and further supports our conclusion that the experimental
approach used here has enabled identification of gene
The Authors (2009)

Research
expression events primarily associated with trans-differentiation

into functional TCs.
Signalling TC induction role for light, auxin and
ethylene?
The extent of wall ingrowth formation in phloem parenchyma
and companion cell TCs of Arabidopsis and pea leaves,
respectively, has been shown to be dependent on incident
light flux densities (Amiard et al., 2005). For the V. faba
cotyledon system, exposure of their adaxial epidermal cells to
white light upon cotyledon removal from seed coats may
initiate a photomorphogenic signal cascade. In this context,
induction of homologues of CONSTANS-like (COL-3; Datta
et al., 2006) and Gravitropic in the Light (GIL1; Allen et al.,
2006) and downregulation of B-EXPANSIN (Tepperman
et al., 2004) is consistent with a phytochrome-driven response
(Tepperman et al., 2004; Tables 2, 3). COL-3, in contrast to
most COLs that function in flowering responses, has been
shown to control vegetative growth patterns (Datta et al.,
2006) that might include cell wall formation. However, rates
of wall ingrowth initiation in adaxial epidermal TCs were
found to be independent of a light signal (Fig. 3). Whether a
light signal affects the extent of wall ingrowth formation in
committed adaxial epidermal TCs (Amiard et al., 2005) remains
to be determined. Indeed, upregulation of GIL1 (Table 2),
that renders auxin transport nonpolar in the dark (Allen et al.,
2006), provides a link between light and auxin signals possibly
mediating induction of wall ingrowth formation.
Elevated auxin levels are known to enhance formation of
TCs in rhizodermal cells of a number of species, including
tomato (Schikora & Schmidt, 2001). An indication that
auxin levels are elevated in adaxial epidermal cells of cultured
cotyledons is provided by the induced expression of a MtN21
homologue (Table 2), a signature gene for elevated levels of
auxin in developing tissues (Busov et al., 2004). Observed
profiles of selective gene expression in adaxial epidermal cells
(Table 2) indicate that elevated auxin levels could arise from
altered transport and/or enhanced biosynthesis. Inhibitory
effects of flavonoids on auxin transport (Peer & Murphy, 2007)
could be relieved by their enhanced metabolism through
induced expression of flavonoid 3-O-galactosyltransferase (Miller
et al., 2002) and glutathione-S-transferase (Smith et al., 2003).
Induction of GIL1 and an aminopeptidase (Table 2) could
impact on auxin transport by randomly relocalizing PIN1
proteins around plasma membranes of cotyledon cells
(Murphy et al., 2005; Allen et al., 2006). These effects on
auxin transport, combined with enhanced auxin biosynthesis
by induced expression of a nitrilase (Table 2), catalysing
hydrolysis of indole-3-acetonitrile into active indole-3-acetic
acid (IAA; Vorwerk et al., 2001), could alter patterns of auxin
distribution to drive wall ingrowth formation. High auxin
concentrations could account for the transient induction of an
early-response auxin gene, GH1 homologue (Table 2), belonging
The Authors (2009)

to the Aux/IAA gene family of transcriptional regulators

(Guilfoyle et al., 1993). The Aux/IAA proteins interact with
auxin response factors (ARFs) to confer various auxin responses
alone or in combination by binding to AuxRe motifs (Guilfoyle
& Hagan, 2007). These motifs are enriched (54 vs 41%)
among Arabidopsis orthologues of the genes identified in our
cDNA-AFLP screen (Tables 2, 3), indicating a potentially
important role for auxin in orchestrating wall ingrowth
formation. This conclusion is supported by finding that PCIB,
an auxin analogue that inhibits auxin action by competitively
binding with auxin receptors (Oono et al., 2003), reduced
numbers of adaxial epidermal cells forming wall ingrowths in
cultured cotyledons (Fig. 3).
The proposition that ethylene may contribute to TC
induction in V. faba cotyledons arises from finding a 2.7-fold
enrichment of ethylene responsive cis-elements in promoter
regions of Arabidopsis orthologues of differentially expressed
V. faba genes (Tables 2 and 3). This proposition is supported
by the finding that 1-aminocyclopropane-1-carboxylic acid
(ACC, an ethylene precursor) enhanced TC formation in root
epidermal cells of tomato (Schikora & Schmidt, 2002) and
adaxial epidermal cells of V. faba cotyledons (F. A. Andriunas
et al., unpublished).
Guided by the presence of AuxRe and GCC-box motifs
(Tables 2, 3), significant downstream targets of auxin and
ethylene signalling pathways inducing TC development could
include cellular metabolism (AuxRe), cell division (AuxRe)
and vesicle trafficking/cell wall biosynthesis (GCC-box). These
phenomena are discussed in the following sections.
Transfer cell induction coincides with increases in cell
division
Induction of TC development in adaxial epidermal cells of
cultured cotyledons involves reinitiation of cell division (Fig. 3f).
Borisjuk et al. (1995) reported that adaxial epidermal cells of
Stage V cotyledons, equivalent to the cotyledons used in
our culture experiments, have ceased division and are fully
committed to cell expansion. After a 3-h culture, however, the
mitotic index of adaxial epidermal cells had increased nearly
15-fold, from 0.5 to 7.4 (Fig. 3f). This rapid increase is
consistent with the endopolyploid status of adaxial epidermal
cells of these cotyledons (Borisjuk et al., 1995), thereby enabling
entry into mitosis without a preceding interphase. This
observation indicates that reinitiation of mitosis represents an
important early event in trans-differentiation of epidermal
TCs, most likely to set in train the substantial genomic
reorganization that accompanies trans-differentiation. Epidermalspecific induction of genes involved in DNA repair (an
endonuclease orthologue of AtMUS81) and chromatin
remodelling (nucleosome/chromatin assembly factor), together
with a mitotic B1 cyclin (Table 2) are consistent with this
possibility. Mitotic cyclin B1 is the noncatalytic partner of
B-type cyclin-dependent protein kinases (CDKs) belonging

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to the B1 subgroup. The B1-CDKs drive the G2/M transition

in mitosis (Francis, 2007), and are expressed preferentially in
epidermal cells (Boudolf et al., 2004). A B1-CDK dependent
arrest at the G2/M phase accounts for the ability of these cells
to rapidly (within 3 h) enter mitosis upon exposure to the
inductive signal (Fig. 3f).
In addition to the epidermal-specific induction of mitosis,
induction of two -1,3-glucanases (Table 2) suggests reinitiation
of cytokinesis during trans-differentiation of epidermal TCs.
Both induced genes are Family 17 glycoside hydrolases (Minic
& Jouanin, 2006), with the Arabidopsis orthologue of V245B
(Table 2) ascribed with an ancestral function in cell division/
cell wall remodelling (Doxey et al., 2007). In this instance, the
-1,3-glucanase may be a candidate for performing a specialized
role during cell plate formation or, alternatively, participating
in wall remodelling events required to achieve the unique
morphology of reticulate wall ingrowths.
Modification of energy metabolism during transfer cell
development
Induced genes selectively expressed in adaxial epidermal
cells contributing to energy metabolism (Table 2) included
components of the mitochondrial electron transport chain
(nad 7, cob, cox1) and Kreb cycle (aconitase, malate dehydrogenase).
Expression of mitochondrial-encoded nad 7, cob and cox1
(Table 2) are insensitive to altered oxygen tensions resulting
from cotyledon excision (Rolletschek et al., 2003) but reflect
expression profiles linked with mitochondrial biogenesis
(Howell et al., 2007). This process is possibly orchestrated by
chromatin assembly factor C (CAF-C; Table 2), which is
known to influence mitochondrial numbers in yeast through
the Ras/cAMP pathway (Ruggieri et al., 1989; Rigoulet et al.,
2004). Consistent with this conclusion, mitochondrial matrix
densities and cristae formation increase along with mitochondrial
numbers in adaxial epidermal cells undergoing wall ingrowth
development (Farley et al., 2000). Induced expression of
NADH-dependent malic enzyme and aconitase (Table 2) is
suggestive that mitochondrial activity has switched to an
anaplerotic mode to meet demand for intermediates consumed
in various synthetic processes underpinning wall ingrowth
construction.
Given that sugar demand exceeds supply during the
trans-differentiation of epidermal TCs in planta (Harrington
et al., 1997) and in vitro (Wardini et al., 2007a), carbon
skeletons are likely to be sourced from reserves. In this
context, a profile of genes potentially involved in remobilization
of storage compounds were induced (Table 2), including
those remobilizing lipids (triacylglycerol lipase, hydroxysteroid
dehydrogenase, aconitase and malate dehydrogenase) and starch
(isoamylase and glyceraldehyde-3-phosphate dehydrogenase).
Oil body breakdown through triacylglyceride lipase activity
would provide free fatty acids to enter glyoxysomes as described
for germinating seeds (Eastmond, 2006). Within glyoxysomes,

fatty acid molecules are oxidized to acetyl-CoA and enter the

glyoxylate cycle to produce C4 precursors which can be used
for energy generation or fed through gluconeogenesis into an
array of biosynthetic pathways, including cell wall component
biosynthesis (see previous section). An alternative energy
source may be derived through starch hydrolysis catalysed by
isoamylases (Hussain et al., 2003) in combination with a
plastid glyceraldehyde-3-phosphate dehydrogenase (Table 2)
forming part of a carbon pathway before plastid/cytosol
exchange of carbon skeletons.
Vesicle trafficking is essential in wall ingrowth
deposition
It is not surprising that genes involved in vesicle trafficking
and cell wall biogenesis are induced in epidermal cells
undergoing trans-differentiation into TCs. Genes induced or
switched off specifically in epidermal TCs (Tables 2, 3) can be
presumed to act as regulators of papillate ingrowth deposition
at defined loci (Talbot et al., 2001; Fig. 3). The absence of key
wall-building genes from Table 2 (listing genes expressed in
epidermal cells but not in storage parenchyma) such as
celluloses and sucrose synthases is explained by their generic
expression in all cells undergoing expansion at this stage of
cotyledon development (Borisjuk et al., 1995).
Modification of vesicle trafficking upon TC induction is
evident through the induction of vesicle-targeting genes ADPribosylation factor 1 (ARF1), a SNARE (YKT61) and a clathrin
coat adaptor subunit (Table 2). The ARFs are considered
central to orchestrating asymmetrical vesicle trafficking to effect
polarity in plant cells (Xu & Scheres, 2005), a characteristic
consistent with wall ingrowth deposition in adaxial epidermal
cells of cotyledons. However, Class 1 ARFs (Table 2) act as
intracellular regulators of trafficking, being primarily localized
to the Golgi and subpopulations of post-Golgi vesicles (Matheson
et al., 2008). Induction of YKT61, a SNARE located in the
cis-Golgi cisternae (Chen et al., 2005), together with ARF1, is
indicative of enhanced protein trafficking between Golgi and
endoplasmic reticulum (ER). Upregulation of ARF1, but not
SAR1, the GTPase responsible for assembly of COP11 protein
coats directing vesicle budding from the ER (Memon, 2004),
points to ARF1 as a key regulator of vesicle trafficking activity
during wall ingrowth formation. This conclusion is supported
by inhibiting this process when cotyledons were cultured in
the presence of Brefeldin A (Fig. 3d,e). Interestingly, this result
suggests that the contribution of cellulose synthase/sucrose
synthase complexes to building papillate wall ingrowths (Talbot
et al., 2007) also depends upon vesicle trafficking.
Modification of trans-Golgi vesicle trafficking in TCs is
indicated by induction of the clathrin coat adaptor AP-3
(Table 2). ARF1 regulates recruitment of AP-3 adaptor
complex to membranes (Ooi et al., 1998) during construction
of clathrin coats, and YKT61 is considered a core component
of SNARE-facilitated fusion at the trans-Golgi network in
The Authors (2009)

Research
Arabidopsis (Chen et al., 2005). Since large numbers of secretory

vesicles are associated with developing wall ingrowths in TCs
(Wardini et al., 2007b), it is possible that, together with the
early transiently upregulated putative ras-GTPase-activating
protein (Table 2), ARF1/YKT61/AP-3 could constitute a
specialized gene complex facilitating increased trans-Golgi vesicle
delivery to the plasma membrane in a polarized manner.
Genes encoding wall components and modifying enzymes
are not well represented in the list of genes specifically induced
or switched-off in epidermal cells (Tables 2, 3), consistent with
wall ingrowths being compositionally equivalent to primary
cell walls (Vaughn et al., 2007). Some exceptions are -Nacetylhexosaminidase (Table 3), UDP-glucosyltransferase and
pectin methylesterase inhibitor (Table 2). Induction of a VATPase (Table 2) in the trans-Golgi network could increase
synthesis of cell wall components and trafficking to the
membrane (Brx et al., 2008). Late induction of a pectin
methylesterase inhibitor (PMEI) is consistent with maintaining
extensibility of developing wall ingrowths. Wall ingrowths are
rich in pectins, and for V. faba cotyledon epidermal TCs these
pectins are esterified (Vaughn et al., 2007 and references cited
therein). Pectin methylesterases (PMEs) have a major role in
pectin remodelling (Pelloux et al., 2007) through de-esterification
decreasing wall extensibility (Rckel et al., 2008). Therefore, late
induction of PMEI (Table 2) suggests a role in maintaining extensibility of developing wall ingrowths as they commence branching
and fusing to form a fenestrated layer (Talbot et al., 2001).
Conclusions
Extensive, rapid and cell-specific transcriptional regulation
underpins trans-differentiation of adaxial epidermal cells of
V. faba cotyledons into TCs. Auxin, possibly in combination
with ethylene, functions as an inductive signal to initiate wall
ingrowth formation. The induction of TCs initiates re-entry
into a division cycle coincidental with modification of vesicle
trafficking and wall assembly machinery specifically in these
cells. The rapid stepped increase in metabolic demand by the
trans-differentiating epidermal cells for intermediates to
support these biosynthetic activities is met by remobilization
of lipid and starch stores processed through an enhanced
anaplerotic pathway in newly formed mitochondria. Inhibition
of pectin de-esterification in wall ingrowths could confer
sufficient mechanical flexibility to form the characteristic
fenestrated wall layers. The insights generated from these
findings open new opportunities for further studies to expand
our understanding of signalling pathways inducing, and
metabolic machinery responsible for constructing, the intricate
wall ingrowths of TCs.
Acknowledgements
We thank Kevin Stokes for raising healthy experimental
material and acknowledge funding of this project from Australian
The Authors (2009)

Research Council Discovery Project grants DP0556217 and

DP0664626.
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Supporting Information
Additional supporting information may be found in the
online version of this article.
Fig. S1 Transcript-derived fragment (TDF) expression patterns
in unamplified cDNA using real-time PCR.
Table S1 Oligonucleotide primer sequences used for cDNA
synthesis and amplification, cDNA-amplified fragment length
polymorphism (AFLP) and real-time PCR verification of
TDF expression
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Early gene expression programs accompanying

New Phytologist (2009) 182: 863877

Key words: cDNA-AFLP, transdifferentiation, transfer cells, Vicia faba,

Transfer cells (TCs) trans-differentiate from differentiated cells by developing

The Authors (2009)

include four defensin-like genes, BETL1-4 (Thompson et al.,

New Phytologist (2009) 182: 863877 863

2000) and thus are not readily accessible for experimental

New Phytologist (2009) 182: 863877

processes as key participants in TC development. Enhanced

Materials and Methods

The Authors (2009)

RNA extraction and cDNA amplification

The Authors (2009)

procedure (Qu et al., 2005). Fragments were visualized on a

New Phytologist (2009) 182: 863877

with gold to a thickness of 20 nm in a sputter-coating unit

New Phytologist (2009) 182: 863877

compared with nanogram amounts retrieved from epidermal

The Authors (2009)

size from 50 bp to 500 bp, from adaxial epidermal cells and

The Authors (2009)

the 112 TDFs showing epidermal-specific, induced expression

New Phytologist (2009) 182: 863877

Cell wall and

The Authors (2009)

Splicing factor Prp8

New Phytologist (2009) 182: 863877

The Authors (2009)

Cell growth, division

AtMUS81; endonuclease/nucleic acid binding

New Phytologist (2009) 182: 863877

Zinc finger, CCHC-type

Cell wall and

The Authors (2009)

New Phytologist (2009) 182: 863877

Fig. 2 Classification of predicted gene

Similar to the cohort of induced genes, promoter analysis

The Authors (2009)

motifs) signalling pathways leading to wall ingrowth induction

New Phytologist (2009) 182: 863877

Fig. 3 Effect of white light, auxin and vesicle

of vesicle formation (Ritzenthaler et al., 2002). Under

New Phytologist (2009) 182: 863877

cotyledons to inductive signal(s). Concurrently there is an

The Authors (2009)

expression events primarily associated with trans-differentiation

The Authors (2009)

to the Aux/IAA gene family of transcriptional regulators

New Phytologist (2009) 182: 863877

to the B1 subgroup. The B1-CDKs drive the G2/M transition

New Phytologist (2009) 182: 863877

fatty acid molecules are oxidized to acetyl-CoA and enter the

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Arabidopsis (Chen et al., 2005). Since large numbers of secretory

The Authors (2009)

Research Council Discovery Project grants DP0556217 and

New Phytologist (2009) 182: 863877

New Phytologist (2009) 182: 863877

Ooi CE, Dell-Angelica EC, Bonifacino JS. 1998. ADP-Ribosylation factor

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