Down-regulation of an ANR gene isolated from white clover that is



Down-regulation of an ANR gene isolated from white clover that is
ANR gene expression in white clover
Graphical abstract
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ANR gene expression in white clover
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Research Article
Spatio-Temporal Profile of the Anthocyanidin Reductase
Gene Expression in White Clover Flowers
Shamila W. Abeynayake1,2,3, Stephen N. Panter1, Nadia Efremova4, Aidyn Mouradov
and German C. Spangenberg1,2
Department of Environment and Primary Industries, Biosciences Research Division, AgriBio,
La Trobe University, Bundoora, Victoria 3083, Australia,
La Trobe University, Bundoora, Victoria, 3083, Australia,
Department of Agroecology, Aarhus University, Forsøgsvej 1, 4200, Slagelse, Denmark
Phytowelt GreenTechnologies GmbH, Stöckheimer Weg 1, D-50829 Köln, Germany
School of Applied Sciences, RMIT University, Bundoora, Victoria 3083, Australia
*Corresponding author: RMIT University, School of Applied Sciences, Bundoora, Victoria
3083, Australia; Tel: 062 3 99257144; Fax: 062 399257100 [email protected]
Proanthocyanidins (PAs) are polymers of flavan 3-ol subunits that are naturally produced by
some plants and ameliorate pasture bloat in livestock. Genes encoding proteins involved in
the biosynthesis, intracellular transport and compartmentalisation of PAs and their
precursors are tightly regulated by transcription factors. In this study, the expression
pattern of an anthocyanidin reductase (ANR) gene was shown to correlate with the
accumulation pattern of PAs and/or their monomers in white clover (Trifolium repens L.).
The sub-cellular localisation patterns of an ANR-GFP fusion protein and proanthocyanidins
and/or monomers are consistent with biosynthesis of flavan 3-ols in the cytoplasm prior to
PA accumulation in the vacuole of cells in flowers. Characterisation of the white clover ANR
gene provides a valuable resource for further work aiming to improve bloat safety in white
clover by elevating PA levels in leaves.
Keywords: condensed tannin, BANYULS, transgenic, in-situ hybridisation
Abbreviations: ANR, anthocyanidin reductase, ER, endoplasmic reticulum, GFP, green
fluorescent protein, LAR, leucoanthocyanidin reductase, PA, proanthocyanidins
ANR gene expression in white clover
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1. Introduction
Flavonoid compounds, including anthocyanins, flavonols and proanthocyanidins, are a large
group of secondary metabolites from plants with a wide range of biological roles (reviewed
by Lepiniec et al. 2006). Proanthocyanidins, or condensed tannins, are bioactive polymers
of flavan 3-ols. Of significant agricultural interest, a threshold level of proanthocyanidins (24% of dry weight) in livestock feed binds to dietary proteins in the rumens of sheep and
cattle, slowing the degradation of protein by gut microbes and the formation of protein
foams that can trap digestive gases (Aerts et al. 1999). This process ameliorates pasture
bloat in sheep and cattle and also reduces the loss of dietary amino acids via conversion to
ammonia in sheep.
The biosynthesis and regulation of proanthocyanidins has been substantiated by
studies of transparent testa (tt) mutants in Arabidopsis that lack oxidised proanthocyanidins
and have seeds that are relatively light in colour (Shirley et al. 1995; Lepiniec et al. 2006).
The proanthocyanidin-specific branch of the flavonoid pathway starts with the conversion of
2,3-flavan 3,4-diols either to anthocyanidins and 2,3-cis-flavan 3-ols by anthocyanidin
synthase (ANS, E.C. and anthocyanidin reductase (ANR, syn BANYULS, E.C. or to 2,3-trans-flavan 3-ols by leucoanthocyanidin reductase (LAR, E.C.
(Abrahams et al. 2003; Tanner et al. 2003; Xie et al. 2003). Anthocyanidins are the branch
point between the biosynthetic pathways leading to production of proanthocyanidins and
anthocyanins, which are generated by O-glycosylation, O-acylation and O-methylation of
anthocyanidins (for a detailed review, see Lepiniec et al. 2006).
An established model for proanthocyanidin biosynthesis, shows that flavan 3-ols are
produced within a complex of flavonoid-pathway enzymes anchored to the cytoplasmic side
of the endoplasmic reticulum and transported to the vacuole by a mechanism involving
several proteins with transport functions or by transport vesicles (for review, see Zhao et al.
2010). Components of the transport machinery for compartmentalisation of flavan 3-ols
include: a glycosyltransferase, a glutathione S-transferase that binds and transports
glycosylated flavan 3-ols across the cytoplasm and a multidrug and toxic compound
extrusion (MATE) transporter coupled to a H+/ATP-ase on the tonoplast to provide active
transport of glycosylated flavan 3-ols into the vacuole. The mechanism for polymerisation of
flavan 3-ols into proanthocyanidins is not well understood, but may involve either laccase
activity or spontaneous condensation within the vacuole (Lepiniec et al. 2006).
The production of proanthocyanidins is spatially restricted and
developmentally controlled at the transcriptional level in many plants, including Arabidopsis,
barrel medic, grapevine and white clover (Lepiniec et al. 2006; Bogs et al. 2005; Pang et al.
2007; Abeynayake et al. 2012). Proanthocyanidin biosynthesis and the subcellular transport
of flavan 3-ols in Arabidopsis have been shown to be regulated by six genes, TT1, TT2, TT8,
TT16 and TTG1. The proteins encoded by three of these genes, TT2 (R2R3-MYB), TT8
(bHLH) and TTG1 (WD40) function as a ternary transcriptional complex regulating the
expression of BANYULS and the tissue- and cell-specific accumulation of proanthocyanidins
(reviewed by Lepiniec et al. 2006). TT2 is expressed only in the innermost cell layer of the
inner seed integument and regulates the expression of DFR, ANS and BANYULS. TT8 also
regulates expression of DFR and BANYULS in siliques, and TTG1 controls BANYULS
ANR gene expression in white clover
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expression, mainly through its effect on the function of TT8. MADS box (TT16), zinc finger
(TT1) and WRKY (TTG2) classes of transcription factors control the expression of BANYULS
indirectly by activating early steps in flavonoid biosynthesis or by regulating the
development of proanthocyanidin-producing cells (reviewed by Lepiniec et al. 2006).
Down-regulation of an ANR gene isolated from white clover that is normally
expressed during early flower development was shown to increase anthocyanin
accumulation, but reduce proanthocyanidin levels in flowers of transgenic white clover
plants (Abeynayake et al. 2012). The current study aimed to characterise this gene further
by identification of putative cis regulatory elements within its promoter and by in situ
hybridization to visualize the pattern of ANR transcript accumulation in floral tissues.
Furthermore, production of the TrANR protein was visualised in specific cells of floral tissues
of transgenic white clover plants expressing a translational fusion between the TrANR
protein and the green fluorescent protein of Aequorea victoria under the control of the
TrANR promoter.
Materials and Methods
Plant growth conditions
White clover plants were vernalised in a controlled growth room for 6 weeks at 5 oC with an
-2 -1
8 h photoperiod and a light intensity of 41+/s at canopy height. Flowering
was then induced in a controlled growth cabinet (Enconair) by growing plants for 4 weeks at
-2 -1
22oC with a 16 hour photoperiod and a light intensity of 240+/-30
s at canopy
Characterisation of the white clover ANR gene
A genomic clone containing TrANR was identified in a custom white clover BAC library by a
PCR-based strategy, using the primers 5`-CACTGCAAAACCACCCACTT-3` and 5`TGCTTGAAACTGAACCCTTCTT-3`. Sequence was obtained from the promoter region of
TrANR using BAC DNA extracted with the Large Construct Kit (QIAGEN) and a primer
walking strategy involving Sanger sequencing. Sequence of the TrANR gene was also
obtained using a GS-20 ‘454’ pyrosequencing strategy according to the manufacturer’s
recommendations and Newbler sequence assembly software, version (Roche).
The genomic sequence of the white clover (Trifolium repens L.) ANR gene (TrANR) has been
deposited in Genbank (accession number GU300807).
Preparation of recombinant plasmids for plant transformation
A 1066 bp PCR fragment containing the coding region of TrANR minus the stop codon was
amplified from a previously-characterised cDNA clone (Sawbridge et al. 2003; Abeynayake
et al. 2012) using the primers 5`-attB1-GCACTAGTGTGTATAAGTTTCTTGG-3` and 5`-attB2ATTCTTCAGTGCCCCCTTAGTCTTA-3`. GATEWAY® cloning was used to insert this PCR
product upstream of and in-frame with the gfp coding region and under the control of an
enhanced CaMV 35S promoter and the CaMV 35S terminator in a plant transformation
ANR gene expression in white clover
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vector. generating p35S:TrANR-GFP (Figure 1). The CaMV 35S promoter in this vector was
replaced by a 1310 bp promoter fragment flanked by AscI and XhoI sites that had been
amplified by PCR from a BAC genomic clone of the white clover ANR gene using the primers
products and the ligation sites in p35S:TrANR-GFP and pTrANR:TrANR-GFP, were verified by
Sanger sequencing (Figure 1). .
Generation of transgenic white clover plants
Transgenic white clover plants (Trifolium repens L. cv Mink) containing pTrANR:TrANR-GFP
were generated by Agrobacterium-mediated transformation using cotyledonary explants and
selection with 50 mg/L kanamycin sulphate (Ding et al. 2003). Transgenic white clover
plants containing a GFP gene, encoding GFP with or without the C-terminal HDEL signal for
retention in the ER, under the control of the CaMV 35S promoter were provided by Emma
Ludlow (Ludlow, 2006 - unpublished data).
Screening of putative transgenic white clover plants
DNA was extracted from leaf tissue of putative transgenic plants using the Wizard DNA
purification kit (Promega) and plants were screened by real-time PCR for the presence of
the npt2 selectable marker gene using the primers 5`-GGCTATGACTGGGCACAACA-3` and
5`-ACCGGACAGGTCGGTCTTG-3` (Dorak et al. 2006). PCR reactions were set up using a
SYBR Green PCR Master Mix (Applied Biosystems), according to the manufacturer’s
thermal cycler (Stratagene) with: 10 mins at 95oC; 40 cycles of 30 s at 95oC, 30 s at 60oC
and 30 s at 72oC; 1 min at 95oC, 30 s at 55oC and 30 s at 95oC.
In-situ hybridisation
Preparation of tissues and in situ hybridisation were performed as previously described, in
order to visualise the pattern of TrANR transcript accumulation within floral tissues of white
clover plants (Efremova et al. 2004). Images were taken under bright–field illumination
using a Zeiss Axiophot microscope equipped with plan-NEOFLURAL objectives, a JVC KY-F70
digital camera and Discus software.
Confocal and epifluorescence microscopy
To examine subcellular localisation of GFP, tissues of transgenic and wild type white clover
plants were hand-sectioned using a scalpel blade and mounted on glass slides in 10% v/v
glycerol and were then examined using a Leica TCS SP2 confocal microscope. For
comparison, Arabidopsis leaves bombarded with the p35S:TrANR-GFP were also examined
after incubation for 24 h on solid MS media (Mathur et al. 2003).
ANR gene expression in white clover
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Sequence characterisation of the TrANR promoter region
In order to identify potential regulatory sequences, the sequence of the putative promoter
region of TrANR obtained from a BAC genomic clone was compared to a public database of
cis-acting promoter elements (
regions identified contained a combination of potential bHLH and MYB-binding sites within 1
kb of the TrANR start codon (Figure 2). The first region, between 340 and 379 bp upstream
of the start codon, contains potential bHLH- and MYB-binding sites located 28 bp apart. The
second region, between 109 and 164 bp upstream of the start codon, contains these two
sites spaced 44 bp apart. Another potential MYB-binding sequence, similar to the highaffinity P-binding site of maize, was found 250 bp upstream of the start codon of TrANR,
between the two MYB/bHLH-binding regions (Grotewold et al. 1994). Other potential ciselements within the TrANR promoter region included a putative LFY-binding site (CCAATGT)
and a number of putative CArG boxes that are targets for MADS-box transcription factors.
TrANR transcripts are present in epidermal cells of immature floral organs in white
Immature, 50% open, and mature inflorescences from white clover plants were divided in
half transversely, allowing flower development to be represented by six stages, the
youngest being the upper half of immature inflorescences (stage 1) and the most developed
being the lower half of mature inflorescences (stage 6). In situ hybridisation was used to
identify cell types of immature white clover flowers in which the TrANR gene is expressed
(Figure 3). The TrANR transcript was not detected in the least developed flowers within
immature inflorescences (stage 1, data not shown). No signal was detected in the negative
control treatment when a sense strand-specific probe was used with stage 2 flowers (Figure
3A). When stage 2 flowers were hybridised to an antisense probe, accumulation of TrANR
transcripts was spatially restricted to epidermal cells of petals and carpels (Figure 3B-E) and
to a lesser extent, to stamens (Figure 3E). Detection of TrANR transcripts in more mature
flowers at stages 3-6 was masked by progressive accumulation of a brown metabolite,
potentially representing oxidised proanthocyanidins and flavan 3-ols within TrANRexpressing cells (Figure 3F-I). Confocal imagines revealed small vesicle-like structures of
between 0.1 and 3 μm in size in the cytoplasm of epidermal cells of fresh petals from
immature flowers (Figure 3J-K).
The TrANR protein is produced in epidermal cells of immature petals, carpels and
PCR analysis showed that five out of the eight white clover lines that were generated as a
result of Agrobacterium-mediated transformation with pTrANR:TrANR-GFP contained the
construct. GFP fluorescence was detected in immature flowers of one of these five
transgenic lines and images of this line were taken using epifluorescence and confocal
scanning laser microscopy (Figure 4). GFP fluorescence was detected only in epidermal cells
of petals, carpels and stamens (Figure 4). GFP fluorescence was initially seen in epidermal
ANR gene expression in white clover
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cells located on the abaxial side of petals and progressed to epidermal cells on the adaxial
side during flower development (Figure 4B). A GFP signal was seen in epidermal cells
located on both the abaxial and adaxial sides of carpels (Figure 4B-C) and in immature
embryos (Figure 4C-D). A mosaic pattern of GFP fluorescence that was similar to the
pattern of proanthocyanidin accumulation detected by DMACA staining was seen in the
epidermal layer of carpels and stamens (Figure 4E-H). Background autofluorescence made it
very difficult to detect GFP fluorescence in trichomes (data not shown).
A reticulate pattern of GFP-fluorescence was seen in optical sections from confocal
images of epidermal cells in floral organs from plants containing pTrANR:TrANR-GFP (Figure
5A-C). The fluorescence pattern seen in transgenic white clover lines ectopically expressing
a C-terminal fusion between GFP and the HDEL signal for retention of proteins in
endoplasmic reticulum membranes (Figure 5D) was similar. When expressed under the
control of a CaMV 35S promoter, GFP fluorescence was not seen in the ER of epidermal cells
of Arabidopsis leaves after particle bombardment (Figure 5E) and was seen in the nucleus
and cytoplasm but not in the ER of cells in transgenic white clover plants (Figure 5F).
Down-regulation of the white clover ANR gene, similar to ANR genes in Arabidopsis and
Medicago truncatula, results in the diversion of intermediates from proanthocyanidin to
anthocyanin production in white clover flowers (Abeynayake et al. 2012). This study showed
that the presence of TrANR transcripts is consistent with the accumulation of
proanthocyanidins and/or their monomers in epidermal cells of petals, stamens, carpels, as
well as in trichomes of sepals in developing white clover flowers. Hence, TrANR is considered
a marker of proanthocyanidin production in white clover, as well as being a target for
engineering bloat safety in this plant (Aerts et al. 1999; Abeynayake et al. 2012).
As in Arabidopsis, proanthocyanidin biosynthesis in white clover is developmentallyregulated, spatially restricted and correlates well with the presence of ANR transcripts. In
Arabidopsis, PA production is restricted to seed coats and appears to be triggered by
fertilisation of flowers. However, PA biosynthesis and TrANR expression are high in
immature flowers of white clover plants and PAs can be detected in both flowers and seed
coats (Figure 3, Abeynayake et al. 2012). Functional analysis of the TrANR promoter, which
contains a similar range of gene regulator binding sites as observed in the promoter of
BANYULS (Debeaujon et al. 2003) showed that gene expression of TrANR is coordinated
spatiotemporally with proanthocyanidin accumulation in white clover flowers (Figures 2, 3
and 4). The presence of a conserved arrangement of MYB- and bHLH-binding motifs in the
TrANR promoter (Figure 2) suggests that this gene, like BANYULS, may be regulated by a
transcription factor complex (Baudry et al. 2004). Recently, Medicago truncatula and
Trifolium arvense genes encoding TT2-like MYB factors have been identified that upregulate
proanthocyanidin production when overexpressed in leaves of transgenic legumes that
otherwise contain only trace levels of proanthocyanidins (Hancock et al. 2012; Verdier et al.
2012). In both cases, enhanced proanthocyanidin production in the transgenic plants was
correlated with ectopic expression of ANR and other genes encoding enzymes required for
proanthocyanidin biosynthesis. Identification of targets for these MYB factors in promoters
ANR gene expression in white clover
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of structural genes, including ANR, and characterisation of the mechanism underlying
asymmetrical pattern of TrANR expression in epidermal layers of floral organs (Figure 3)
would improve our understanding of proanthocyanidin pathway regulation in white clover.
The characterised TrANR promoter region lacks motifs that would be expected to direct
asymmetrical gene expression (eg: Watanabe and Okada, 2003), but trans-activation is still
a possibility.
A widely accepted model for compartmentalisation of flavonoids involves the
organisation of the flavonoid enzymes in metabolons that are anchored to the cytoplasmic
face of the ER membrane (Hrazdina and Wagner, 1985). Confocal imaging of transgenic
white clover flowers expressing a TrANR-GFP fusion construct clearly showed localisation of
the fusion protein within the ER in epidermal cells of petals (Figure 5). Unlike most of the
known ER-targeted proteins, TrANR is predicted to be a soluble protein without known
signal peptides for transport into any cellular compartment, including the ER. Also, transient
expression of MtANR-GFP (Pang et al. 2007) and TrANR-GFP fusion proteins (Figure 5E) in
epidermal cells of Arabidopsis leaves did not result in GFP fluorescence in the ER and this
was consistent with the lack of functional proanthocyanidin biosynthesis machinery. This
suggests that interaction between TrANR and membrane-bound enzymes of the flavonoid
pathway,, such as C4H, is required for localisation of TrANR to the ER (Hrazdina and Wagner
1985) .
In this study, spherical vesicle-like structures were seen in the cytoplasm of
proanthocyanidin-accumulating epidermal cells of petals within immature white clover
flowers (Figure 3J-K). Although biochemical confirmation is needed, the structures might
represent transport vesicles facilitating the movement of flavan 3-ols or proanthocyanidins
from the cytoplasm to the vacuole. Some studies have provided evidence for vesiclemediated transport of proanthocyanidins from the endoplasmic reticulum to the vacuole in
plants (eg: Parham and Kaustinen 1977; Zobel, 1986).
ANR gene expression is thus a useful marker for the presence of proanthocyanidin
biosynthetic machinery in both Arabidopsis and white clover plants. Isolation of specific cell
types that express TrANR and accumulate proanthocyanidins from transgenic plants with
the pTrANR:TrANR-GFP construct should allow co-ordinately regulated genes involved in
proanthocyanidin biosynthesis to be identified. This finding should facilitate metabolic
engineering to reconstitute the proanthocyanidin biosynthesis pathway in white clover
leaves for enhanced bloat safety.
The authors thank Matthew Hayes, Noel Cogan, Daniel Isenegger and Ulrik John for critical
comments on the manuscript and are grateful to Rob Glaisher and Catherine Li from La
Trobe University for technical help with the confocal and electron microscopy and to Megan
McKenzie for GS-20 ‘454’ pyrosequencing of the TrANR BAC clone. The authors would also
like to thank Emma Ludlow for providing transgenic white clover plants containing chimeric
GFP constructs. This work was supported by the Molecular Plant Breeding Cooperative
Research Centre and the Department of Primary Industries, Victoria.
ANR gene expression in white clover
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CaMV 35S
nos CaMV 35S
prom prom
nos TrANR
prom prom
CaMV 35S
Figure 1. Schematic diagram showing the T-DNA regions of transformation vectors
for expression of TrANR-GFP translational fusions
The p35S:TrANR-GFP (A) and pTrANR:TrANR-GFP (B) transformation constructs were
designed to allow the production of a C-terminal translational fusion between the white
clover anthocyanidin reductase protein, encoded by TrANR, and green fluorescent protein
under the control of an enhanced CaMV 35S promoter and a TrANR promoter, respectively.
TrANR prom - Trifolium repens anthocyanidin reductase gene promoter; CaMV 35S prom –
enhanced cauliflower mosaic virus 35S promoter; TrANR – Trifolium repens anthocyanidin
reductase gene coding region; gfp – green fluorescent protein gene coding region; CaMV
35S term – cauliflower mosaic virus 35S terminator; ocs term – octopine synthase gene
terminator; nptII – neomycin phosphotransferase 2 gene coding region; nos prom –
nopaline synthase gene promoter; RB – right border of T-DNA region; LB – left border of TDNA region.
ANR gene expression in white clover
-379 -340
P a g e | 13
-149 -101
Figure 2. Cis-acting elements in the promoter regions of anthocyanidin reductase
Positions of selected MYB and bHLH cis-acting elements are shown relative to the start
codons of the Trifolium repens and Arabidopsis thaliana anthocyanidin reductase genes
(TrANR and BANYULS, respectively). haPBS, high-affinity P-binding site (after Debeaujon et
al. 2003).
ANR gene expression in white clover
P a g e | 14
Figure 3. Cell-specific accumulation of TrANR transcripts in white clover flowers
In-situ hybridisation was used to visualise TrANR transcripts in fixed tissue sections from
floral organs of white clover cv ‘Mink’. Negative control, involving a sense strand-specific
probe and a section through an immature flower (A, stage 2). Transcript accumulation in
carpels (B-C) and petals (D-E) of an immature flower (stage 2). Accumulation of a brown
metabolite in epidermal cells of a petal (F) and a carpel (G-H) and a multicellular trichome
(I) from maturing flowers (stage 5). Vesicles in epidermal cells of petals from fresh flowers
collected at stages 3 (J) and 6 (K), viewed by confocal scanning laser microscopy. Crosssections (A,C,E). Longitudinal sections (B,D, F-K). ab, abaxial side; ad, adaxial side; st,
stamen; ca, carpel; pe, petals; sp, sepal; tr, trichome. Refer to text and Abeynayake et al.
ANR gene expression in white clover
P a g e | 15
Figure 4. Cell-specific production of a TrANR-GFP fusion protein in white clover
A fusion construct containing a translational fusion between the white clover anthocyanidin
reductase (TrANR) coding region and the gfp coding region, under the control of the TrANR
promoter, was expressed in white clover plants.
A longitudinal section of petals from a transgenic plant viewed using bright field conditions
(A) and with a GFP fluorescence filter set (B). Cross-section of a flower from a transgenic
plant viewed with a GFP fluorescence filter set (C). Carpel of a flower from a transgenic
plant viewed with a GFP fluorescence filter set (D-E). Carpel from a non-transgenic flower
stained for the presence of proanthocyanidins and/or 2,3-flavan 3-ols with DMACA (F).
Epidermal cells from a stamen tube of a transgenic plant viewed with a GFP fluorescence
filter set (G). Stamen tube of a non-transgenic plant stained with DMACA (H). ab, abaxial;
ad, adaxial; e, embryo; ov, ovule; pe, petals; sp, sepal.
ANR gene expression in white clover
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Figure 5. Intracellular localisation of a TrANR-GFP fusion protein in white clover
flowers and Arabidopsis leaves
Visualisation of GFP fluorescence by confocal microscopy (single optical sections): GFP
fluorescence in epidermal cells of petals from transgenic white clover plants expressing a
TrANR:TrANR-GFP construct (A-C); petal epidermal cells from a transgenic white clover
plant expressing a 35S:GFP-HDEL construct (D); a leaf epidermal cell from Arabidopsis
thaliana after particle bombardment with a 35S:TrANR-GFP construct (E); GFP fluorescence
in epidermal cells of petals from transgenic white clover plants expressing a 35S:GFP
construct (F). nc –

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