Study of uptake of cell penetrating peptides and their cargoes in permeabilized wheat immature embryos Archana Chugh and Francois Eudes ¸ Lethbridge Research Centre, Agriculture and Agri-Food Canada, Alberta, Canada 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 February 2008, accepted 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 treatment of the embryos with toluene ⁄ ethanol (1 : 20, v ⁄ v with permeabilization buffer) resulted in a remarkably higher uptake of cell-penetrating peptides, whereas nonpermeabilized embryos failed to show significant cellpenetrating 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 comparable fluorescence levels even in permeabilized embryos A glucuronidase histochemical assay revealed that Tat peptides can efficiently deliver functionally 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 suggest 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 LipofectamineÔ 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 Cell-penetrating peptides (CPPs) comprise a fast growing class of short length peptides that differ in sequence, size and charge but share a common characteristic ability to translocate across the plasma mem- brane It has been demonstrated that CPPs can act efficiently as nonviral delivery vehicles for macromolecules that are much larger in size than their own and lack the self-potential to enter living cells due to the 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 CPPs and their cargo in wheat permeability barrier posed by the plasma membrane [1,2] A range of molecules, including some of the largest 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 modifications 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 indicating 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 extracellular 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 translocation 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 vascular 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 internalized by various plant tissues of triticale seedlings [27] Macropinocytosis dependent transduction of fluorescent proteins by arginine-rich intracellular delivery (AID) and Tat-protein transduction domain has been reported in onion and corn root tip cells [28,29] Cationic 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 amenability towards tissue culture procedures and a high efficacy for plant regeneration We investigated the 2404 A Chugh and F Eudes 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 prerequisite 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-glucuronidase (GUS) enzyme more efficiently in permeabilized embryos than the nonpermeabilized embryos A commercially 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 translocation 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 complex formation of Tat dimer with plasmid DNA carrying the GUS gene using gel retardation assay, DNaseI protection assay and confocal laser microscopy 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 permeabilizing 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 permeabilizing agent-toluene ⁄ ethanol (1 : 20, v ⁄ v with permeabilization 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) 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 A Chugh and F Eudes CPPs and their cargo in wheat Table List of CPPs employed in the present study FI, fluoresceination at the N-terminal amino group Sequence a Tat Tat2 M-Tat Transportanb pVECc Peptide length Reference FI-RKKRRQRRR-amide FI-RKKRRQRRRRKKRRQRRR-amide FI-AKKRRQRRR-amide FI-GWTLNSAGYLLGKINLKALAALAKKIL-amide FI-LLIILRRRIRKQAHAHSK-amide Peptide 18 27 18 [50] [26,51] [51] [52] [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 a b c d e f g a’ b’ c’ d’ e’ f’ g’ -T/E +T/E M-Tat Tat pVEC Tat2 Transportan ta or sp an Tr n tra Cell-penetrating peptide D ex n EC pV t2 Ta t Ta -T lp su M te l tro on at Without toluene:ethanol With toluene:ethanol 4000 3000 2000 1000 C B Dextran sulphate Relative fluorescence uptake Control Fig 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, Tat2, pVEC and transportan for h in the presence or absence toluene ⁄ ethanol (1 : 20, v ⁄ v with permeabilization buffer) Control: no treatment; negative controls: FITC-dextran sulfate (nonpeptidic in nature, molecular mass = 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) 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 permeabilized 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 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 CPPs and their cargo in wheat A Chugh and F Eudes significantly lower than the other CPPs investigated (Fig 1B) to 51.8% in nonpermeabilized embryos Similarly, the percentage of embryos showing Tat-mediated GUS enzyme uptake increased from 22.6% to 66.5% (2.9fold 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 permeabilized embryos than the nonpermeabilized embryos (Fig 2B) The negative control, M-Tat, showed a lower intensity of blue colour than the other Tat peptides, indicating peptide sequence dependent GUS enzyme delivery in immature embryos GUS enzyme alone was unable to translocate efficiently in the immature embryos (Fig 2A,B) 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 nonlabeled 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 : ratio (w ⁄ w) in the presence and absence of cell permeabilizing agent toluene ⁄ 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 histochemical assay demonsrated that the number of permeabilized embryos showing Tat2-mediated GUS enzyme uptake increased to 92.2% (1.7-fold higher) compared A a b c d e f b’ c’ d’ e’ f’ Sc Ge –T/E a’ +T/E GUS only Tat + GUS 120 Tat2 + GUS Chariot + GUS Without toluene/ethanol 100 With toluene/ethanol 80 60 40 20 t rio t t2 C Ta Ta at -T M on G U S tro up on ex tra n sl C D ly te l B M-Tat + GUS GUS +ve embryos (%) Control Cell-penetrating peptide Fig Noncovalent GUS enzyme transduction by Tat peptides and Chariot in wheat immature embryos Peptide–GUS enzyme complex was prepared at an optimal ratio of : (w ⁄ w) and incubated for h The embryos were incubated with the peptide–GUS protein complex for 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 performed 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 (Tat2) and Chariot–GUS enzyme complex (B) The number of embryos showing exogenous GUS enzyme activity also increased with permeabilization treatment 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 A Chugh and F Eudes CPPs and their cargo in wheat Effect of inhibitors on Tat2-mediated GUS enzyme delivery in permeabilized embryos Peptide–DNA complex formation as observed under confocal laser microscope The influence of low temperature, endocytosis and macropinocytosis inhibitors was evaluated by employing 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 (4 °C) 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) Further experiments were conducted to confirm the optimal ratio of Tat2 and plasmid DNA for transfecting 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, : 1, : and : (w ⁄ w) Image merging (yellow) showed that the optimum ratio for a complex formation was : The complex size observed at : varied from as small as 0.85 lm up to lm after h of incubation (Fig 4C) 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, : 1, : 1, : 1, w ⁄ w) A gel retardation assay showed that the mobility shift began at a : ratio of the Tat2 and GUS enzyme The fluorescence diminished at : and higher ratios, indicating 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 : and higher ratios whereas, at lower ratios (0.5 : 1, : 1, : 1), it showed various extents of degradation by the nuclease (Fig 4B) – – + A B C +++ D Tat2-mediated plasmid DNA delivery: transient GUS gene expression in the immature embryos Tat2–plasmid DNA (pAct-1GUS) complex was prepared at the optimal ratio of : (w ⁄ w) and added to the immature embryos In the presence of permeabilizing agent toluene ⁄ ethanol, transient GUS gene expression increased from 2.5% to 8.3% Further addition of 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 fluorescence was observed under the fluorescence microscope – + – – E F G H Fig Effect of inhibitors A GUS histochemical assay showed inhibition of uptake of Tat2–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 Tat2–GUS protein cargo complex at °C (D) Permeabilized embryos incubated with Tat2–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 mM sodium azide and 10 lM nocodazole, respectively) (G, H) Permeabilized embryos incubated with Tat2–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 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 A 0.5:1 1:1 2:1 3:1 4:1 5:1 0.5:1 A Chugh and F Eudes 1:1 2:1 3:1 4:1 5:1 CPPs and their cargo in wheat B C a Tat2-fluorescein b c pAct-1GUS-rhodamine Merge Fig Tat2–plasmid DNA complex formation for DNA transfection studies in permeabilized immature embryos Various concentrations of nonlabeled Tat2 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 microscopy; complexes of various sizes (in the range 0.85–4 lm) were formed when fluorescein labeled Tat2 and rhodamine labeled circular pAct-1GUS DNA were incubated for h at the optimum ratio : (w ⁄ w) giving a yellow colour in the merged image The complex size was determined using the IMAGEJ software A a 20 15 10 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 2408 LF A+ A Treatment Ta t2 +D N Ta t 2+ A- D N E T/ N A Ta t2 +D N -T at +D N A M D N A- T/ tro on C D E l Transient GUS gene expression (%) B b Fig Transfection studies using Tat2 as carrier peptide for GUS gene delivery in permeabilized 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 Tat2 and 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 showing transient GUS gene expression with different treatments of Tat2 and plasmid DNA The treatment was carried out in permeabilized embryos unless otherwise noted LF, LipofectamineÔ 2000 The percentage of transient GUS gene expression was calculated as the number of embryos showing transient GUS gene expression ⁄ total number of embryos in a treatment · 100 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] 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 A Chugh and F Eudes We speculated that the use of membrane permeabilizing 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 penetration in the embryos at the various concentrations investigated (0.1–2 mgỈL)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 peptides In the mammalian system, penetration of fluorescently 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 acetone ⁄ 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 efficiency 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 permeabilization 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 carrier peptide) for protein delivery in mammalian cell lines delivered the GUS enzyme in wheat embryos efficiently 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 Tatprotein transduction domain and AID proteins in corn and onion root tip cells In the present study, we demonstrate that, in permeabilized wheat embryos, low molecular mass Tat peptides (1.37–2.7 kDa) have the potential of a noncovalently transducing macromolecule (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- CPPs and their cargo in wheat ous studies have also demonstrated a higher permeation 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 significantly low even in permeabilized embryos, suggesting that the carrier peptide sequence also plays an important 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 Tat2mediated 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] Accordingly, we investigated the effect of endocytosis as well as macropinocytosis inhibitors on Tat2 peptide-mediated 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 conclusive data emerged that would enable us to determine 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 experiments between endocytosis inhibitors, we observed that sodium azide was a more effective inhibitor than nocodazole, whereas EIPA (a specific macropinocytic inhibitor) 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 presented), suggesting that endocytosis is not involved in the translocation of CPPs alone In our previous studies, 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- 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 CPPs and their cargo in wheat trostatically, resulting in a complex formation that can be employed for gene delivery in plant cells The complex size at the optimal ratio (4 : 1) of Tat2 and plasmid 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 complexes of 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 transfection efficiency in a rat fibroblast cell line than complexes 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 studies 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 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 versions 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 (indicated by embryo germination) of CPPs alone or of their cargo on embryos Similarly, previous studies 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 germination 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 answer2410 A Chugh and F Eudes ing questions such as how the combined use of cell permeabilizing 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 barrier 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 permeabilized 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, simple 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 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 min, and then washed four times for 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 temperature 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 nonpeptidic 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 : 20 ratio (v ⁄ v) with 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 A Chugh and F Eudes permeabilization buffer To this, 400 lL of lm fluorescein labeled CPP was added After incubation for 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 at room temperature Embryos were washed two to three times with permeabilization 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 °C The supernatant was collected in a fresh tube and relative fluorescence 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 delivery of GUS enzyme in wheat embryos Tat peptide and GUS enzyme were first prepared in separate microcentrifuge tubes Nonlabeled Tat peptide (4 lg) was added to sterile water (with the final volume made up to 100 lL) Similarly, 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 : peptide ⁄ protein ratio in the mixture The mixture was incubated for h at room temperature and then added to the isolated immature embryos (in a mL microcentrifuge tube) in the presence or absence of permeabilizing agent toluene ⁄ ethanol (1 : 40, v ⁄ v with the total volume of the peptide ⁄ protein mixture) After h of incubation at room temperature, embryos were washed twice with the permeabilization buffer and subjected to trypsin treatment [1 : (v ⁄ v) with permeabilization buffer] for at room temperature The embryos were washed twice with permeabilization buffer followed by GUS histochemical analysis of the embryos For delivery of lg of GUS enzyme by the Chariot protein transduction kit (Active Motif, Carlsbad, CA, USA), the manufacturer’s protocol was followed Permeabilized and nonpermeabilized embryos were incubated with Chariot–GUS complex for h All post-incubation steps were the same as that described for Tat peptides CPPs and their cargo in wheat 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, 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 treated 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 °C in permeabilization buffer (pH 7.1) for 45 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 h at °C Similarly, embryos were pre-incubated with either the endocytosis 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 pAct1GUS, 7.2 kb) was mixed with different concentrations of Tat2 to give ratios of 0.5 : 1, : 1, : 1, : 1, : and : 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 h for complex formation and subjected to electrophoresis on 1% agarose 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, lL of DNaseI (RNase-Free DNase set; Qiagen, Valancia, CA, USA) was added to the mixture volume (50 lL) and incubated at 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 CPPs and their cargo in wheat room temperature for 15 and then incubated on ice for Plasmid–peptide dissociation and plasmid purification was carried out with a commercially available DNA purification kit (QIAquick PCR purification kit; Qiagene) DNA was eluted in sterile water An aliquot of 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 Tat2 and DNA were employed to observe the complex 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 imagej software (NIH, Bethesda, MD, USA) Transfection of permeabilized immature embryos with Tat2–plasmid DNA complex The complex was prepared at an optimal ratio : (w ⁄ w) of Tat2 and 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 h at room temperature As an optional step, lg of LipofectamineÔ 2000 (Invitrogen) was added to the mixture and the mix incubated for another 30 for complex formation The mixture (total volume of 200 lL) was added to the sterilized embryos along with the permeabilizing agent (toluene ⁄ ethanol : 40, v ⁄ v with the mixture) The embryos were incubated with Tat2–plasmid DNA complex for h at room temperature followed by two washings with permeabilization buffer (pH 7.1) The embryos were plated on germination medium containing 250 lgỈmL)1 cefotaxime at 25 °C in the dark for 3–4 days Transient GUS gene expression was studied by incubating the immature embryos in GUS histochemical buffer as described above The percentage of embryos showing transient 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 appreciated The Alberta Peptide Institute provided high 2412 A Chugh and F Eudes 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 Neuroscience (CCBN, University of Lethbridge) We thank Dr Ray Wu (Cornell University) for plasmid pAct1GUS A.C thanks the Natural Science and Engineering Research Council of Canada (NSERC) for the award of Visiting Fellowship The authors acknowledge financial support from Matching Investment Initiative (MII) program and Alberta Agriculture Research Institute (AARI) References Rolland A (2006) Nuclear gene delivery: the Trojan horse approach Expert Opin Drug Deliv 3, 1–10 Wagstaff KM & Jans DA (2006) Protein transduction: cell penetrating peptides and their therapeutic applications Curr Med Chem 13, 1371–1387 Gait MJ (2003) Peptide-mediated cellular delivery of antisense oligonucleotides and their analogues Cell Mol Life Sci 60, 844–853 Jarver P & Langel U (2004) The use of cell-penetrating peptides as a tool for gene regulation Drug Discov Today 9, 395–402 Simeoni F, Morris MC, Heitz F 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Kihlmark M, Hallbrink M, Fernaeus S, Raid R, Land T, Hallberg E, Bartfai T & Langel U (2001) Cellular translocation of proteins by transportan FASEB J 15, 1451–1453 52 Elmquist A & Langel U (2003) In vitro uptake and stability study of pVEC and its all-D analog Biol Chem 384, 387–393 53 Mahalakshmi A, Chugh A & Khurana P (2000) Exogenous DNA uptake via cellular permeabilization and expression of foreign gene expression in wheat zygotic embryos Plant Biotechnol 17, 235–240 54 Kosugi S, Ohashi Y, Nakajima K & Arai Y (1990) An improved assay for b-glucuronidase in transformed cells: methanol almost completely suppresses a putative endogenous b-glucuronidase activity Plant Sci 70, 133– 140 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 ... studied in wheat immature embryos The role of cell permeabilizing agent in enhancing the uptake of CPPs alone and cargo complex in the immature embryos was evaluated Cellular uptake of CPPs is... the combined use of cell permeabilizing agent and cell- penetrating peptides results in the delivery of such large sized cargoes in plant cells involving endocytic ⁄ macropinocytic pathways In conclusion,... 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