Báo cáo khoa học: Study of uptake of cell penetrating peptides and their cargoes in permeabilized wheat immature embryos pot

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

studied in wheat immature embryos. Interestingly, permeabilization treat-
ment of the embryos with toluene ⁄ ethanol (1 : 20, v ⁄ v with permeabiliza-
tion buffer) resulted in a remarkably higher uptake of cell-penetrating
peptides, whereas nonpermeabilized embryos failed to show significant cell-
penetrating peptide uptake, as observed under fluorescence microscope and
by fluorimetric analysis. Among the cell-penetrating peptides investigated,
Tat monomer (Tat) showed highest fluorescence uptake (4.2-fold greater)
in permeabilized embryos than the nonpermeabilized embryos. On the
other hand, mutated-Tat serving as negative control did not show compa-
rable fluorescence levels even in permeabilized embryos. A glucuronidase
histochemical assay revealed that Tat peptides can efficiently deliver func-
tionally active b-glucuronidase (GUS) enzyme in permeabilized immature
embryos. Tat
2
-mediated GUS enzyme delivery showed the highest number
of embryos with GUS uptake (92.2%) upon permeabilization treatment
with toluene ⁄ ethanol (1 : 40, v ⁄ v with permeabilization buffer) whereas
only 51.8% of nonpermeabilized embryos showed Tat
2
-mediated GUS
uptake. Low temperature, endocytosis and macropinocytosis inhibitors
reduced delivery of the Tat
2
–GUS enzyme cargo complex. The results sug-
gest that more than one mechanism of cell entry is involved simultaneously
in cell-penetrating peptide-cargo uptake in wheat immature embryos. We
also studied Tat
2
-plasmid DNA (carrying Act-1GUS) complex formation
by gel retardation assay, DNaseI protection assay and confocal laser

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

cargoes still remains an enigma. There are reports indi-
cating that cellular translocation of CPPs is energy as
well as endocytosis independent and there is direct
transfer of the peptides through the lipid bilayer by
inverted micelle formation [9–14]. Another report,
however, proposes an energy dependent mechanism of
cell entry of CPPs [15], which may also involve extra-
cellular heparan sulfate and various endocytosis and
macropinocytosis pathways [16–23]. It is also suggested
that classical and nonclassical endocytosis pathways
may be associated simultaneously with CPP transloca-
tion depending upon the biophysical properties of
CPPs and their cargo [7,14,24].
Interestingly, most investigations involving CPPs
have been carried out in mammalian cell lines and
there are only few reports on translocation of CPPs in
plant cells. Penetratin, transportan and peptide vascu-
lar endothelial-cadherin (pVEC) have been shown to
internalize in tobacco suspension derived protoplasts
[25]. We have shown translocation and accumulation
of fluorescently labeled Tat monomer (Tat) and dimer
(Tat
2
) in the nuclei of triticale mesophyll protoplasts
[26]. Labeled pVEC and transportan are also internal-
ized by various plant tissues of triticale seedlings [27].
Macropinocytosis dependent transduction of fluores-
cent proteins by arginine-rich intracellular delivery
(AID) and Tat-protein transduction domain has been
reported in onion and corn root tip cells [28,29]. Cat-

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

substitution of first arginine with an alanine in Tat
basic domain) served as negative control for translo-
cation studies of CPPs alone and CPP-mediated
macromolecule delivery in the embryos. The effect of
low temperature, endocytosis and macropinocytosis
inhibitors was also investigated on Tat
2
–GUS
enzyme cargo delivery in wheat embryos. The com-
plex formation of Tat dimer with plasmid DNA car-
rying the GUS gene using gel retardation assay,
DNaseI protection assay and confocal laser micros-
copy was investigated. Furthermore, transient GUS
gene expression in permeabilized wheat immature
embryos transfected with Tat
2
–plasmid DNA
complex was studied.
Results
In the present study, the uptake of five cell-penetrating
peptides differing in their sequence and length (Tat,
Tat
2
, M-Tat, pVEC, transportan; Table 1) was studied
in wheat immature embryos. The role of cell permeabi-
lizing agent in enhancing the uptake of CPPs alone
and cargo complex in the immature embryos was
evaluated.
Cellular uptake of CPPs is enhanced remarkably
by permeabilization treatment of immature

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

observed in the embryos subjected to permeabilization
treatment. Fluorescence uptake for negative controls
[M-Tat and fluorescein isothiocyanate (FITC)-dextran
sulfate] also doubled in permeabilized embryos
but their relative fluorescence uptake level remained
0
1000
2000
3000
4000
Control
Dextran sulphate
M-Tat
Tat
Tat2
pVEC
Transportan
Cell-penetrating peptide
Relative fluorescence
uptake
Without toluene:ethanol
With toluene:ethanol
a
A
B
g f e d b c
a’ b’ c’ d’ e’ f’ g’
-T/E
+T/E
Control Dextran

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]

a
Source: HIV-1 TAT protein transduction domain (49–57).
b
Source: chimeric peptide including 12 amino acids from the neuropeptide galanin
in the N-terminus connected with Lys13 to 14 amino acids from the wasp venom mastoparan in the C-terminus.
c
Source: derived from
murine vascular endothelial cadherin (amino acids 615–632).
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
2405
significantly lower than the other CPPs investigated
(Fig. 1B).
GUS enzyme delivery by Tat peptides in
immature embryos
Because the uptake of fluorescently labeled peptides
was significantly increased in permeabilized immature
embryos, we further investigated the potential of non-
labeled Tat monomer and dimer to deliver GUS
enzyme in the permeabilized immature embryos. The
embryos were incubated with peptide and GUS
enzyme complex prepared in 4 : 1 ratio (w ⁄ w) in the
presence and absence of cell permeabilizing agent tolu-
ene ⁄ ethanol (1 : 40, v ⁄ v with permeabilization buffer
optimised for cargo delivery). The delivery of the GUS
enzyme increased remarkably in the presence of the
permeabilizing agent (Fig. 2A,B). A GUS histochemi-
cal assay demonsrated that the number of permeabi-
lized embryos showing Tat

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

f
a’ b’ c’ d’ e’ f’
0
20
40
60
80
100
120
Control
Dextran sluphate
GUS only
M-Tat
Tat
Tat2
Chariot
Cell-penetrating peptide
GUS +ve embryos (%)
Without toluene/ethanol
With toluene/ethanol
–T/E
+T/E
Control GUS only M-Tat + GUS Tat + GUS Tat
2
+ GUS Chariot + GUS
Ge
Sc
Fig. 2. Noncovalent GUS enzyme transduction by Tat peptides and Chariot in wheat immature embryos. Peptide–GUS enzyme complex
was prepared at an optimal ratio of 4 : 1 (w ⁄ w) and incubated for 1 h. The embryos were incubated with the peptide–GUS protein complex
for 1 h in the presence and absence of permeabilizing agent toluene ⁄ ethanol (1 : 40, v ⁄ v with permeabilization buffer, pH 7.1). For GUS

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

internalize in permeabilized embryos incubated with
macropinocytosis inhibitors [cytochalasin D (also
mediates many endocytic pathways) and 5-(N-ethyl
N-isopropyl) amirolide (EIPA)] because significantly
low or no blue colour intensity was observed (Fig. 3).
Gel retardation and DNaseI protection assay for
Tat
2
–plasmid DNA complex
The ability of nonlabeled Tat
2
to bind the plasmid
DNA carrying GUS gene was tested at different ratios
while keeping concentration of the plasmid constant
(0.5 : 1, 1 : 1, 2 : 1, 3 : 1, 4 : 1, 5 : 1, w ⁄ w). A gel
retardation assay showed that the mobility shift began
at a 1 : 1 ratio of the Tat
2
and GUS enzyme. The fluo-
rescence diminished at 3 : 1 and higher ratios, indicat-
ing that Tat
2
completely covered the plasmid DNA
(Fig. 4A).
A DNaseI protection assay further showed that
DNA was protected by Tat
2
and, thus, was not
degraded by DNaseI at 3 : 1 and higher ratios
whereas, at lower ratios (0.5 : 1, 1 : 1, 2 : 1), it showed

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

gene expression in permeabilized embryos (12.7%).
Lipofectamine alone did not result in efficient plasmid
DNA delivery in permeabilized embryos (4.8%).
M-Tat serving as the negative control did not deliver
plasmid DNA in permeabilized embryos (Fig. 5).
Discussion
Until recently, CPPs were assumed to possess the
inherent property of translocation across cells in a cell
type-independent manner. However, in the present
study, when wheat immature embryos were incubated
with the fluorescein labeled CPPs, very low fluores-
cence was observed under the fluorescence microscope
–– – ––+++++
FCBA HDE G
Fig. 3. Effect of inhibitors. A GUS histochemical assay showed inhibition of uptake of Tat
2
–GUS enzyme cargo complex in the permeabilized
immature embryos incubated at low temperature (4 °C) or treated with endocytosis or macropinocytosis inhibitors. (A, B) Negative controls,
toluene ⁄ ethanol (1 : 40, v ⁄ v with permeabilization buffer), GUS protein only, respectively. (C) Permeabilized embryos treated with Tat
2
–GUS
protein cargo complex at 4 °C. (D) Permeabilized embryos incubated with Tat
2
–GUS enzyme cargo complex at room temperature with no
inhibitors added to the permeabilization buffer. (E, F) Permeabilized embryos incubated with Tat
2
–GUS enzyme cargo complex in the
presence of endocytosis inhibitors (5 m
M sodium azide and 10 lM nocodazole, respectively). (G, H) Permeabilized embryos incubated with
Tat

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

4:1
3:1
2:1
1:1
5:1
0
Tat
2
-fluorescein pAct-1GUS-rhodamine Merge
Fig. 4. Tat
2
–plasmid DNA complex forma-
tion for DNA transfection studies in permea-
bilized immature embryos. Various
concentrations of nonlabeled Tat
2
were
tested for interaction with purified circular
plasmid Act-1GUS on ethidium bromide
stained 1% agarose gel observed under UV
light. (A) Gel retardation assay. (B) DNaseI
protection assay. (C) Confocal laser micros-
copy; complexes of various sizes (in the
range 0.85–4 lm) were formed when fluo-
rescein labeled Tat
2
and rhodamine labeled
circular pAct-1GUS DNA were incubated for
1 h at the optimum ratio 4 : 1 (w ⁄ w) giving
a yellow colour in the merged image. The

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

–
plasmid DNA (pAct-1GUS) cargo complex.
The complex was prepared by mixing 20 lg
of Tat
2
and 5 lg of plasmid DNA (for further
details, see Experimental procedures). (a)
Control. (b) Permeabilized embryo treated
with Tat
2
–plasmid DNA (pAct-1GUS) cargo
complex. (B) Percentage of embryos show-
ing transient GUS gene expression with dif-
ferent treatments of Tat
2
and plasmid DNA.
The treatment was carried out in permeabi-
lized embryos unless otherwise noted. LF,
LipofectamineÔ 2000. The percentage of
transient GUS gene expression was calcu-
lated as the number of embryos showing
transient GUS gene expression ⁄ total
number of embryos in a treatment · 100.
CPPs and their cargo in wheat A. Chugh and F. Eudes
2408 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
We speculated that the use of membrane permeabi-
lizing agents such as saponin and toluene ⁄ ethanol may
aid the cellular entry of CPPs into wheat immature
embryos. We observed that saponin, a mild detergent

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-

zation treatment of the immature embryos not only
increased the number of embryos carrying GUS
enzyme, but also the intensity of the blue color was
distinctly higher than the nonpermeabilized embryos.
A commercially available Chariot kit (pep-1 as the car-
rier peptide) for protein delivery in mammalian cell
lines delivered the GUS enzyme in wheat embryos effi-
ciently. The Chariot kit has been also used for the
direct delivery of bacterial avirulence proteins into
resistant Arabidopsis protoplasts (single wall-less plant
cells), resulting in hypersensitive cell death in a gene
for gene specific manner [37].
Chang et al. [29] have demonstrated noncovalent
transduction of 27 kDa fluorescent proteins by Tat-
protein transduction domain and AID proteins in corn
and onion root tip cells. In the present study, we dem-
onstrate that, in permeabilized wheat embryos, low
molecular mass Tat peptides (1.37–2.7 kDa) have the
potential of a noncovalently transducing macromole-
cule (GUS enzyme, molecular mass = 270 kDa) that
is 100-fold greater than their own size. The results also
indicate that CPPs can deliver GUS enzyme in its
functionally active form in plant tissue. In the present
study, Tat
2
showed maximum efficiency for GUS
enzyme delivery in permeabilized embryos. Our previ-
ous studies have also demonstrated a higher perme-
ation potential of Tat dimer than the monomer in
triticale mesophyll protoplasts [26]. We also observed

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

sodium azide was a more effective inhibitor than noco-
dazole, whereas EIPA (a specific macropinocytic inhib-
itor) showed greater inhibition of GUS uptake in the
embryos than cytochalasin D (an inhibitor for both
endocytosis ⁄ macropinocytosis). Our results strongly
indicate that more than one mechanism is involved
simultaneously in the uptake of the cargo complex in
plant cells and may involve both endocytosis and
macropinocytosis pathways. We also observed that
uptake of Tat
2
alone (labeled) in permeabilized
embryos was not inhibited in the presence of any
endocytosis ⁄ macropinocytosis inhibitor (data not pre-
sented), suggesting that endocytosis is not involved in
the translocation of CPPs alone. In our previous stud-
ies, triticale mesophyll protoplasts treated with various
labeled CPPs indicated direct translocation of peptides
in plant cells. Investigations in mammalian cell lines
have also shown that translocation of CPPs alone may
involve entirely different mechanism(s) of entry than
their macromolecular cargo complexes [14,39,40].
Besides GUS enzyme delivery, the efficacy of Tat
2
peptide in delivering plasmid DNA carrying the GUS
gene was studied in permeabilized wheat embryos.
Being arginine rich, Tat
2
can bind anionic DNA elec-
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
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

can efficiently
deliver plasmid DNA in plant cells. GUS gene
expression was further enhanced by the addition of
cationic transfecting agent LipofectamineÔ 2000.
Very recently, AID-mediated delivery of plasmid was
demonstrated in mungbean and soyabean root cells.
In the mammalian system, Tat and its branched ver-
sions have been shown to deliver plasmid DNA in
various cell lines [44–47]. Oligomers of Tat peptides
in combination with cationic transfecting agents have
also shown enhanced transfection efficiency in human
bronchoepithelial cells [48].
We noted that there were no cytotoxic effects (indi-
cated by embryo germination) of CPPs alone or of their
cargo on embryos. Similarly, previous studies do not
indicate any cytotoxic effects of CPPs on plant cells
[26–29], and the developed technique of CPP-mediated
gene delivery in the present study holds tremendous
potential for the genetic manipulation of crop plants.
Permeabilization treatment, however, reduces the ger-
mination of immature embryos by 3–5%.
To extend Tat-mediated gene delivery on a larger
scale and for stable genetic engineering in plants, it
may be important to gain insight into various factors
such as the optimum peptide ⁄ DNA concentration and
complex size required, the release of DNA from the
complex in the cellular milieu and its further fate in
plant cells. Future investigations will focus on answer-
ing questions such as how the combined use of cell per-
meabilizing agent and cell-penetrating peptides results

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

germination medium [49] for 24 h in the dark at room tem-
perature prior to CPP translocation studies.
Peptide synthesis and fluorophore labelling
Peptides (Table 1) [26,50–53] were custom synthesised and
fluoresceinated at the N-terminal amino group (Alberta
Peptide Institute, Edmonton, Canada). FITC-dextran sulfate
(4 kDa; Sigma Aldrich, Oakville, Canada) served as a non-
peptidic negative control in the initial experiments.
Translocation of fluorescein labeled cell
penetrating peptides in zygotic embryos using
cell membrane permeabilizing agent
Isolated and sterilized embryos (15–20) were imbibed
in total volume of 420 lL of permeabilization buffer, as
previously described [54] (15 mm sodium chloride, 1.5 mm
sodium citrate, pH 7.1) containing cellular permeabilizing
agent toluene ⁄ ethanol (1 : 4, v ⁄ v) in 1 : 20 ratio (v ⁄ v) with
CPPs and their cargo in wheat A. Chugh and F. Eudes
2410 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
permeabilization buffer. To this, 400 lLof5lm fluorescein
labeled CPP was added. After incubation for 1 h in the
dark at room temperature, embryos were washed twice with
the permeabilization buffer followed by treatment with
trypsin: EDTA (0.25% solution; Sigma-Aldrich) (1 : 1, v ⁄ v
with permeabilization buffer) for 5 min at room tempera-
ture. Embryos were washed two to three times with per-
meabilization buffer and subsequently used either for
fluorescence microscopy or fluorimetric analysis.
Fluorescence microscopy
For visual fluorescence, the embryos were observed under

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]

for 5 min at room temperature. The embryos were washed
twice with permeabilization buffer followed by GUS histo-
chemical analysis of the embryos.
For delivery of 1 lg of GUS enzyme by the Chariot pro-
tein transduction kit (Active Motif, Carlsbad, CA, USA),
the manufacturer’s protocol was followed. Permeabilized
and nonpermeabilized embryos were incubated with Char-
iot–GUS complex for 1 h. All post-incubation steps were
the same as that described for Tat peptides.
GUS histochemical assay
For GUS histochemical analysis, embryos were incubated
in GUS buffer as described previously [54] (500 mm
NaH
2
PO4, 100 mm EDTA, 0.3 m mannitol, 2 mm X-gluc,
pH 7.0) at 37 °C in the dark for 4–5 h. Methanol (20%)
was added to suppress endogenous GUS expression, if
any.
The percentage of number of embryos showing GUS
enzyme activity was calculated as the number of embryos
showing GUS enzyme activity (blue colour) as observed
under the light microscope ⁄ total number of embryos trea-
ted with peptide–enzyme complex · 100.
Effect of inhibitors on delivery of Tat
2
–GUS enzyme
complex
For low temperature treatment, immature embryos were
pre-incubated at 4 °C in permeabilization buffer (pH 7.1)
for 45 min. The buffer was removed and Tat

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

and the DNA were prepared separately in 25 lLof
sterile water. The mixture was incubated for 1 h for com-
plex formation and subjected to electrophoresis on 1% aga-
rose gel stained with ethidium bromide (10 lL aliquot of
each ratio was loaded along with 200 ng of pure plasmid
DNA).
DNaseI protection assay
Tat
2
–plasmid DNA complex was prepared as described for
the gel retardation assay. For DNaseI assay, 5 lL of DNa-
seI (RNase-Free DNase set; Qiagen, Valancia, CA, USA)
was added to the mixture volume (50 lL) and incubated at
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
2411
room temperature for 15 min and then incubated on ice for
5 min. Plasmid–peptide dissociation and plasmid purifica-
tion was carried out with a commercially available DNA
purification kit (QIAquick PCR purification kit; Qiagene).
DNA was eluted in sterile water. An aliquot of 6 lL was
subjected to 1% agarose gel electrophoresis.
Confocal laser microscopy
The complex was prepared as described for gel retardation
and DNaseI protection assay except that fluorescently
labeled Tat
2
and DNA were employed to observe the com-
plex formation under the confocal laser microscope (Nikon

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

2
–plasmid
DNA complex for 1 h at room temperature followed by
two washings with permeabilization buffer (pH 7.1). The
embryos were plated on germination medium containing
250 lgÆmL
)1
cefotaxime at 25 °C in the dark for 3–4 days.
Transient GUS gene expression was studied by incubat-
ing the immature embryos in GUS histochemical buffer as
described above. The percentage of embryos showing tran-
sient GUS gene expression was calculated as the number of
treated embryos expressing GUS activity (blue colour)
as observed under the light microscope ⁄ total number of
treated embryos · 100.
Acknowledgements
Eric Amundsen raised wheat plants in the growth
chamber and skillfully hand dissected immature
embryos for the experiments. His help is greatly appre-
ciated. The Alberta Peptide Institute provided high
quality custom synthesised peptides. We are thankful
to Dr Fran Leggett (Lethbridge Research Centre) for
her excellent help with confocal laser microscopy and
size determination of the peptide–DNA complex.
We also acknowledge the support of Doug Bray
(University of Lethbridge) for use of the microscope
facility at the Canadian Centre for Behavioural Neuro-
science (CCBN, University of Lethbridge). We thank
Dr Ray Wu (Cornell University) for plasmid pAct-
1GUS. A.C. thanks the Natural Science and Engineer-

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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2414 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
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