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

Tat2-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 buf

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: eudesf@agr.gc.ca

(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, Tat2, 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 Tat2-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 Tat2-mediated GUS uptake Low temperature, endocytosis and macropinocytosis inhibitors reduced delivery of the Tat2–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 Tat2-plasmid DNA (carrying Act-1GUS) complex formation

by gel retardation assay, DNaseI protection assay and confocal laser microscopy Permeabilized embryos transfected with Tat2–plasmid DNA complex showed 3.3-fold higher transient GUS gene expression than the nonpermeabilized embryos Furthermore, addition of cationic transfecting agent Lipofectamine 2000 to the Tat2–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.

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

(Tat2) 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), Tat2, 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 (Tat2) 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 Tat2–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 Tat2–plasmid DNA complex was studied

Results

In the present study, the uptake of five cell-penetrating peptides differing in their sequence and length (Tat, Tat2, 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, Tat2, 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 (Tat2, pVEC and transportan)

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

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

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

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.cSource: derived from murine vascular endothelial cadherin (amino acids 615–632).

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 Tat2-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

a’ b’ c’ d’ e’ f’

0

20

40

60

80

100

120

Control Dextran sluphate

GUS only

Cell-penetrating peptide

Without toluene/ethanol With toluene/ethanol

–T/E

+T/E

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.

Effect of inhibitors on Tat2-mediated GUS

enzyme delivery in permeabilized embryos

The influence of low temperature, endocytosis and

macropinocytosis inhibitors was evaluated by

employ-ing Tat dimer (Tat2) as the carrier peptide because it

had demonstrated the highest GUS enzyme uptake in

the permeabilized immature embryos

The delivery of Tat2–GUS enzyme complex (4 : 1,

w⁄ w) was distinctly inhibited at low temperatures

(4C) in the immature embryos (Fig 3) Furthermore,

the presence of endocytosis inhibitors (sodium azide

and nocadazole) inhibited the uptake of Tat2–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

Tat2–plasmid DNA complex

The ability of nonlabeled Tat2 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 Tat2and GUS enzyme The

fluo-rescence diminished at 3 : 1 and higher ratios,

indicat-ing that Tat2 completely covered the plasmid DNA

(Fig 4A)

A DNaseI protection assay further showed that

DNA was protected by Tat2 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 Tat2 and plasmid DNA for transfect-ing permeabilized embryos Fluorescein labeled Tat2

(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 lm up to 4 lm after 1 h of incubation (Fig 4C)

Tat2-mediated plasmid DNA delivery: transient GUS gene expression in the immature embryos Tat2–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 Lipofectamine 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 fluoresfluores-cence microscope – – + +++ – + – –

F C

B

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 Tat2–GUS enzyme cargo complex in the presence of endocytosis inhibitors (5 m M sodium azide and 10 l M nocodazole, respectively) (G, H) Permeabilized embryos incubated with Tat 2 –GUS enzyme cargo complex in the presence of macropinocytosis inhibitors (50 l M cytochalasin D and 100 l M EIPA, respectively) +, blue colour intensity (indicator of GUS enzyme activity); ), no blue colour.

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

Tat2-fluorescein pAct-1GUS-rhodamine Merge

Fig 4 Tat 2 –plasmid DNA complex forma-tion for DNA transfecforma-tion 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 Tat2and 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

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 Tat2– plasmid DNA (pAct-1GUS) cargo complex The complex was prepared by mixing 20 lg

of Tat2and 5 lg of plasmid DNA (for further details, see Experimental procedures) (a) Control (b) Permeabilized embryo treated with Tat2–plasmid DNA (pAct-1GUS) cargo complex (B) Percentage of embryos show-ing transient GUS gene expression with dif-ferent treatments of Tat2and plasmid DNA The treatment was carried out in permeabi-lized embryos unless otherwise noted LF, Lipofectamine 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.

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 concentrapenetra-tions

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 (Tat2) 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, Tat2 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 Tat-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, Tat2 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 (4C) treatment

of permeabilized embryos resulted in low GUS enzyme activity, indicating that endocytosis is involved in Tat2 -mediated cargo translocation, because temperatures below 10C 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 Tat2 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 Tat2 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 Tat2

peptide in delivering plasmid DNA carrying the GUS gene was studied in permeabilized wheat embryos Being arginine rich, Tat2 can bind anionic DNA

elec-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 Tat2 can efficiently

deliver plasmid DNA in plant cells GUS gene

expression was further enhanced by the addition of

cationic transfecting agent Lipofectamine 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 (Tat2) 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

permeabilization buffer To this, 400 lL of 5 lm 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 4C 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, Tat2, 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 NaH2PO4, 100 mm EDTA, 0.3 m mannitol, 2 mm X-gluc,

pH 7.0) at 37C 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 Tat2–GUS enzyme complex

For low temperature treatment, immature embryos were pre-incubated at 4C in permeabilization buffer (pH 7.1) for 45 min The buffer was removed and Tat2–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 4C 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 Tat2–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 Tat2–plasmid DNA complex formation studies Gel retardation assay

The purified supercoiled plasmid DNA (1 lg of pAct-1GUS, 7.2 kb) was mixed with different concentrations of Tat2 to give ratios of 0.5 : 1, 1 : 1, 2 : 1, 3 : 1, 4 : 1 and

5 : 1 of Tat2 and plasmid DNA However, before mixing, Tat2 and the DNA were prepared separately in 25 lL of 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 Tat2–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

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 Tat2and 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) Tat2 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

imagejsoftware (NIH, Bethesda, MD, USA)

Transfection of permeabilized immature embryos with

Tat2–plasmid DNA complex

The complex was prepared at an optimal ratio 4 : 1 (w⁄ w)

of Tat2and plasmid DNA Tat2(20 lg) and plasmid DNA

pAct-1GUS (5 lg) were separately prepared in 100 lL of

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 Lipofectamine 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 Tat2–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)1cefotaxime at 25C 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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