Study of uptake of cell penetrating peptides and their
cargoes in permeabilized wheat immature embryos
Archana Chugh and Franc¸ois Eudes
Lethbridge Research Centre, Agriculture and Agri-Food Canada, Alberta, Canada
Cell-penetrating peptides (CPPs) comprise a fast grow-
ing class of short length peptides that differ in
sequence, size and charge but share a common charac-
teristic ability to translocate across the plasma mem-
brane. It has been demonstrated that CPPs can act
efficiently as nonviral delivery vehicles for macromole-
cules that are much larger in size than their own and
lack the self-potential to enter living cells due to the
Keywords
cell membrane permeabilization;
cell-penetrating peptide; endocytosis;
macropinocytosis; nanocarrier
Correspondence
F. Eudes, Lethbridge Research Centre,
Agriculture and Agri-Food Canada, PO Box
3000, 5403 1st Avenue South, Lethbridge,
Alberta T1J 4B1, Canada
Fax: +1 403 382 3156
Tel: +1 403 317 3338
E-mail:
(Received 16 November 2007, revised 8
February 2008, accepted 3 March 2008)
doi:10.1111/j.1742-4658.2008.06384.x
The uptake of five fluorescein labeled cell-penetrating peptides (Tat, Tat
2
,
mutated-Tat, peptide vascular endothelial-cadherin and transportan) was

microscopy. Permeabilized embryos transfected with Tat
2
–plasmid DNA
complex showed 3.3-fold higher transient GUS gene expression than the
nonpermeabilized embryos. Furthermore, addition of cationic transfecting
agent LipofectamineÔ 2000 to the Tat
2
–plasmid DNA complex resulted in
1.5-fold higher transient GUS gene expression in the embryos. This is the
first report demonstrating translocation of various cell-penetrating peptides
and their potential to deliver macromolecules in wheat immature embryos
in the presence of a cell membrane permeabilizing agent.
Abbreviations
AID, arginine-rich intracellular delivery; CPP, cell-penetrating peptide; EIPA, 5-(N-ethyl N-isopropyl) amirolide; b-GUS, b-glucuronidase;
M-Tat, mutated-Tat; pVEC, peptide vascular endothelial-cadherin.
FEBS Journal 275 (2008) 2403–2414 ª 2008 Her Majesty the Queen in Right of Canada, as represented by the Minister of Agriculture and Agri-Food Canada
Journal compilation ª 2008 FEBS
2403
permeability barrier posed by the plasma membrane
[1,2]. A range of molecules, including some of the larg-
est known proteins, oligonucleotides and plasmids,
have been delivered in the cells in their bioactive form
by CPPs [3–7]. Low cytotoxicity, high cargo delivery
efficiency and versatility to undergo diverse modifica-
tions without losing their translocation property make
CPPs an attractive tool for the intracellular delivery of
therapeutic molecules [8]. However, even though many
milestones have been achieved in this relatively new
field of CPP-mediated macromolecule delivery, the
mechanism of cell entry of CPPs alone or with their

ionic oligopeptides such as polyarginine have been
shown to deliver dsRNA to induce post-transcriptional
gene silencing in tobacco suspension cells [30]. AID
has been reported to deliver plasmid DNA in plant
root cells [31].
In the present study, wheat zygotic immature
embryos were chosen as the system for investigation
because they are an important tissue in which to
study various biochemical processes during seed
development. They also serve as a model tissue for
genetic transformation studies owing to their amena-
bility towards tissue culture procedures and a high
efficacy for plant regeneration. We investigated the
uptake of five fluorescently labeled CPPs [Tat (49–
57), Tat
2
, mutated-Tat (M-Tat), pVEC, transportan]
in wheat immature embryos. We demonstrate that
permeabilization of immature embryos is a prerequi-
site to achieve efficient translocation of CPPs and
their macromolecular cargoes. Further investigations
show that nonlabeled Tat monomer (Tat) and dimer
(Tat
2
) are able to deliver a large protein-b-glucuroni-
dase (GUS) enzyme more efficiently in permeabilized
embryos than the nonpermeabilized embryos. A com-
mercially available Chariot kit for protein delivery in
mammalian cell lines has also been shown to deliver
GUS enzyme in the immature embryos. M-Tat (with

embryos
The embryos were treated with fluorescently labeled
Tat, Tat
2
, M-Tat, pVEC or transportan. Fluorescence
observed under a fluorescence microscope indicated
that all the tested CPPs showed significantly higher
uptake in the immature embryos treated with permea-
bilizing agent-toluene ⁄ ethanol (1 : 20, v ⁄ v with per-
meabilization buffer) than the nonpermeabilized
embryos (Fig. 1A). Interestingly, the fluorescence
uptake of the Tat monomer was accentuated in the
germ area of the permeabilized immature embryos,
whereas other peptides (Tat
2
, pVEC and transportan)
CPPs and their cargo in wheat A. Chugh and F. Eudes
2404 FEBS Journal 275 (2008) 2403–2414 ª 2008 Her Majesty the Queen in Right of Canada, as represented by the Minister of Agriculture and Agri-Food Canada
Journal compilation ª 2008 FEBS
also showed fluorescence in the scutellum region of
permeabilized embryos.
As revealed by fluorimetric analysis, the effect of cell
permeabilization was most noteable with uptake of
Tat monomer (4.2-fold higher) and dimer (3.1-fold
higher) followed by pVEC (2.9-fold higher) in permea-
bilized embryos (Fig. 1B). Fluorimetric analysis also
indicated that transportan had relatively more penetra-
tion ability in the nonpermeabilized embryos than the
Tat peptides and pVEC; nonetheless, an increase in
uptake of labeled transportan (1.9-fold) was also

sulphate
M-Tat Tat Tat
2
pVEC Transportan
Fig. 1. Fluorescence microscopy showing the increase in the uptake of various fluorescently labeled cell-penetrating peptides in wheat
immature embryos treated with cell permeabilizing agent toluene ⁄ ethanol. (A) Embryos were incubated in permeabilization buffer (pH 7.1)
with fluorescein labeled Tat, Tat
2
, pVEC and transportan for 1 h in the presence or absence toluene ⁄ ethanol (1 : 20, v ⁄ v with permeabiliza-
tion buffer). Control: no treatment; negative controls: FITC-dextran sulfate (nonpeptidic in nature, molecular mass = 4 kDa) and M-Tat (first
arginine of HIV-1 Tat basic domain is substituted by an alanine). Ge, germ; Sc, scutellum area of wheat immature embryos. (B) Fluorimetric
analysis showing relative fluorescence uptake of various labeled CPPs in the presence and absence of cell permeabilizing agent toluene ⁄
ethanol (1 : 20, v ⁄ v with permeabilization buffer, pH 7.1).
Table 1. List of CPPs employed in the present study. FI, fluoresceination at the N-terminal amino group.
Peptide Sequence
Peptide
length Reference
Tat
a
FI-RKKRRQRRR-amide 9 [50]
Tat
2
FI-RKKRRQRRRRKKRRQRRR-amide 18 [26,51]
M-Tat FI-AKKRRQRRR-amide 9 [51]
Transportan
b
FI-GWTLNSAGYLLGKINLKALAALAKKIL-amide 27 [52]
pVEC
c
FI-LLIILRRRIRKQAHAHSK-amide 18 [53]

2
-mediated GUS enzyme
uptake increased to 92.2% (1.7-fold higher) compared
to 51.8% in nonpermeabilized embryos. Similarly, the
percentage of embryos showing Tat-mediated GUS
enzyme uptake increased from 22.6% to 66.5% (2.9-
fold higher) in the presence of permeabilization agent.
Nonlabeled M-Tat served as a negative control and
demonstrated a significantly lower percentage of
embryos with GUS enzyme activity (35%) even in the
presence of permeabilizing agent.
Chariot, a commercially available cell-penetrating
peptide for transducing proteins in mammalian cell
lines, was also able to deliver GUS enzyme in wheat
immature embryos (Fig. 2A:f,f¢). Chariot–GUS
enzyme complex uptake was 1.3-fold higher in per-
meabilized embryos than the nonpermeabilized
embryos (Fig. 2B).
The negative control, M-Tat, showed a lower inten-
sity of blue colour than the other Tat peptides, indicat-
ing peptide sequence dependent GUS enzyme delivery
in immature embryos. GUS enzyme alone was unable
to translocate efficiently in the immature embryos
(Fig. 2A,B).
a
A
B
b
c d
e

enzyme transduction by commercially available Chariot kit, the manufacturer’s protocol was followed. A GUS histochemical assay was per-
formed after trypsin treatment and washings with permeabilization buffer. Embryos were incubated in GUS histochemical buffer containing
20% methanol [43] for 4–5 h in the dark at 37 °C. (A) Permeabilized embryos treated with Tat monomer (Tat), dimer (Tat
2
) and Chariot–GUS
enzyme complex. (B) The number of embryos showing exogenous GUS enzyme activity also increased with permeabilization treatment.
CPPs and their cargo in wheat A. Chugh and F. Eudes
2406 FEBS Journal 275 (2008) 2403–2414 ª 2008 Her Majesty the Queen in Right of Canada, as represented by the Minister of Agriculture and Agri-Food Canada
Journal compilation ª 2008 FEBS
Effect of inhibitors on Tat
2
-mediated GUS
enzyme delivery in permeabilized embryos
The influence of low temperature, endocytosis and
macropinocytosis inhibitors was evaluated by employ-
ing Tat dimer (Tat
2
) as the carrier peptide because it
had demonstrated the highest GUS enzyme uptake in
the permeabilized immature embryos.
The delivery of Tat
2
–GUS enzyme complex (4 : 1,
w ⁄ w) was distinctly inhibited at low temperatures
(4 °C) in the immature embryos (Fig. 3). Furthermore,
the presence of endocytosis inhibitors (sodium azide
and nocadazole) inhibited the uptake of Tat
2
–GUS
enzyme complex. The cargo complex also failed to

various extents of degradation by the nuclease
(Fig. 4B).
Peptide–DNA complex formation as observed
under confocal laser microscope
Further experiments were conducted to confirm the
optimal ratio of Tat
2
and plasmid DNA for transfect-
ing permeabilized embryos. Fluorescein labeled Tat
2
(green) at different concentrations was incubated with
fixed concentration of rhodamine labeled plasmid
DNA (red) in the ratios of 1 : 1, 2 : 1, 3 : 1, 4 : 1 and
5:1 (w⁄ w). Image merging (yellow) showed that the
optimum ratio for a complex formation was 4 : 1. The
complex size observed at 4 : 1 varied from as small as
0.85 lmupto4lm after 1 h of incubation (Fig. 4C).
Tat
2
-mediated plasmid DNA delivery: transient
GUS gene expression in the immature embryos
Tat
2
–plasmid DNA (pAct-1GUS) complex was pre-
pared at the optimal ratio of 4 : 1 (w ⁄ w) and added to
the immature embryos. In the presence of permeabiliz-
ing agent toluene ⁄ ethanol, transient GUS gene expres-
sion increased from 2.5% to 8.3%. Further addition of
5 lg LipofectamineÔ 2000 (Invitrogen, Gaithersburg,
MD, USA) to the complex enhanced transient GUS

2
–GUS enzyme cargo complex in the presence of macropinocytosis inhibitors (50 lM cytochalasin D and 100 lM EIPA, respectively). +,
blue colour intensity (indicator of GUS enzyme activity); ), no blue colour.
A. Chugh and F. Eudes CPPs and their cargo in wheat
FEBS Journal 275 (2008) 2403–2414 ª 2008 Her Majesty the Queen in Right of Canada, as represented by the Minister of Agriculture and Agri-Food Canada
Journal compilation ª 2008 FEBS
2407
as well as by fluorimetric analysis. In mammalian cells,
reports pertaining to a plasma membrane-mediated
permeability barrier to the Tat basic domain in well
differentiated epithelial cells have emerged [32,33].
Limited intercellular transduction of VP22-GFP full
length proteins in human carcinoma A549 cells, H1299
and monkey Cos-1 cells has been also reported [34].
Tat-eGFP and VP-22 linked N-terminus of diptheria
toxin A fragment have shown restricted translocation
in muscle and Vero cells, respectively [35,36].
a
A
C
B
b c
0.5:1
4:1
3:1
2:1
1:1
5:1
0
0.5:1

complex size was determined using the
IMAGEJ software.
a
A
B
b
0
5
10
15
20
Control
DNA-T/E
DNA
M-Tat+DNA
Tat2+DNA-T/E
Tat2+DNA
Tat2+DNA+LF
Treatment
Transient GUS gene
expression (%)
Fig. 5. Transfection studies using Tat
2
as
carrier peptide for GUS gene delivery in per-
meabilized wheat immature embryos. (A)
GUS histochemical assay showing transient
GUS gene expression in the permeabilized
immature embryos incubated with Tat
2

and a plant glucoside, was ineffective for CPP penetra-
tion in the embryos at the various concentrations
investigated (0.1–2 mgÆL
)1
, data not shown). However,
when the embryos were incubated with labeled CPPs
in the presence of toluene ⁄ ethanol (1 : 20, v ⁄ v with
permeabilization buffer), an inducer of transient pore
formation in plasma membrane, there was remarkable
increase in the fluorescence uptake for labeled Tat pep-
tides. In the mammalian system, penetration of fluores-
cently labeled peptides in Madin–Darby canine kidney
renal epithelial cells and CaCo-2 colonic carcinoma
cells has been achieved by treatment with the cell
membrane permeabilizing agent digitonin and ace-
tone ⁄ methanol [32]. The effect of permeabilization
treatment was most distinct for Tat monomer (Tat)
and dimer (Tat
2
) followed by pVEC and transportan.
Substitution of the first arginine residue by alanine in
M-Tat significantly reduced the internalization effi-
ciency of the peptide, suggesting that differential
uptake of CPPs in the same tissue can be a function of
the sequence and length of the peptide.
Tat peptides (Tat, Tat
2
and M-Tat) were further
chosen as carrier peptide to investigate GUS enzyme
delivery in wheat immature embryos. The permeabili-

that the delivery of GUS enzyme by M-Tat was signifi-
cantly low even in permeabilized embryos, suggesting
that the carrier peptide sequence also plays an impor-
tant role in macromolecular cargo delivery.
We observed that low temperature (4 °C) treatment
of permeabilized embryos resulted in low GUS enzyme
activity, indicating that endocytosis is involved in Tat
2
-
mediated cargo translocation, because temperatures
below 10 °C are known to inhibit endocytosis pathway
in the cells. Recent reports in mammalian cells further
suggest that macropinocytosis may be involved in the
cellular translocation of CPPs [18,21,23,38]. Accord-
ingly, we investigated the effect of endocytosis as well
as macropinocytosis inhibitors on Tat
2
peptide-medi-
ated GUS enzyme delivery in permeabilized embryos.
Both type of inhibitors caused a reduction in GUS
enzyme activity. Several experiments were conducted
to determine which inhibitor reduced the uptake of the
cargo complex by the greatest extent; however, no con-
clusive data emerged that would enable us to deter-
mine the involvement of a specific pathway in the
uptake of the cargo complex in immature embryos.
The macropinocytosis pathway has been suggested as
a mechanism of peptide-fluorescent protein uptake in
root tip cells [29]; however, based on our repeat experi-
ments between endocytosis inhibitors, we observed that

FEBS Journal 275 (2008) 2403–2414 ª 2008 Her Majesty the Queen in Right of Canada, as represented by the Minister of Agriculture and Agri-Food Canada
Journal compilation ª 2008 FEBS
2409
trostatically, resulting in a complex formation that can
be employed for gene delivery in plant cells. The com-
plex size at the optimal ratio (4 : 1) of Tat2 and plas-
mid DNA was in the range 0.85–4 lm. It has been
reported that the size of the peptide–DNA complex
continuously increases with time. Recently, Choi et al.
[41] reported that the initial size of the complex was
0.4 lm and that it can reach up to 26 lm with an
increasing time of incubation of the R15 peptide with
the plasmid DNA expressing b-gal gene. Larger com-
plexes of 6 lm in size resulted in higher b-gal gene
expression in 293T cells than the smaller complexes.
Previous reports have also shown that small complexes
of polyarginine-DNA (1–2 lm) exhibit reduced trans-
fection efficiency in a rat fibroblast cell line than com-
plexes of larger size (10 lm) [42]. Ogris et al. [43]
reported that larger particles (0.03–0.06 lm) of
DNA ⁄ transferrin-PEI complexes showed 100- to
500-fold higher luciferase gene expression in Neuro2A
neuroblastoma cells and erythromyeloid K562 cells
than smaller complex particles (30–60 nm). These stud-
ies indicate that complex size may play an important
role in determining the transfection efficiency of a
polycationic peptide.
Transient GUS gene expression in permeabilized
immature embryos showed that Tat
2

in the delivery of such large sized cargoes in plant cells
involving endocytic ⁄ macropinocytic pathways.
In conclusion, the present study demonstrates many
significant findings with respect to CPP–plant cell
interaction. Besides showing that the permeation bar-
rier for CPP uptake in wheat immature embryos can
be overcome by cell permeabilization, this is the first
report to show that Tat peptide (Tat
2
) can efficiently
deliver large protein as well as plasmid DNA in per-
meabilized wheat immature embryos. Our studies also
suggest diverse applications of CPPs in the area of
plant biotechnology. As the information from plant
genome sequencing projects is constantly growing, sim-
ple and time saving techniques based on CPP-mediated
macromolecule delivery will benefit protein–protein
interaction and gene expression studies in plants
immensely.
Experimental procedures
Isolation and surface sterilization of wheat
immature embryos
Embryos were isolated from spikes 2 weeks post-anthesis
(scutellum diameter 1–2 mm; Triticum aestivum cv. Superb
or Fielder). Immature caryopses were surface sterilised with
70% ethanol for 30 s followed by treatment with 10%
hypochlorite (Clorox, Brompton, Canada) and a drop of
Tween 20 for 3 min, and then washed four times for 1 min
each in sterile water. The embryos were hand dissected
under sterile conditions. Isolated embryos were placed on

fluorescence microscope (GFP filter; excitation
470 nm ⁄ emission 525 nm; Leica Inc., Wetzlar, Germany).
Fluorimetric analysis
The embryos were treated with 4% Triton X-100 (prepared
in permeabilization buffer, pH 7.1) for 30 min, at 4 °C. The
supernatant was collected in a fresh tube and relative fluo-
rescence uptake by the embryos with different CPPs was
estimated by a VersaFluor fluorimeter (excitation
490 nm ⁄ emission 520 nm; Bio-Rad, Hercules, CA, USA).
Preparation and delivery of CPP – GUS enzyme
cargo complex in wheat immature embryos
Tat peptides (Tat, Tat
2
, M-Tat) were employed for deliv-
ery of GUS enzyme in wheat embryos. Tat peptide and
GUS enzyme were first prepared in separate microcentri-
fuge tubes. Nonlabeled Tat peptide (4 lg) was added to
sterile water (with the final volume made up to 100 lL).
Similarly, 1 lg of GUS enzyme (Sigma Aldrich) was
added to sterile water to give a final volume of 100 lL.
The contents of the two tubes were mixed together, giving
a 4 : 1 peptide ⁄ protein ratio in the mixture. The mixture
was incubated for 1 h at room temperature and then
added to the isolated immature embryos (in a 2 mL
microcentrifuge tube) in the presence or absence of per-
meabilizing agent toluene ⁄ ethanol (1 : 40, v ⁄ v with the
total volume of the peptide ⁄ protein mixture). After 1 h of
incubation at room temperature, embryos were washed
twice with the permeabilization buffer and subjected to
trypsin treatment [1 : 1 (v ⁄ v) with permeabilization buffer]

2
–GUS enzyme
complex and permeabilizing agent toluene ⁄ ethanol (1 : 40,
v ⁄ v with enzyme complex mixture) were added to the
embryos followed by incubation period of 1 h at 4 °C. Sim-
ilarly, embryos were pre-incubated with either the endocy-
tosis inhibitors (5 mm sodium azide or 10 lm nocodazole)
or macropinocytosis inhibitors (50 lm cytochalasin D or
100 lm EIPA) followed by treatment with Tat
2
–GUS
enzyme complex in the presence of permeabilizing agent.
Trypsin treatment, washing steps and GUS histochemical
assay were performed as described above.
CPP–plasmid DNA complex uptake by
permeabilized immature embryos
Tat
2
–plasmid DNA complex formation studies
Gel retardation assay
The purified supercoiled plasmid DNA (1 lgofpAct-
1GUS, 7.2 kb) was mixed with different concentrations of
Tat
2
to give ratios of 0.5 : 1, 1 : 1, 2 : 1, 3 : 1, 4 : 1 and
5 : 1 of Tat
2
and plasmid DNA. However, before mixing,
Tat
2

C1+ confocal Nikon Eclipse TE2000U microscope with
epifluorescence; Nikon, Tokyo, Japan). Tat
2
was labeled
with fluorescein (green, excitation wavelength 488 nm) and
DNA was rhodamine labeled (red, excitation wavelength
546 nm; Mirus Label IT, CX-Rhodamine DNA labeling
kit; Mirus, Madison, WI, USA). The images at the different
wavelength were merged (yellow colour) to analyse the
complex formed. The complex size was determined using
imagej software (NIH, Bethesda, MD, USA).
Transfection of permeabilized immature embryos with
Tat
2
–plasmid DNA complex
The complex was prepared at an optimal ratio 4 : 1 (w ⁄ w)
of Tat
2
and plasmid DNA. Tat
2
(20 lg) and plasmid DNA
pAct-1GUS (5 lg) were separately prepared in 100 lLof
sterile water. The two were then mixed by gentle tapping
and incubated for 1 h at room temperature. As an optional
step, 5 lg of LipofectamineÔ 2000 (Invitrogen) was added
to the mixture and the mix incubated for another 30 min
for complex formation. The mixture (total volume of
200 lL) was added to the sterilized embryos along with the
permeabilizing agent (toluene ⁄ ethanol 1 : 40, v ⁄ v with the
mixture). The embryos were incubated with Tat

ing Research Council of Canada (NSERC) for the
award of Visiting Fellowship. The authors acknowl-
edge financial support from Matching Investment Ini-
tiative (MII) program and Alberta Agriculture
Research Institute (AARI).
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