fisiologia

An epidermal-specific ethylene signal cascade regulates
trans-differentiation of transfer cells in Vicia faba
cotyledons
Yuchan Zhou*, Felicity Andriunas*, Christina E. Offler, David W. McCurdy and John W. Patrick
School of Environmental & Life Sciences, The University of Newcastle, Callaghan, NSW 2308, Australia
Author for correspondence:
John W. Patrick
Tel: +61 2 49 21 5712
Email: John.Patrick@newcastle.edu.au
Received: 22 July 2009
Accepted: 1 November 2009
New Phytologist (2010) 185: 931–943
doi: 10.1111/j.1469-8137.2009.03136.x
Key words: ethylene, signal transduction,
transfer cells (TCs), Vicia faba (faba bean),
wall ingrowths.
Summary
• Transfer cells (TCs) trans-differentiate by developing extensive wall ingrowths
that facilitate enhanced plasma membrane transport of nutrients. Signal(s) and sig-
nalling cascades responsible for initiating this trans-differentiation event are poorly
understood. We tested the hypothesis that ethylene functions as a key inductive
signal for wall ingrowth formation in epidermal cells of Vicia faba cotyledons.
• Scanning electron microscopy of epidermal cells monitored their propensity for
wall ingrowth formation. Spatial and temporal expression profiles of ethylene bio-
synthetic enzymes and key elements of ethylene signalling cascades (ethylene
insensitive 3 (EIN3) and ethylene response factors (ERFs)) were determined.
• Wall-ingrowth formation responded positively to manipulation of ethylene bio-
synthesis and perception. It was preceded by a cell-specific burst in ethylene bio-
synthesis accompanied by a co-localized post-translational up-regulation of
VfEIN3-1 and differential expression of three VfERF genes. Blocking ethylene pro-
duction arrested ongoing wall ingrowth development. Wound-induced ethylene in
pod walls and seed coats caused an in planta activation of ethylene biosynthetic
genes in adaxial epidermal cells that coincidentally formed wall ingrowths.
• A cell-specific burst of ethylene biosynthesis functions as an inductive signal ini-
tiating and sustaining trans-differentiation to a TC morphology in vitro. These
events are reproduced for developing V. faba seeds in planta.
Introduction
Transfer cells (TCs) are characterized by wall ingrowths that
protrude into the cytoplasm to form 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). This structural⁄functional organization con-
fers the capacity to support high plasma membrane fluxes of
nutrients at symplasmic ⁄apoplasmic boundaries located at
key nutrient exchange sites, including sites of apoplasmic
phloem loading (e.g. Amiard et al., 2007; Maeda et al.,
2008) and maternal⁄filial interfaces of developing seeds
(Zhang et al., 2007). TCs form exclusively as a result of
trans-differentiation from a range of cell types (Offler et al.,
2003). Despite their key physiological significance in nutri-
ent transport and plant productivity (Offler et al., 2003),
the signal(s) and signalling cascades responsible for initiat-
ing this trans-differentiation event are poorly understood.
In many cases, inductive signal(s) responsible for initiat-
ing TC development appear to be stress-associated. For
instance, TC formation occurs in, or proximal to, plant tis-
sues subjected to wounding caused by invasion of patho-
gens⁄symbionts or to mineral ion deficiencies and excess
salt (see reviews by Thompson et al., 2001; Offler et al.,
2003). More recently, wall ingrowth formation in phloem
parenchyma and companion cells of leaf minor veins was
*These authors contributed equally to this work.
Accession numbers: VfACO1 (EU543653), VfACO2 (EU543654),
VfACS1 (EU543655), VfACS2 (EU543656), VfEIN3-1 (EU543657),
VfEIN3-2 (EU543658), VfERF1 (EU543659), VfERF2 (EU543660)
and VfERF3 (EU543661).
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Ó The Authors (2010)
Journal compilation Ó New Phytologist (2010)
New Phytologist (2010) 185: 931–943 931
www.newphytologist.org

shown to be enhanced by exposure to high light (Amiard
et al., 2007) or cold (Maeda et al., 2008). As trans-differen-
tiation to a TC morphology in various circumstances can be
linked to exposure to biotic and abiotic stress episodes, a
common stress-responsive signal may function as a key
developmental trigger. In this context, ethylene (Schikora
& Schmidt, 2002; Thiel et al., 2008; Dibley et al., 2009)
and jasmonic acid (Amiard et al., 2007) have been impli-
cated in stimulating wall ingrowth formation of rhizoder-
mal, barley (Hordeum vulgare) aleurone, cotyledon
epidermal (ethylene) and phloem parenchyma (jasmonic
acid) TCs. Whether either hormone serves as a primary
inductive signal initiating wall ingrowth development is not
clear. Responses to inhibitors of ethylene biosynthesis or
action suggested a facilitating rather than inductive role for
ethylene in regulating trans-differentiation of rhizodermal
TCs (Schikora & Schmidt, 2002). Similar conclusions were
reached for jasmonic acid whereby the extent of wall
ingrowth formation in phloem parenchyma cells was
increased by treating leaves with methyl jasmonate and was
depressed in jasmonic acid synthesis or signalling-insensitive
mutants of Arabidopsis (Amiard et al., 2007).
Progress in discovering inductive signal(s) and signalling
cascade(s) initiating trans-differentiation to TCs requires a
system that provides experimental access to a large popula-
tion of developmentally uncommitted cells capable of being
induced to undergo synchronous trans-differentiation into a
TC morphology. Adaxial epidermal cells of cultured cotyle-
dons of faba bean (Vicia faba) meet these criteria (Offler
et al., 1997). In planta, unlike their abaxial counterparts,
adaxial epidermal cells of faba bean cotyledons do not form
a TC morphology. However, when isolated cotyledons are
cultured, adaxial epidermal cells are induced to form wall
ingrowths, with ingrowth papillae first appearing within
3 h of excision (Wardini et al., 2007b). Several thousand
adaxial epidermal cells are readily accessible for visualization
and experimental manipulation (Talbot et al., 2001; Dibley
et al., 2009), enabling TC induction to be studied with rel-
ative ease, in a population of normally uncommitted cells
(Offler et al., 1997; Farley et al., 2000). Furthermore, com-
pression of induction to 15 h (Wardini et al., 2007b)
increases developmental synchronicity, allowing temporal
patterns to be followed with confidence. In addition, in vi-
tro formed adaxial epidermal TCs of faba bean cotyledons
mimic their in vivo abaxial counterparts in terms of wall
ingrowth morphology (Talbot et al., 2001) and transport
function (Farley et al., 2000). Thus the faba bean cotyledon
experimental system provides a validated platform for
extrapolation of in vitro observations to developmental pro-
cesses occurring in planta.
This paper reports findings from a series of cell and
molecular studies designed to test the role of ethylene and
ethylene signal cascades in inducing trans-differentiation of
adaxial epidermal cells of cultured faba bean cotyledons into
epidermal TCs. Perturbation of ethylene biosynthesis and
perception indicated that ethylene is required not only for
induction but also for ongoing development of wall in-
growths. Temporal and spatial expression patterns of ethyl-
ene biosynthetic genes correlated with ethylene production
and rates of wall ingrowth induction. Protein abundance
and gene expression profiles of key downstream ethylene
signalling components, ethylene insensitive 3 (VfEIN3-1)
and ethylene response factors (ERFs), respectively, showed
that ethylene-regulated signalling events occur rapidly and
before wall ingrowth induction. Significantly, these events
were spatially restricted to adaxial epidermal cells forming
wall ingrowths. Finally, our hypothesis was tested in planta,
with results indicating that endogenously produced ethylene
is capable of inducing wall ingrowth formation in adaxial
epidermal cells of intact developing cotyledons.
Materials and Methods
Plant growth conditions
Faba bean (Vicia faba L. cv. Fiord) plants were raised ini-
tially under glasshouse conditions before being transferred
to environmentally controlled growth cabinets as described
by Dibley et al. (2009).
Cotyledon culture
Cotyledons isolated from seed coats were cultured asepti-
cally on agar containing Murashige & Skoog (1962) med-
ium (MS; Sigma Australia; and for more details see Dibley
et al., 2009). Sister cotyledon pairs were divided between
media with and without additions, at specified final concen-
trations, of aminoethyoxyvinylglycine (AVG), aminocyclo-
propane carboxylic acid (ACC), silver thiosulphate (STS),
or p-chlorophenoxyisobutyric acid (PCIB). Cultures were
placed in darkness at 22°C or 26°C for 15 h unless speci-
fied otherwise. Slowed induction of wall ingrowths at 22°C
permitted stimulatory effects to be examined (Fig. 2c),
while more rapid induction at 26°C was useful to detect
inhibition of the process.
Scanning electron microscopy
Epidermal peels were removed from the adaxial surface of
each cotyledon and washed in 2% (v⁄v) NaOCl for 3 h at
room temperature (21–22°C) and subsequently dehydrated
at 4°C through a 10% step-graded ethanol⁄dH2O series,
changed at 30-min intervals. Peels were critical-point dried
with liquid CO2 in a critical-point drier (Balzers Union
Ltd, Balzers, Liechtenstein), and held outer face down onto
sticky tabs to reveal the cytoplasmic face of their outer peri-
clinal cell walls. Samples were sputter-coated with gold to a
thickness of 20 nm in a sputter-coating unit (SPI Supplies,
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West Chester, PA, USA), and viewed at 15 kV with a Phi-
lips XL30 SEM. For each epidermal peel, eight regions were
randomly chosen and in each region 20 cells were scored for
the presence⁄absence of wall ingrowths, with wall ingrowth
presence scored irrespective of papilla number per cell. As
there was no evidence of any spatial influence on the
sequence of wall ingrowth induction, this sampling proce-
dure provided representative estimates of the entire cell
population for each peel. Numbers of cells induced to form
wall ingrowths were expressed as a percentage of total cells
scored (150–200 cells for each replicate cotyledon). From
these data, the mean percentage of cells containing wall in-
growths, or the mean percentage of cells within a wall
ingrowth stage of development, was computed.
Measurement of ethylene concentrations
Ethylene concentrations produced by cultured cotyledons
were measured using a gas chromatograph (Varian Star
3400CXB; Varian Inc, San Francisco, CA, USA) equipped
with a flame-ionization detector with a Porapak Q (Chro-
malytic Technology, Boronia, Australia) packed column.
Approximately 50 cotyledons were cultured in sealed Petri
dishes on standard medium ± 100 lM ACC and 100 lM
AVG. At specified times, 1-ml air samples were removed
from each Petri dish using a gastight syringe and injected
immediately into a gas chromatograph for ethylene mea-
surement.
Cloning ACC synthase (ACS), ACC oxidase (ACO),
EIN3 and ERF cDNAs
Total RNA was extracted from epidermal peels, discs of
storage parenchyma or whole cotyledons using the RNeasy
kit followed by on-column DNase digestion (Qiagen) (Dib-
ley et al., 2009). Extracted RNA was reverse-transcribed
with Superscript reverse transcriptase and oligo (dT) (Invi-
trogen). Resulting cDNA was subjected to degenerate
PCR using specified primer sets (Supporting Information
Table S1). About 40 clones were sequenced for each PCR
product. Full-length ACS, EIN3 and ERF sequences from
total RNA were obtained by 5¢ and 3¢ SMARTÔ Rapid
(d)
(a)
(c)
(e) (f)
(b)
250 25
20
15
10
5
0
200
Numberofcellscontaining
wallingrowths(%ofcontrol)
150
Ethyleneproduction
(nll–1
g–1
FW)
100
50
0
ACC ETH AVG STS
**
**
**
**
*
*
Control
Control ACC AVGAVG +
ACC
Fig. 1 Ethylene biosynthesis ⁄ perception and
wall ingrowth induction. (a–d) Scanning elec-
tron microscope (SEM) photomicrographs of
outer periclinal walls of adaxial epidermal
cells of cotyledons of Vicia faba (faba bean)
cultured on standard medium (a, b), or
medium containing 100 lM aminoethyoxy-
vinylglycine (AVG) (c, d). Arrowheads, wall
ingrowth papillae; arrows, starch grains. Bars:
(a, c) 20 lm; (b, d) 2 lm. (e) Percentage
(compared with control) of adaxial epidermal
cells containing wall ingrowths in cotyledons
cultured for 15 h on medium containing
± 100 lM AVG, 100 lM AVG + 100 lM
aminocyclopropane carboxylic acid (ACC),
10 lM silver thiosulphate (STS) or 100 lM
ACC or in the presence of 0.1 ll l)1
ethylene
(ETH). To detect stimulatory effects of ACC
and ETH, cotyledons were cultured at 22°C;
in these cotyledons 45 ± 4.5% of adaxial
epidermal cells contained wall ingrowths at
15 h. For all other treatments, cotyledons
were cultured at 26°C; in these cotyledons
90 ± 1.3% of adaxial epidermal cells
contained wall ingrowths at 15 h. Values
are mean ± SE of at least six replicate
cotyledons. **P < 0.001. (f) Ethylene
production by cotyledons cultured for 15 h
on medium containing ± 100 lM AVG or
100 lM ACC. Values are mean ± SE of three
replicate cotyledon cultures. *P < 0.05.
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Amplification of cDNA ends (RACE) RT-PCR followed by
full-length amplification (Clontech, Mountain View, CA,
USA). Sequences were analysed using sequencher (version
4.1; Gene Codes Corporation, Ann Arbor, MI, USA).
Quantitative real-time PCR
At harvest, cotyledons were immediately immersed in ice-
cold ethanol and acetic acid (3 : 1, v⁄v) and fixed for 1 h.
Epidermal peels and 7-mm-diameter discs of storage paren-
chyma tissues were removed from fixed cotyledons (Dibley
et al., 2009) and stored in liquid nitrogen. Four epidermal
peels or four storage parenchyma discs were pooled for each
total RNA isolation. Total RNA from various tissues,
including cotyledons, seed coats and pod walls, was reverse-
transcribed to cDNA as described in the previous section.
Real-time PCR was performed on a Corbett Research
Rotor-Gene 6000 cycler with the QuantiFast SYBR Green
PCR Kit (Qiagen). Thermocycling was initiated with a
5-min incubation at 95°C, followed by 45 cycles (95°C for
10 s; 60°C for 30 s) with fluorescence acquisition at the
end of each cycle through the green channel (see Table S1
for primer sets). The specificity of amplification was con-
firmed by high-resolution melt curve analysis at the end of
each run. The efficiency of each primer set was evaluated by
producing a standard curve using serial dilutions of plasmid
DNA or cDNA samples containing its amplified regions.
Each reaction was carried out in duplicate (technical
repeat), with nuclease-free water or non-reverse-transcribed
cDNA (RT–) as negative controls. The expression of each
gene was an average of three biological replicates. The tran-
script abundance was normalized to Vicia faba elongation
factor a (VfEFa). In specified cases, relative gene expression
units were presented as the fold change by setting the
expression of one control replicate to a value of 1.
Western blotting
The highly conserved sequence of VfEIN3-1 (Fig. S4)
between residues 185 and 205 (CDPPQRKFPLEKGVPPP
WWPT) was synthesized and the resulting peptide used
to raise antibodies in a rabbit (Mimotopes, Melbourne,
Australia). Polyclonal anti-VfEIN3 was purified by affinity
chromatography.
Total soluble proteins were extracted from cultured
whole cotyledons, epidermal peels and storage parenchyma
discs, and protein concentrations determined by the dot-
METRIC protein assay (Gene Technology, St Louis, MO,
USA). The protein extracts were resolved on 10% SDS-
PAGE gels (15 lg protein per lane except for epidermal
peels with 10 lg protein per lane) and electroblotted onto
nitrocellulose membranes followed by immuno-detection as
described by Tegeder et al. (1999). Blots were probed with
pre-immune serum as negative controls. Colour develop-
ment was performed using an alkaline phosphatase-conju-
gated secondary antibody with Western blue (Promega) as
substrate. Each blot was first probed with anti-VfEIN3 anti-
body, then stripped with 0.2 N glycine, pH 2.5, and re-
probed with anti-actin (Sigma). The ratio of the band
intensity of the EIN3 and that of the corresponding actin
was analysed by quantity one software in the GelDoc sys-
tem (BioRad).
Statistical analyses
The statistical significance of treatment effects or temporal
changes in gene expression was determined using a t-test.
(a)
(b)
(c)
0
1
2
VfACO1 EP
VfACO1 SP
VfACO2 EP
VfACO2 SP
0
0.5
1
1.5
0 5 10 15
Ethylene(nll–1g–1FWh–1)
Culture period (h)
0
5
10
15
Relativeexpression(10–1)
VfACS1 EP
VfACS1 SP
VfACS2 EP
VfACS2 SP
Fig. 2 Vicia faba aminocyclopropane carboxylic acid synthase
(VfACS) and aminocyclopropane carboxylic acid oxidase (VfACO)
expression, ethylene production and transfer cell (TC) induction. (a,
b) Temporal expression profiles of VfACS1 and VfACS2 (a) and
VfACO1 and VfACO2 (b) in cotyledon epidermal (EP) and storage
parenchyma (SP) tissues of Vicia faba (faba bean). Arrows indicate
the 3-h time-point at which wall ingrowths first appear (Wardini
et al., 2007b). Values are mean ± SE of three biological replicates.
(c) Ethylene production rate in cultured cotyledons. Data represent
mean ± SE of three replicate cotyledon cultures; 50 cotyledons per
culture.
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Results
Manipulation of ethylene biosynthesis or perception
affects wall ingrowth formation
To determine if ethylene regulates induction of wall
ingrowths in adaxial epidermal cells of faba bean cotyle-
dons, ethylene biosynthesis and perception were manipu-
lated during cotyledon culture. Induced cells exhibited
densely formed papillate wall ingrowths (Fig. 1a,b), while
noninduced cells contained smooth outer periclinal cell
walls (Fig. 1c). Exposure to the ethylene precursor ACC
elicited a 1.8-fold increase and exposure to ethylene elicited
a 2-fold increase in the percentage of epidermal cells with
wall ingrowths (Fig. 1e). By contrast, treatment with the
ethylene biosynthesis inhibitor AVG suppressed induction
of wall ingrowths 4-fold (Fig. 1e). In those few cells of
AVG-treated cotyledons that contained wall papillae, papil-
lae morphologies were identical to those found in control
cotyledons but their cell densities were much lower (Fig. 1d
vs Fig. 1b). AVG suppression of wall ingrowth formation
was recovered through exposure to ACC (Fig. 1e). Treat-
ment with ACC and AVG significantly enhanced and
reduced, respectively, the rate of ethylene production dur-
ing cotyledon culture (Fig. 1f). The ethylene receptor
antagonist STS suppressed wall ingrowth induction by 2.3-
fold (Fig. 1e), suggesting that the ethylene effects require
ethylene to interact with a receptor. Taken together, these
results suggest that ethylene functions as a positive regulator
of wall ingrowth induction.
Ethylene biosynthesis genes are up-regulated during
transfer cell induction
To identify the enzyme(s) rate-limiting ethylene biosynthe-
sis and its (their) tissue localization, genes encoding ACS
and ACO were isolated from cultured cotyledons and their
temporal and spatial expression patterns determined. Two
ACS genes, VfACS1 and VfACS2 (Fig. S1), and two ACO
genes, VfACO1 and VfACO2 (Fig. S2), were identified.
To determine if temporal and spatial expression profiles
of cloned ethylene biosynthetic genes correlated with
induction of wall ingrowths, real-time PCR was conducted
on cDNA obtained from adaxial epidermal peels and
storage parenchyma discs harvested at various times across
cotyledon culture (Dibley et al., 2009). The VfACS2 tran-
script was rapidly and selectively up-regulated in adaxial
epidermal cells by 1 h, with a 25-fold increase compared
with 0 h and 25-fold higher expression than that of storage
parenchyma tissues (Fig. 2a). An autoregulatory response
appeared to contribute to this rapid increase in levels of
VfACS2 transcript (Fig. S3). Thereafter, VfACS2 expres-
sion gradually declined, although transcript levels at 3 and
15 h were 7-fold greater than at 0 h (Fig. 2a). By contrast,
storage parenchyma expression levels of VfACS2 were much
lower and showed a different temporal pattern to that of
epidermal cells (Fig. 2a). Relative expression of VfACS1
was 105
lower than that of VfACS2 throughout the culture
period and did not change significantly (Fig. 2a).
VfACO2 expression declined 5-fold during the first hour
of culture, but thereafter increased 23-fold in an epidermal-
(a) (b)
(c) (d)
100 3
2
1
0
80
**
*
60
40
Mean%cellsWI
Relativeexpression
20
0
Control ControlPCIB PCIBPCIB +
ACC
ACC
Fig. 3 Interaction between ethylene and
auxin during induction of wall ingrowths
(WIs) in adaxial epidermal cells of Vicia faba
(faba bean). (a, b) Scanning electron micro-
scope (SEM) photomicrographs of outer peri-
clinal walls of adaxial epidermal cells of
cotyledons cultured on medium containing
(a) 100 lM p-chlorophenoxyisobutyric acid
(PCIB) or (b) 100 lM PCIB + 100 lM amino-
cyclopropane carboxylic acid (ACC). Arrow-
heads, wall ingrowth papillae; arrows, starch
grains. Bars, 20 lm. (c) Percentage of adaxial
epidermal cells containing wall ingrowths in
cotyledons cultured for 15 h on medium con-
taining ± 100 lM PCIB, 100 lM ACC, or
100 lM PCIB + 100 lM ACC. All values
represent mean ± SE of six biological repli-
cates (**P < 0.001). (d) Expression of V.
faba ACC synthase 2 (VfACS2) following 1 h
of culture on standard medium or medium
containing 100 lM PCIB. All values represent
mean ± SE of four biological replicates
(*P < 0.02).
New

specific manner (Fig. 2b). By contrast, its expression in stor-
age parenchyma tissues remained relatively stable and low
throughout this period (Fig. 2b). Similar to VfACS1, rela-
tive expression of VfACO1 was extremely low and barely
detectable throughout the entire induction period (Fig. 2b).
Rates of ethylene production correlated with VfACS2
expression, with high rates at 1 h, decreasing to 3 h and
then stabilizing between 3 and 15 h (Fig. 2c). Thus a rapid
burst in ethylene biosynthetic capacity and production
occurs before the appearance of wall ingrowth papillae at
3 h (Fig. 2; Wardini et al., 2007b).
Auxin acts upstream of ethylene and regulates expres-
sion of VfACS2
The question arises as to what induces the spatially specific
and enhanced expression of VfACS2 (Fig. 2a) and hence
ethylene biosynthesis (Fig. 2c). Auxin is a strong candidate
as it has been shown to regulate wall ingrowth induction
(Schmidt & Schikora, 2001; Dibley et al., 2009). Culturing
cotyledons in the presence of PCIB, a competitive inhibitor
of auxin action (Oono et al., 2003), resulted in a 2-fold
reduction in the percentage of epidermal cells with wall in-
growths (Fig. 3a,c). Papilla morphologies were unaltered
but their densities were lower in induced cells treated with
PCIB (Fig. 3a) compared with those of control cotyledons
(Fig. 1a). Including ACC in the culture medium com-
pletely recovered wall ingrowth formation to control levels
in terms of cell numbers induced (Fig. 3c) and papilla
densities and morphologies (Fig. 3b vs Fig. 1a). This result
suggests that auxin acts upstream of ethylene in regulating
wall ingrowth deposition.
Auxin effects on ethylene biosynthetic gene expression
(Swarup et al., 2002) were investigated to determine
whether auxin action elicits the burst in ethylene biosynthe-
sis preceding wall ingrowth induction. VfACS2 was chosen
for expression analysis, as it appears to drive the high rate of
ethylene production observed at 1 h of culture (Fig. 2a,c).
Culturing cotyledons in the presence of PCIB caused a
6-fold reduction in VfACS2 transcript levels in adaxial epi-
dermal cells (Fig. 3d), consistent with auxin inducing
VfACS2 expression in adaxial epidermal cells before induc-
tion of wall ingrowths.
EIN3 accumulated rapidly during TC induction
Temporal and spatial accumulation of downstream ethyl-
ene-signalling components was mapped during induction
of wall ingrowths, which is initiated at 3 h and reaches
completion at 15 h of cotyledon culture (Wardini et al.,
2007b) We first focused on the EIN3 family, key transcrip-
tional regulators in ethylene signalling (Stepanova &
Alonso, 2005). Two genes encoding EIN3⁄EIN3-like (EIL)
proteins, VfEIN3-1 and VfEIN3-2, were identified from
freshly harvested cotyledons (Fig. S4), but only VfEIN3-1
was expressed in cultured cotyledons. Expression of
VfEIN3-1 demonstrated little quantitative change during
early cotyledon culture (0–3 h) but a 4-fold increase in
expression occurred specifically in adaxial epidermal cells
between 15 and 63 h (Fig. 4a).
EIN3 abundance increased significantly in whole cotyle-
dons and adaxial epidermal cells by 3 h but not in storage
parenchyma tissues (Fig. 4b). To demonstrate the regula-
tory effect of ethylene on EIN3, the effect of ACC or AVG
treatments on EIN3 protein abundance was determined
during induction (15 h) and ongoing wall ingrowth devel-
opment (63 h). ACC increased EIN3 protein accumulation
at both 15 and 63 h, while AVG decreased protein accumu-
lation in both cases (Fig. 4c). These results correlate directly
with the response of wall ingrowth formation to ethylene
manipulation (compare Fig. 1e with Fig. 4c).
15-h whole cotyledons 63-h whole cotyledons
EIN3
Actin
EIN3/
actin
EIN3/
actin
(b)
(a)
(c)
Epidermal peels Storage parenchyma
EIN3
0 h
Control ACC AVG Control ACC AVG
7.8 ± 2.0 0.9 ± 0.4 0.3 ± 0.238.4 ± 6.1
7.1 ± 2.0 1.7 ± 0.6 8.2 ± 1.2 35.2 ± 5.7 0.6 ± 0.322.5 ± 3.6
3 h 0 h 3 h
Actin
2
1
Relativeexpression(10–3
)
0
0 20
Culture period (h)
40 60
Fig. 4 Analyses of Vicia faba ethylene insensitive 3 (VfEIN3-1)
transcript and protein abundance. (a) Temporal profile of VfEIN3-1
expression in adaxial epidermal (EP) (closed circles) and storage
parenchyma (SP) tissues (open circles) of Vicia faba (faba bean). (b)
Comparison of EIN3 protein abundance at 0 and 3 h of culture in
peels of epidermal tissue and discs of storage parenchyma tissues.
(c) Effect of aminocyclopropane carboxylic acid (ACC) or amino-
ethyoxyvinylglycine (AVG) on the EIN3 protein abundance in whole
cotyledons cultured for 15 and 63 h. All values represent mean ± SE
of three biological replicates.
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Expression of ERFs changes during wall ingrowth
induction
Direct targets of EIN3 are a large family of transcription
factors termed ERFs (Alonso & Stepanova, 2004). Three
ERF genes, VfERF1–3, containing a single conserved
APETALA2 DNA-binding domain (Ohme-Takagi &
Shinshi, 1995), were cloned from faba bean cotyledons.
Phylogenetic analysis demonstrated that VfERF3, contain-
ing an EAR motif, (L⁄F)DLN(L⁄F)(x)P, at its C-terminus
(Fig. S5a), clustered with predicted Arabidopsis ERF
repressors while VfERF1 and VfERF2 exhibited close
relationships with predicted transcription activators (Ohta
et al., 2001; Fig. S5b).
VfERF1 and VfERF3 expression was significantly and
selectively up-regulated in adaxial epidermal cells before
induction of wall ingrowths at 1 h (Fig. 5a). Thereafter,
transcript levels decreased to 3 h and then remained rela-
tively stable to 15 h (Fig. 5a). This temporal expression pat-
tern directly correlated with that of ethylene production
(Fig. 2c). By contrast, VfERF2 was rapidly and selectively
down-regulated in adaxial epidermal cells at 1 h of culture
while being up-regulated in storage parenchyma tissue
(Fig. 5b). Following this initial change, a slight increase in
expression of VfERF2 was observed to 3 h with no change
thereafter to 15 h for both epidermal and storage paren-
chyma tissues (Fig. 5b).
Ethylene responsiveness of the ERFs was examined by
exposing cotyledons to ACC or AVG during a 1-h culture
period when rates of ethylene production were high
(Fig. 2c). AVG significantly suppressed VfERF1 and VfERF3
expression, but substantially enhanced that of VfERF2
(Fig. 5c). By contrast, ACC up-regulated expression of
VfERF3, but had no significant effect on VfERF1 or VfERF2
expression at 1 h (Fig. 5c). The inability of ACC to further
increase VfERF1 expression or reduce VfERF2 expression at
1 h (Fig. 5c) may be explained by saturating concentrations
of ethylene present in control cotyledons (Fig. 2c).
Ethylene sustains ongoing wall ingrowth development
A large increase in ethylene production (Table 1) coincided
with up-regulated expression of VfACS2 from 15 to 63 h
and maintenance of the high expression level of VfACO2 in
adaxial epidermal cells (Table 1). Similar to the induction
phase (Fig. 2a,b), VfACS1 and VfACO1 expression levels
were extremely low and showed no significant change
between 15 and 63 h (Table 1). Together, these data sug-
gest that a second wave of ethylene production was linked
0
4
8
12
Relativeexpression(10–3
)Relativeexpression(10–2
)Relativeexpression(102
)
VfERF1 EP
VfERF1 SP
VfERF3 EP
VfERF3 SP
(a)
(c)
(b)
0
2
4
6
0 5 10 15
Culture period (h)
VfERF2 EP
VfERF2 SP
0
1
2
3
VfERF1 VfERF2 VfERF3
*
*
*
*
Fig. 5 Expression analyses of Vicia faba ethylene response factors
(VfERFs). (a, b) Expression profile of VfERFs in adaxial epidermal
(EP) and storage parenchyma (SP) tissues of V. faba (faba bean). (c)
Effect of aminocyclopropane carboxylic acid (ACC; black bars) or
aminoethyoxyvinylglycine (AVG; grey bars) treatment on VfERF
expression in epidermal tissues after 1 h of culture (white bars;
control). All values represent mean ± SE of three biological replicates
(*P < 0.05).
Table 1 Ethylene production and ethylene biosynthetic gene
expression during ongoing development of wall ingrowths in Vicia
faba (faba bean )
Culture period (h)
15 63
Relative expression (10)1
)
VfACS1 Epidermal peels 0.09 ± 0.02 0.13 ± 0.02
Storage parenchyma 0.00 ± 0.00 0.00 ± 0.00
VfACS2 Epidermal peels 3.19 ± 0.10 7.24 ± 0.30
VfACO1 Epidermal peels 0.00 ± 0.00 0.00 ± 0.00
VfACO2 Epidermal peels 2.70 ± 0.04 3.05 ± 0.21
Ethylene production
(nl l)1
g)1
FW h)1
)
2.92 ± 0.78 32.38 ± 3.89
All values represent mean ± SE of three biological replicates.
ACS, aminocyclopropane carboxylic acid synthase; ACO, aminocy-
clopropane carboxylic acid oxidase .
New

with ongoing development of wall ingrowths in adaxial epi-
dermal TCs. This possibility was investigated by culturing
cotyledons for 15 h on standard medium before their trans-
fer to medium with or without AVG for a further 48 h of
culture (63 h total). By 15 h, induction of adaxial epider-
mal cells is completed (Wardini et al., 2007b) and those
induced cells had formed discrete wall ingrowth papillae
(Fig. 6a). Further development was restricted to construc-
tion of the wall labyrinth (Fig. 6b) which was slowed in the
presence of AVG (Fig. 6c). Indeed, most AVG-treated cells
(99%), cultured for 63 h, did not progress beyond stage 1
wall ingrowths (Fig 6d; Wardini et al., 2007a), which were
evident for 100% of cells in control cotyledons at 15 h
(Fig. 6e). By contrast, 81% of cells had progressed to stages
2 and 3 in control cotyledons by 63 h of culture (Fig. 6d;
Wardini et al., 2007a), with only 19% of cells containing
stage 1 wall ingrowths (Fig. 6e). Hence, adding AVG at
15 h significantly arrested progressive development of wall
ingrowths.
Wounding induces wall ingrowths in adaxial epidermal
cells of in planta cotyledons
To expose in planta adaxial epidermal cells of cotyledons to
increased ethylene concentrations, pod walls and seed coats
of faba beans were wounded to increase their rates of ethyl-
ene production (e.g. Argueso et al., 2007). As early as 6 h
following wounding, wall ingrowth papillae were observed
in 8% of adaxial epidermal cells (Fig. 7c). Numbers of
induced cells increased gradually, reaching 68% by 4 d fol-
lowing the wounding event (Fig. 7c). At this time, papilla
density within induced cells (Fig. 7a) and papilla morphol-
ogy (Fig. 7b) were comparable to those of cotyledons cul-
tured for 15 h (Fig. 1a,b, respectively).
(a) (b) (c)
Stage 2(d)
(e)
Stage 1
*
*
Stage 3
120
** **
100
80
Mean%cells
60
40
20
0
15 h 63 h 63 h with AVG added at 15 h
Fig. 6 Effect of aminoethyoxyvinylglycine (AVG) on ongoing wall ingrowth development in Vicia faba (faba bean). (a–c) Scanning electron
microscope (SEM) images of outer periclinal cell walls of adaxial epidermal cells of cotyledons cultured on standard medium for 15 h (a) before
being transferred for a further 48 h of culture to either standard medium (b) or medium containing 100 lM AVG (c). Arrowheads, wall
ingrowth papillae; arrow, starch grain. Bar, 10 lm. (d) SEM images of outer periclinal cell walls of adaxial epidermal cells exhibiting defined
stages of wall ingrowth development (Wardini et al., 2007a). Stage 1, wall ingrowths occur as single discrete papillae (arrowheads) that may
be beginning to elongate or fuse (double arrowhead). Stage 2, ingrowths have formed a continuous fused network. Stage 3, ingrowths have
formed a fenestrated layer (*) and may have initiated a second layer of papillae (arrowheads). Bar, 2 lm. (e) Mean percentage of cells exhibit-
ing defined stages of wall ingrowth development (black bars, stage 1; grey bars, stage 2; white bars, stage 3). Cotyledons were cultured on
standard medium for 15 h before being transferred to medium containing ± 100 lM AVG for a further 48 h. All values represent mean ± SE
of six biological replicates (**P < 0.001 for AVG treatment against 63-h controls).
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To establish a link between wounding, ethylene biosynthe-
sis and wall ingrowth induction, temporal and spatial expres-
sion patterns of VfACS1 and VfACS2 were analysed.
Significant up-regulation of VfACS2 expression was detected
in pod walls, seed coats and adaxial epidermal cells of cotyle-
dons but not in cotyledon storage parenchyma tissues
(Fig. 7d,e). In adaxial epidermal cells, up-regulated expres-
sion of VfACS2 occurred at 3 h (Fig. 7d), before the first
appearance of wall ingrowth papillae at 6 h (Fig. 7c). By con-
trast, but similar to cultured cotyledons (Fig. 2a), VfACS1
transcript levels remained very low in adaxial epidermal cells
(Fig. 7d). Interestingly, there was a significant response in
expression of VfACS2 to wounding in seed coats with tran-
script abundance increasing 10-fold by 3 h and quantita-
tively exceeding that in other tissues examined (Fig. 7e).
Discussion
We have used a faba bean cotyledon culture system to test
the role of ethylene as a TC inductive signal. The study was
based on the finding that an enhanced proportion of genes,
selectively induced in cells undergoing trans-differentiation
to a TC morphology, carry ethylene-responsive elements in
promoter regions of orthologous genes (Dibley et al.,
2009). This discussion commences by exploring ethylene as
an inductive signal initiating formation of wall ingrowth
papillae at both cell and molecular levels. Thereafter we
address the question of an ongoing regulatory role for ethyl-
ene in sustaining construction of the wall labyrinth com-
prised of multiple fenestrated layers (Talbot et al., 2001).
Finally, the findings are assembled into a model describing
the in planta generation of an ethylene signal and attendant
signal cascade to initiate and sustain trans-differentiation of
abaxial epidermal cells of faba bean cotyledons to fully func-
tional TCs.
Ethylene induces trans-differentiation of TCs
At the cell level, alterations to TC induction were moni-
tored by determining impacts of specified cotyledon treat-
ments upon relative numbers of their adaxial epidermal
cells forming wall ingrowth papillae (Wardini et al.,
(c)
(e)
(d)
80
60
40
Mean%cellsWIRelativeexpression
Relativeexpression(10–2)
20
0
30
20
10
0
0 3
Time from wounding (h)
6
0 24 48
72 96 0 24
20
VfACS1 EP VfACS1 SP
VfACS2 SPVfACS2 EP
VfACS1 SC VfACS1 PW
VfACS2 PWVfACS2 SC
15
10
5
0
48
72
(a) (b)
Fig. 7 In planta induction of wall ingrowths
(WIs) and expression of Vicia faba aminocy-
clopropane carboxylic acid synthase (VfACS)
genes. (a, b) Scanning electron microscope
(SEM) images of V. faba (faba bean) outer
periclinal cell walls of adaxial epidermal cells
induced to form wall ingrowth papillae
(arrowheads) in planta. (a) Low magnifica-
tion; bar, 20 lm; (b) high magnification; bar,
2 lm. (c) Time course of wall ingrowth for-
mation. Data represent mean ± SE for six
replicate cotyledons. (d) Relative expression
of VfACS1 and VfACS2 in adaxial epidermal
cells (EP) and storage parenchyma (SP) tis-
sue. (e) Relative expression of VfACS1 and
VfACS2 in seed coats (SC) and pod walls
(PW). Data represent mean ± SE of three
biological replicates.
New

2007b). Exposure of cultured cotyledons to elevated doses
of ethylene, directly or indirectly through the ethylene pre-
cursor ACC (Fig. 1f), enhanced induction of wall in-
growths (Fig. 1e). By contrast, slowing ethylene production
by AVG (Fig. 1f) dampened papilla formation and this
inhibitory action could be relieved by ACC (Fig. 1e). Col-
lectively these data are consistent with ethylene rate-limiting
induction of wall ingrowths via binding to an STS-sensitive
receptor (Fig. 1e). These findings are broadly consistent
with those reported for induction of rhizodermal TCs in
tomato (Solanum lycopersicum) (Schikora & Schmidt,
2002).
Temporal and spatial analysis of ethylene production in
cultured cotyledons provided additional support for the
conclusion that ethylene initiates the cascade of selective
gene expression in adaxial epidermal cells leading to their
trans-differentiation into TCs (Dibley et al., 2009). First, a
burst in ethylene production (Fig. 2c) coincided with adax-
ial epidermal cells becoming competent to form wall
ingrowth papillae by 1 h of cotyledon culture (Fig. 2; War-
dini et al., 2007b). Secondly, the burst in ethylene produc-
tion was primarily localized to adaxial epidermal cells which
are the targets for trans-differentiation. This conclusion is
based on using the spatial pattern of VfACS2 expression
(Fig. 2a) as a surrogate measure of ethylene biosynthesis
(Wang et al., 2002) on the grounds that, in this time win-
dow, the temporal profile of ethylene biosynthesis most
strongly correlated with that of VfACS2 expression
(Fig. 2a–c).
The spike in ethylene production acting as the trigger
to induce TC formation is consistent with storage paren-
chyma cells not forming TCs (Offler et al., 1997). This
observation raises the question of what induces the ethyl-
ene burst and localizes it to the adaxial epidermal cells of
cultured cotyledons; a question that focuses on the regu-
lation of VfACS2 expression as argued above. One candi-
date that fulfils this role is auxin. This phytohormone is
well known to induce ethylene biosynthesis (Swarup
et al., 2002) and up-regulate expression of PsACS2 (Peck
& Kende, 1998) and is a key player in driving selective
expression of auxin-responsive genes leading to wall
ingrowth formation in adaxial epidermal cells of faba
bean cotyledons (Dibley et al., 2009). This claim was
supported by the finding that PCIB (Oono et al., 2003)
suppressed wall ingrowth induction (Fig. 3c). The fact
that ACC restored this suppression to control levels
(Fig. 3c) demonstrated that auxin regulation of wall
ingrowth induction occurs through effects on ethylene.
Furthermore, the finding that PCIB inhibited VfACS2
expression at 1 h of culture in adaxial epidermal cells
(Fig. 3d) supports the observation that auxin acts
upstream of ethylene, and plays a role in inducing the
epidermal-specific ethylene burst before wall ingrowth
induction.
An ethylene signalling cascade is localized to and
precedes wall ingrowth formation in adaxial epidermal
cells
Primary transcriptional regulation by ethylene is mediated
through the nuclear localized transcription factor EIN3 or
its paralogues, EIN3-like proteins (EILs; see Alonso &
Stepanova, 2004). The only expressed EIN3⁄EIL gene
detected, VfEIN3-1, displayed little change during early
cotyledon culture (Fig. 4a). However, consistent with a
post-transcriptional regulation of EIN3 (Etheridge et al.,
2005), its protein accumulated rapidly and selectively in
adaxial epidermal cells during the first 3 h of culture
(Fig. 4b). EIN3 protein levels were regulated by ethylene
(Yanagisawa et al., 2003) as demonstrated by their
responses to ACC and AVG (Fig. 4c). Thus, it is likely that
this spatial pattern of EIN3 accumulation is driven by dif-
ferential exposure of epidermal and storage parenchyma
cells to the ethylene burst before wall ingrowth formation
(Fig. 2a).
Downstream gene expression is regulated by EIN3⁄EILs
family members specifically binding to a primary ethylene
response element (PERE) sequence in promoter regions of
ERFs (Solano et al., 1998). In this context, three ERFs,
VfERF1–3 (Fig. S5), exhibited significant expression
changes (VfERF1 and VfERF3 were up-regulated and
VfERF2 was down-regulated) specifically in adaxial epider-
mal cells before wall ingrowth formation (Fig. 5a,b). These
expression events were likely to be driven by ethylene as
they coincided with the ethylene burst at 1 h of culture
(Figs 2c, 5a,b) and responded to ACC and AVG treatments
(Fig. 5c). Because of the rapid response of VfERF expres-
sion to ethylene (Fig. 5c), VfERFs are likely to be EIN3 tar-
gets (Solano et al., 1998). Therefore, preceding wall
ingrowth induction in adaxial epidermal cells, ethylene dif-
ferentially regulated activator-type (VfERF1 and VfERF2)
and repressor-type (VfERF3) ERFs (Fig. S5), providing fur-
ther evidence that an epidermal-specific ethylene signalling
cascade is activated before induction of wall ingrowths.
Moreover, such a proposal is consistent with the high fre-
quency of an ethylene response element (GCC-box; Bu¨tt-
ner & Singh, 1997) in promoters of orthologous genes
selectively up-regulated in adaxial epidermal cells (Dibley
et al., 2009).
Together, the results of our study demonstrate that ethyl-
ene activates key components of the ethylene-signalling cas-
cade not only within the timeframe of TC induction but
specifically in cells forming wall ingrowths. Here the pattern
of ethylene biosynthesis correlated temporally and spatially
with EIN3 accumulation and ERF expression profiles. Fur-
thermore, ERFs may in turn regulate expression of suites of
genes required to orchestrate wall ingrowth deposition.
Consistent with this conclusion is a recent finding that
genes encoding enzymes for ethylene metabolism and ethyl-
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ene responsive transcription factors are up-regulated in bar-
ley aleurone TCs (Thiel et al., 2008).
Ongoing wall ingrowth development depends upon
the continued presence of ethylene
Development of the fenestrated layers of a reticulate wall
labyrinth (Talbot et al., 2001; Fig. 6d) is a continuous pro-
cess involving ongoing deposition of new wall ingrowths.
Therefore it is plausible that an inductive signal is required
across the entire wall-building process, as found for cotton
(Gossypium hirsutum) fibre elongation (Shi et al., 2006) and
tracheary element differentiation (Church & Galston,
1988). This proposition is supported by the finding that
progressive wall ingrowth development was arrested by
AVG (Fig. 6e).
Consistent with the above findings was ongoing expres-
sion of ethylene biosynthetic genes accompanied by ethyl-
ene production. The rise in ethylene production between
15 and 63 h of culture was probably regulated by ACO
expression (Zhong & Burns, 2003), as indicated by the
presence of high VfACO2 transcript levels (Table 1). Signif-
icantly, this high gene expression was confined to adaxial
epidermal cells (Table 1), indicating a localized action of
ethylene to sustain wall ingrowth development. Further-
more, the downstream ethylene-signalling component,
EIN3, responded to ethylene during this period (Fig. 4c),
in part through substantially up-regulated expression of
VfEIN3-1 in adaxial epidermal cells (Fig. 4a). Thus, ethyl-
ene and its signalling cascade up to EIN3 are required not
only for induction of wall ingrowth papillae but also for
constructing the wall labyrinth (Fig. 6).
In planta model for induction of TCs
As culture-induced adaxial epidermal cells of faba bean cot-
yledons are morphologically (Talbot et al., 2001) and func-
tionally (Farley et al., 2000) comparable to their in planta
formed abaxial epidermal counterparts, findings from cul-
tured cotyledons can be used with some confidence to
inform events linked to in planta induction of abaxial epi-
dermal TCs. This assertion is strengthened substantially by
the demonstration that wounding of pod walls and seed
coats resulted in trans-differentiation of normally uncom-
mitted adaxial epidermal cells to a TC morphology in
planta (Fig. 7c). Wounding caused up-regulated expression
of VfACS2 in the seed coat (Fig. 7e) and hence presumably
raised endogenous ethylene concentrations (e.g. Argueso
et al., 2007). Significantly, spatial and temporal expression
patterns of VfACS2 in cotyledons mimicked those observed
in vitro (cf. Fig. 7d with Fig. 2a). Increased expression of
VfACS2 in adaxial epidermal cells of cotyledons must have
resulted from autoregulated expression (Chang et al., 2008;
Fig. S3) induced by ethylene emanating from the wounded
pod walls and seed coats.
The above findings provide insights into the possible
sequence of events leading to the abaxial epidermal cells
of cotyledons being induced to undergo TC development
in planta (Offler et al., 1997). Here, wall ingrowth forma-
tion coincides with the expanding cotyledon surface con-
tacting and crushing the innermost cell layers of the
abutting seed coat (Harrington et al., 1997). Sensitivity of
VfACS2 expression in seed coats to wounding (Fig. 7e)
suggests that crushing of their innermost cell layers
in planta (Harrington et al., 1997) could cause a spike in
ethylene production (Fig. 8). In turn, seed-coat-derived
ethylene might drive an accompanying ethylene burst in
the opposing abaxial epidermal cells (Fig. 8) through
autoregulated expression of VfACS2 in these cells
(Fig. S3). This latter burst in ethylene production may set
in train a signalling cascade through the EIN3 pathway
leading to an up-regulated cohort of ERFs orchestrating
selective expression of a gene compliment responsible for
constructing wall ingrowths (Fig. 8) as deduced from our
in vitro observations (Figs 1,2,4,5).
Acknowledgements
We thank Kevin Stokes for raising healthy experimental
material and acknowledge funding of this project from Aus-
tralian Research Council Discovery Project grants
DP0556217 and DP0664626. F.A. is supported by an Aus-
tralian Postgraduate Award.
Fig. 8 Model of ethylene induction of wall ingrowth formation in
abaxial epidermal cells of Vicia faba (faba bean) cotyledons. Sc, seed
coat; Cot, cotyledon; ETR, ethylene receptor.
New

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Supporting Information
Additional supporting information may be found in the
online version of this article.
Fig. S1 Alignment of deduced Vicia faba aminocyclo-
propane carboxylic acid synthase (VfACS) amino acid
sequences with pea (Pisum sativum) ACS.
Fig. S2 Alignment of deduced Vicia faba aminocyclopropane
carboxylic acid oxidase (VfACO) amino acid sequences with
pea (Pisum sativum) and Medicagotruncatula ACO.
Fig. S3 Effect of aminoethyoxyvinylglycine (AVG) on Vicia
faba aminocyclopropane carboxylic acid synthase 2
(VfACS2) expression in adaxial epidermal cells.
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Fig. S4 Alignment of deduced Vicia faba ethylene insensi-
tive 3 (VfEIN3) amino acid sequences with Arabidopsis
EIN3⁄EIN3-like (EIL) proteins.
Fig. S5 Sequence comparison and phylogenetic relation-
ships of Vicia faba ethylene response factors (VfERFs).
Table S1 Primer sequences used in this study
Please note: Wiley-Blackwell are not responsible for the
content or functionality of any supporting information sup-
plied by the authors. Any queries (other than about missing
material) should be directed to the New Phytologist Central
Ofﬁce.
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