Research
© The Authors (2009) New Phytologist (2009) 182: 863–877
863 Journal compilation © New
Phytologist (2009) www.newphytologist.org
863
BlackwellPublishingLtdOxford, UKNPHNew
Phytologist0028-646X1469-8137© The Authors
(2009).Journalcompilation© New Phytologist
(2009)282210.1111/j.1469-8137.2009.02822.xMarch200900863???877???OriginalArticle XX
XX
Early gene expression programs accompanying trans-differentiation
of epidermal cells of Vicia faba cotyledons into transfer
cells
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
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.
Email:
[email protected]
Key words: cDNA-AFLP, trans- differentiation, transfer cells, Vicia
faba, wall ingrowths.
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
include four defensin-like genes, BETL1-4 (Thompson et
al ., 2001), a novel cell wall-related protein, MEG1
(Thompson et al ., 2001; Gutiérrez-Marcos etal ., 2004),
and a transcriptional activator, ZmMRP-1 (Gómez et al .,
2002). The transcriptional activator has been shown to activate
BETL and MEG1 promoters (Gutiérrez-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
(Muñiz et al ., 2006). While these studies provide
important insights, our wider understanding of the molecular
processes underlying TC development remains poor.
www.newphytologist.org Journal compilation © New
Phytologist (2009)
Research864
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 trans - differentiate 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 cDNA-
amplified 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
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
etal ., 2001). At harvest, cotyledons of 80–120 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 4°C 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.
Journal compilation © New Phytologist (2009)
www.newphytologist.org
Research 865
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 phenol–chloroform 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-α
(VfGAPDH1-FP/RP and VfEF1α-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 Mse I 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 streptavidin- coated
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 94°C for 10 min followed by 13 cycles of 94°C for 30
s, 65°C for 30 s and 72°C for 1 min, with the annealing
temperature dropping by 0.6°C each cycle. The reactions were
completed by 23 cycles of 94°C for 30 s, 56°C for 30 s and 72°C 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
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 µl pipette tip and incubated
in 15 µl of 10 mm Tris-HCl (pH 8.0) for 30 min at room
temperature. Each fragment was reamplified using 5 µl of the
fragment extract as template and subjected to the selective PCR
cycle program with an additional seven cycles at an annealing
temperature of 56°C. 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 (O’Conner 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-α (VfEF1-α ) 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 4°C through a 10% step-graded ethanol–distilled 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
www.newphytologist.org Journal compilation © New
Phytologist (2009)
Research866
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 µg ml−1
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) p- phenylenediamine. Tissue
was viewed with a Zeiss Axiophot epifluorescence microscope
equipped with a 50 W short-arc mercury lamp and a UV (365–420 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 µg 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
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
Number (%) of TDFs
s p e c
i f i c
Induced 69 (15) Late-Induced 22 (5) Early Transient-Induced 21 (4)
Up-Regulated 30 (6) Rapidly Switched-Off 102 (22) Gradually
Switched-Off 20 (4)
E p
a n
p a r e
n c
h y m a
Induced 116 (25) Late-Induced 13 (3) Early Transient-Induced 19 (4)
Up-Regulated 19 (4) Rapidly Switched-Off 26 (6) Gradually
Switched-Off 14 (3)
Total number of TDFs 471 (100)
Journal compilation © New Phytologist (2009)
www.newphytologist.org
Research 867
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 Mse I (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 cDNA- AFLP 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 epidermal- specific 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 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 ≤ 10–3) 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 (O’Conner et al ., 2005) for the presence of
auxin- and ethylene-regulatory cis -element sequences. Of the
48 Arabi- dopsis 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, respec- tively, found by searching all
promoter regions in the Arabi- dopsis 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 . 150–400 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).
Journal compilation © New Phytologist (2009)
www.newphytologist.org
Research 871
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 cDNA- AFLP 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
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
www.newphytologist.org Journal compilation © New
Phytologist (2009)
Research872
of vesicle formation (Ritzenthaler et al ., 2002). Under
these conditions, wall ingrowth formation was abolished (93%
inhibition; Fig. 3e), demonstrating an absolute require- ment 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 trans - differentiation 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
Journal compilation © New Phytologist (2009)
www.newphytologist.org
Research 873
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
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
www.newphytologist.org Journal compilation © New
Phytologist (2009)
Research874
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 twoβ-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 β-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 ADP-
ribosylation 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.
Journal compilation © New Phytologist (2009)
www.newphytologist.org
Research 875
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 β-N - acetylhexosaminidase (Table 3),
UDP-glucosyltransferase and pectin methylesterase inhibitor (Table
2). Induction of a V- ATPase (Table 2) in the trans-Golgi
network could increase synthesis of cell wall components and
trafficking to the membrane (Brüx 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. fabacotyledon
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
(Röckel et al ., 2008). Therefore, late induction of PMEI
(Table 2) suggests a role in maintaining exten- sibility 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
Research Council Discovery Project grants DP0556217 and
DP0664626.
References
Allen T, Ingles PJ, Praekelt U, Smith H, Whitelam GC.
2006.
Phytochrome-mediated agravitropism in Arabidopsis hypocotyls
requires
GIL1 and confers a fitness advantage. Plant Journal 46:
641–648.
Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller
W,
Lipman DJ. 1997. Gapped blast and psi-blast: a new generation
of
protein database search programs. Nucleic Acids Research 25:
3389–3402.
Amiard V, Much KE, Demmig-Adams B, Ebbert V, Turgeon R,
Adams
WWI. 2005. Anatomical and photosynthetic acclimation to
the light
environment in species with differing mechanisms of phloem
loading.
Proceedings of the National Academy of Sciences, USA 102:
12968–12973.
Bachem CWB, Oomen RJFJ, Visser RGF. 1998. Transcript imaging
with
cDNA-AFLP: a step-by-step protocol. Plant Molecular Biology
Reporter 16:
157–173.
Barrero C, Royo J, Grijota-Martinez C, Faye C, Paul W, Sanz S,
Steinbiss
H-H, Hueros G. 2009. The promoter of ZmMRP-1, a maize
transfer
cell-specific transcriptional activator, is induced at solute
exchange surfaces
and responds to transport demands. Planta 229:
235–247.
Borisjuk L, Weber H, Panitz R, Manteuffel R, Wobus U. 1995.
Embryogenesis of Vicia faba L.: histodifferentiation in relation to
starch
and storage protein synthesis. Journal of Plant
Physiology 147: 203–218.
Boudolf V, Barrôco R, Engler JDA, Verkest A, Beeckman T, Nandts
M,
Inzé D, De Veylder L. 2004. B1-type cyclin-dependent kinases
are
essential for the formation of stomatal complexes in
Arabidopsis thaliana . Plant Cell 16:
945–955.
Brüx A, Liu T-Y, Krebs M, Stierhof Y-D, Lohmann JU, Miersch
O,
Wasternack C, Schumacher K. 2008. Reduced V-ATPase activity
in the
trans -Golgi network causes oxylipin-dependent hypocotyl
growth
inhibition in Arabidopsis . Plant Cell 20:
1088–1100.
Busov VB, Johannes E, Whetten RW, Sederoff RR, Spiker SL,
Lanz-Garcia
C, Goldfarb B. 2004. An auxin-inducible gene from loblolly
pine (Pinus taeda L.) is differentially expressed in mature and
juvenile-phase shoots and
encodes a putative transmembrane protein. Planta 218:
916–927.
Chen Y, Shin YK, Bassham DC. 2005. YKT6 is a core constituent
of
membrane fusion machineries at the Arabidopsis trans -Golgi
network.
Journal of Molecular Biology 350: 92–101.
Datta S, Hettiarachchi GHCM, Deng X-W, Holm M. 2006.
Arabidopsis CONSTANS-LIKE3 is a positive regulator of red
light signaling and root
growth. Plant Cell 18: 70–84.
Demura T, Tashiro G, Horiguchi G, Kishimoto N, Kubo M, Matsuoka
N,
Minami A, Nagata-Hiwatashi M, Nakamura K, Okamurua Y et
al .
2002. Visualization by comprehensive microarray analysis of
gene
expression programs during transdifferentiation of mesophyll cells
into
xylem cells. Proceedings of the National Academy of Sciences, USA
99:
15794–15799.
Doxey AC, Yaish MW, Moffatt BA, Griffith M, McConkey BJ.
2007.
Functional divergence in the Arabidopsis β-1,3-glucanase gene
family
inferred by phylogenetic reconstruction of expression
states. Molecular Biology & Evolution 24:
1045–1055.
Eastmond PJ. 2006. SUGAR-DEPENDENT1encodes a patatin domain
triacylglycerol lipase that initiates storage oil breakdown in
germinating
Arabidopsis seeds. Plant Cell 18: 665–675.
Farley SJ, Patrick JW, Offler CE. 2000. Functional transfer cells
differentiate
in cultured cotyledons of Vicia faba L. seeds.
Protoplasma 214: 102–117.
Francis D. 2007. The plant cell cycle – 15 years on.New
Phytologist 174:
261–278.
Gómez E, Royo J, Guo Y, Thompson R, Hueros G. 2002. Establishment
of
cereal endosperm expression domains: identification and properties
of a
maize transfer cell-specific transcription factor, ZmMRP-1.
Plant Cell 14:
599–610.
www.newphytologist.org Journal compilation © New
Phytologist (2009)
Research876
Guilfoyle TJ, Hagan G. 2007. Auxin response factors. Current
Opinion in Plant Biology 10: 453–460.
Guilfoyle TJ, Hagen G, Li Y, Ulmasov T, Liu ZB, Strabala T, Gee M.
1993.
Auxin-regulated transcription. Australian Journal of
Plant Physiology 20:
489–502.
Gutiérrez-Marcos JF, Costa LM, Biderre-Petit C, Khbaya B,
O’Sullivan
DM, Wormwald M, Perez P, Dickinson HG. 2004. maternally expressed
gene1 is a novel maize endosperm transfer cell-specific gene
with a maternal
parent-of-origin pattern of expression. Plant Cell 16:
1288–1301.
Haritatos E, Medville R, Turgeon R. 2000. Minor vein structure and
sugar
transport in Arabidopsis thaliana .
Planta 211: 105–111.
Harrington GN, Nussbaumer Y, Wang X-D, Tegeder M, Franceschi
VR,
Frommer WB, Patrick JW, Offler CE. 1997. Spatial and temporal
expression of sucrose transport-related genes in developing
cotyledons of
Vicia faba L. Protoplasma 200: 35–50.
Hertzberg M, Sievertzon M, Aspeborg H, Nilsson P, Sandberg G,
Lundeburg J. 2001. cDNA microarray analysis of small plant
tissue
samples using a cDNA tag target amplification protocol. Plant
Journal 25:
585–591.
Howell KA, Cheng K, Murcha MW, Jenkin LE, Millar H, Whelan J.
2007.
Oxygen initiation of respiration and mitochondrial biogenesis in
rice.
Journal of Biological Chemistry 282:
15619–15631.
Hussain H, Mant A, Seale R, Zeeman S, Hinchliffe E, Edwards
A,
Hylton C, Bornemann S, Smith AM, Martin C et al . 2003.
Three
isoforms of isoamylase contribute different catalytic properties
for the
debranching of potato glucans. Plant Cell 15:
133–149.
Matheson LA, Suri SS, Hanton SL, Chatre L, Brandizzi F. 2008.
Correct
targeting of plant ARF GTPases relies on distinct protein domains.
Traffic 9: 103–120.
Memon AR. 2004. The role of ADP-ribosylation factor and SAR1
in
vesicular trafficking in plants. Biochimica et Biophysica
Acta 1664:
9–30.
Milioni D, Sado P-E, Stacey NJ, Roberts K, McCann MC. 2002. Early
gene
expression associated with the commitment and differentiation of a
plant
tracheary element is revealed by cDNA-amplified fragment
length
polymorphism analysis. Plant Cell 14: 2813–2824.
Miller KD, Stommer J, Taylor LP. 2002. Conservation in
divergent
solanaceous species of the unique gene structure and enzyme
activity of a
gametophytically-expressed flavonol
3-O -galactosyltransferase. Plant Molecular
Biology 48: 233–242.
Minic Z, Jouanin L. 2006. Plant glycoside hydrolases involved in
cell
wall polysaccharide degradation. Plant Physiology &
Biochemistry 44: 435–449.
Muñiz LM, Royo J, Gómez E, Barrero C, Bergareche D, Hueros G.
2006.
The maize transfer cell-specific type-A response
regulator ZmTCRR-1 appears to be involved in intercellular
signalling. Plant Journal 48:
17–27.
Murphy AS, Bandyopadhyay A, Holstein SE, Peer WA. 2005.
Endocytotic cycling of PM proteins. Annual Review of Plant
Biology 56:
221–251.
Nakazono M, Qiu F, Borsuk LA, Schnable PS. 2003.
Laser-capture
microdissection, a tool for the global analysis of gene expression
in specific
plant cell types: identification of genes expressed differentially
in epidermal
cells or vascular tissues of maize. Plant Cell 15:
583–596.
O’Conner TR, Dyreson C, Wyrick JJ. 2005. Athena: a resource
for rapid
visualization and systematic analysis of Arabidopsis promoter
sequences.
Bioinformatics 21: 4411–4413.
Offler CE, Liet E, Sutton EG. 1997. Transfer cell induction in
cotyledons
of Vicia faba L. Protoplasma 200: 51–64.
Offler CE, McCurdy DW, Patrick JW, Talbot MJ. 2003. Transfer
cells:
cells specialized for a special purpose. Annual Review of
Plant Biology 54:
431–454.
proteins that interact with an ethylene-responsive element. Plant
Cell 7:
173–182.
1 (ARF1) regulates recruitment of the AP-3 adaptor complex to
membranes. Journal of Cell Biology 142:
391–402.
Oono Y, Ooura C, Rahman A, Aspuria ET, Hayashi K, Tanaka A,
Uchimiya H. 2003. p-Chlorophenoxyisobutyric acid impairs
auxin
response in arabidopsis root. Plant Physiology 133:
1135–1147.
Peer WA, Murphy AS. 2007. Flavonoids and auxin transport:
modulators or
regulators? Trends in Plant Science 12: 556–563.
Pelloux J, Rustérucci C, Mellerowicz EJ. 2007. New insights into
pectin
methylesterase structure and function. Trends in Plant
Science 12:
267–277.
Pruitt RE, Vielle-Calzada J-P, Ploense SE, Grossniklaus U, Lolle
SJ. 2000.
FIDDLEHEAD , a gene required to suppress epidermal cell
interactions in
Arabidopsis , encodes a putative lipid biosynthetic
enzyme. Proceedings of the National Academy of Sciences,
USA 97: 1311–1316.
Qu LJ, Li XY, Wu GQ, Yang N. 2005. Efficient and sensitive method
of
DNA silver staining in polyacrylamide gels.
Electrophoresis 26: 99–101.
Rigoulet M, Aguilaniu H, Avéret N, Bunoust O, Camougrand N,
Grandier-Vazeille X, Larsson C, Pahlman I-L, Manon S, Gustafsson
L.
2004. Organization and regulation of the cytosolic NADH metabolism
in
the yeast Saccharomyces cerevisiae . Molecular and
Cellular Biochemistry 256–257: 73–81.
Ritzenthaler C, Nebenführ A, Movafeghi A, Stussi-Garaud C, Behnia
L,
Pimpl P, Staehelin LA, Robinson DG. 2002. Reevaluation of the
effects
of brefeldin A on plant cells using tobacco bright yellow 2 cells
expressing
Golgi-targeted green fluorescent protein and COPI antisera. Plant
Cell 14:
237–261.
Röckel N, Wolf S, Kost B, Rausch T, Greiner S. 2008. Elaborate
spatial
patterning of cell-wall PME and PMEI at the pollen tube tip
involves
PMEI endocytosis, and reflects the distribution of esterified
and
de-esterified pectins. Plant Journal 53: 133–143.
Rolletschek H, Weber H, Borisjuk L. 2003. Energy status and its
control on
embryogenesis of legumes. Embryo photosynthesis contributes to
oxygen
supply and is coupled to biosynthetic fluxes. Plant
Physiology 132:
1196–1206.
Ruggieri R, Tanaka K, Nakafuku M, Kaziro Y, Toh-EA, Matsumoto
K.
1989. MSI1, a negative regulator of the RAS-cAMP pathway in
Saccharomyces cerevisiae . Proceedings of the National Academy
of Sciences, USA 86: 8778–8782.
Schikora A, Schmidt W. 2001. Acclimative changes in root
epidermal cell
fate in response to Fe and P deficiency: a specific role for
auxin?
Protoplasma 218: 67–75.
Schikora A, Schmidt W. 2002. Formation of transfer cells and
H+-ATPase
expression in tomato roots under P and Fe deficiency.
Planta 215:
304–311.
Smith AP, Nourizadeh SD, Peer WA, Xu J, Bandyopadhyay A, Murphy
AS,
Goldsbrough PB. 2003. Arabidopsis AtGSTF2 is regulated by
ethylene and
auxin, and encodes a glutathione S-transferase that interacts
with
flavonoids. Plant Journal 36: 433–442.
Suh MC, Samuels AL, Jetter R, Kunst L, Pollard M, Ohlrogge J,
Beisson F.
2005. Cuticular lipid composition, surface structure, and gene
expression
in Arabidopsis stem epidermis. Plant Physiology 139:
1649–1665.
Talbot MJ, Franceschi VR, McCurdy DW, Offler CE. 2001. Wall
ingrowth
architecture in epidermal transfer cells of Vicia faba
cotyledons.
Protoplasma 215: 191–203.
Talbot MJ, Wasteneys GO, Offler CE, McCurdy DW. 2007.
Cellulose
synthesis is required for deposition of reticulate wall ingrowths
in transfer
cells. Plant & Cell Physiology 48: 147–158.
Tepperman JM, Hudson ME, Khanna R, Zhu T, Chang SH, Wang X,
Quail PH. 2004. Expression profiling of phyB mutant
demonstrates
substantial contribution of other phytochromes to
red-light-regulated
gene expression during seedling de-etiolation. Plant
Journal 38:
725–739.
Thompson RD, Hueros G, Becker HA, Maitz M. 2001. Development
and
Journal compilation © New Phytologist (2009)
www.newphytologist.org
Research 877
Vaughn KC, Talbot MJ, Offler CE, McCurdy DW. 2007. Wall
ingrowths
in epidermal transfer cells of Vicia faba cotyledons are modified
primary
walls marked by localized accumulations of arabinogalactan
proteins.
Plant & Cell Physiology 48: 159–168.
Vorwerk S, Biernacki S, Hillebrand H, Janzik I, Müller A,
Weiler EW,
Piotrowski M. 2001. Enzymatic characterization of the
recombinant
Arabidopsis thaliana nitrilase subfamily encoded by the NIT
2/NIT 1/NIT 3-gene cluster. Planta 212: 508–516.
Wardini T, Talbot MJ, Offler CE, Patrick JW. 2007a. Role of
sugars in
regulating transfer cell development in cotyledons of developing
Vicia faba seeds. Protoplasma 230: 75–88.
Wardini T, Wang X-D, Offler CE, Patrick JW. 2007b. Induction
of wall
ingrowths of transfer cells occurs rapidly and depends upon
gene
expression in cotyledons of developing Vicia faba seeds.
Protoplasma 231:
15–23.
FACTOR 1 function on epidermal cell polarity. Plant
Cell 17:
525–536.
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
Please note: Wiley-Blackwell are not responsible for the content or
functionality of any supporting information supplied by the
authors. Any queries (other than missing material) should be
directed to the New Phytologist Central Office.
About New Phytologist
• New Phytologist is owned by a non-profit-making
charitable trust dedicated to the promotion of plant
science, facilitating projects from symposia to open access for our
Tansley reviews. Complete information is available at
www.newphytologist.org .
• Regular papers, Letters, Research reviews, Rapid reports and both
Modelling/Theory and Methods papers are encouraged. We are
committed to rapid processing, from online submission through to
publication ‘as-ready’ via Early View – our average submission
to decision time is just 29 days. Online-only colour is free, and
essential print colour costs will be met if necessary. We also
provide 25 offpr ints as well as a PDF for each article.
• For online summaries and ToC alerts, go to the website and click
on ‘Journal online’. You can take out a personal
subscription to the journal for a fraction of the
institutional price. Rates start at £139 in Europe/$259 in the USA
& Canada for the online edition (click on ‘Subscribe’ at the
website).