Conjugated polymer nanoparticles for effective siRNA delivery to tobacco BY-2 protoplasts
BMC Plant Biology
Conjugated polymer nanoparticles for effective siRNA delivery to tobacco BY-2 protoplasts
Asitha T Silva 1
Alien Nguyen 0
Changming Ye 1
Jeanmarie Verchot 1
Joong Ho Moon 0
0 Department of Chemistry and Biochemistry, Florida International University , Miami, FL 33199 , USA
1 Department of Entomology and Plant Pathology, Oklahoma State University , Stillwater, OK, USA 74078
Background: Post transcriptional gene silencing (PTGS) is a mechanism harnessed by plant biologists to knock down gene expression. siRNAs contribute to PTGS that are synthesized from mRNAs or viral RNAs and function to guide cellular endoribonucleases to target mRNAs for degradation. Plant biologists have employed electroporation to deliver artificial siRNAs to plant protoplasts to study gene expression mechanisms at the single cell level. One drawback of electroporation is the extensive loss of viable protoplasts that occurs as a result of the transfection technology. Results: We employed fluorescent conjugated polymer nanoparticles (CPNs) to deliver siRNAs and knockdown a target gene in plant protoplasts. CPNs are non toxic to protoplasts, having little impact on viability over a 72 h period. Microscopy and flow cytometry reveal that CPNs can penetrate protoplasts within 2 h of delivery. Cellular uptake of CPNs/siRNA complexes were easily monitored using epifluorescence microscopy. We also demonstrate that CPNs can deliver siRNAs targeting specific genes in the cellulose biosynthesis pathway (NtCesA-1a and NtCesA1b). Conclusions: While prior work showed that NtCesA-1 is a factor involved in cell wall synthesis in whole plants, we demonstrate that the same gene plays an essential role in cell wall regeneration in isolated protoplasts. Cell wall biosynthesis is central to cell elongation, plant growth and development. The experiments presented here shows that NtCesA is also a factor in cell viability. We show that CPNs are valuable vehicles for delivering siRNAs to plant protoplasts to study vital cellular pathways at the single cell level.
Post transcriptional gene silencing (PTGS) is a cellular
mechanism that regulates gene expression in the
cytoplasm [1,2]. In this mechanism, mRNA is reverse
transcribed to produce long double-stranded RNA which is
then digested by the Dicer enzyme to produce smaller
fragments of discrete sizes. There are two classes of
silencing RNAs, known as microRNA (miRNA) and
small interfering RNA (siRNA) [2-4]. miRNAs are
endogenous noncoding small RNAs that are 18 to 25
nucleotide (nt) long and function to repress mRNA translation
or target mRNA for degradation . miRNAs contribute
to the regulation of gene expression for development,
responses to external stressors, and cell cycle control .
siRNAs are 21 to 24 nt long and derive from mRNAs or
viral RNAs . Endoribonuclease-containing complexes,
known as RNA-induced silencing complexes (RISCs),
incorporate the miRNAs and siRNAs which act to guide
the RISCs to homologous cellular mRNAs, targeting
them for degradation [8-10]. PTGS acts to prevent
translation of targeted gene products and effectively knock out
PTGS has been harnessed by plant biologists as a tool
to knock down expression of essential genes during
investigations of their role in metabolism in whole
plants and protoplasts . Viral vectors are commonly
used for delivery of siRNAs or miRNAs into plants.
Viral vectors offer the advantage of transiently and
directly expressing the siRNA without relying on plant
transformation. The most widely used vector for delivery
of siRNAs is the bipartite Tobacco rattle virus (TRV)
[12,13]. The Cabbage leaf curl virus (CbLCV) vector
was recently developed for expressing synthetic and
endogenous miRNAs in plants . For TRV and
CbLCV vectors, the genomic cDNA was introduced into
T-DNA vectors and used for Agrobacterium tumefaciens
delivery by infiltration into leaves [12,14,15]. Entire or
partial gene sequences are expressed from the TRV
vector while artificial miRNA precursors have been
expressed from the CbLCV genome which share
homology to the host gene targeted for silencing. As the virus
spreads systemically, virus-derived siRNAs or miRNAs
guide the RISC complex to degrade target transcripts.
Because most viruses are limited in their host range,
additional viral vectors are being developed for small
RNA delivery to diverse plant species. Another
drawback of viral vectors is that they do not uniformly infect
all tissues, although they might spread systemically. In
addition, the phenotype attributed to PTGS is mixed
with the onset of virus symptoms which include mosaic
pattern of disease and mild leaf curling.
Protoplasts are isolated from plant suspension cells or
intact tissues by treating them with cell wall degrading
enzymes. Protoplasts have intact plasma membranes but
are fragile because of the loss of the cell wall.
Protoplasts are typically employed in cell culture assays for
physiological, biochemical, and molecular studies of
plant cell functions. They can survive for up to 72 h in
culture with some loss of viability, but in suitable media,
cultured protoplasts can regenerate cell walls, undergo
cell division, and even regenerate plants [16-18]. Gene
transfer or siRNA delivery into protoplasts is typically
achieved using electroporation which involves applying
an electric field to protoplasts held in a cuvette.
Electroporation increases the plasma membrane permeability
and enables nucleic acid penetration [19,20]. One
important drawback is the significant loss of viable
protoplasts during electroporation. Depending on the
source of protoplasts (i.e. plant species as well as the
source tissues such as leaf, cotyledons, young shoots,
suspension cells) and the voltage applied, losses of 50%
viable protoplasts can occur [19,21].
CPNs are intrinsically fluorescent nanoparticles
fabricated by ultrafiltration of amine-containing conjugated
polymers (CPs) treated with an organic acid in aqueous
phases [22,23]. These organic nanoparticles are stably
suspended in water (without evidence of precipitation)
for several months under ambient storage condition.
The spectral properties of CPN were previous described
and the absorption maximum is centered at 438 nm and
emission maximum is at 483 nm . Dynamic light
scattering measurements revealed the hydrodynamic
radius of CPNs is around 60-80 nm, depending on
molecular weights and organic acid treatments. CPNs are
positively charged, and exhibit affinity with negatively
charged biological substances such as nucleic acids .
CPNs have the potential to act as a protective, efficient
and self-tracking transfection agent for RNA interference
experiments. The complexation of siRNA with CPNs can
improve RNA stability by protecting them from RNAse
degradation, as reported for other polymeric siRNA
delivery systems . In addition, various mammalian
cells take up CPNs without toxic effects. Given their
ability to traverse cellular membranes we postulated that
CPNs can be used to visually monitor siRNA
internalization using a simple complexation between CPNs and
In this study, we examine the delivery of siRNA to
protoplasts using CPNs. We employed siGLO Red siRNA,
which is a commercially available, red fluorescent
dye-labeled siRNA. We found that CPN is a potent
transfection agent that can be used to deliver and visually
monitor the uptake of abundant siRNAs to plant
protoplasts. We also demonstrate that CPNs can deliver
siRNAs targeting specific genes in the cellulose biosynthesis
pathway (NtCesA-1a and NtCesA-1b). Cellulose synthase
is a multigene family that is not fully characterized in
tobacco. NtCesA-1a and NtCesA-1b are related
accessions but NtCesA-2 is a distinct gene with 80% homology
to NtCesA-1a. In a prior report, a PVX vector containing
CesA-1a gene fragments were delivered to intact plants.
The outcomes of CesA-1a silencing included reduced
cellulose content of the plant cell walls, but this was also
accompanied by an increase in homogalacturonan and
decreased esterification of pectic polysaccharides in
silenced plants . Here, we show that CPNs deliver
NtCesA-1 siRNAs that effectively knockdown cell wall
biosynthesis during the early stages of synthesis in
protoplasts indicating that NtCesA-1 is crucial. Therefore,
CPNs provide an attractive alternative for siRNA delivery
and gene knockout in cultured protoplasts.
CPNs are taken up by BY-2 protoplast but not by intact
BY-2 protoplasts were incubated with various
concentrations of CPNs (5, 10, 15, and 25 μM) for either 2 or 24 h
followed by counting cells to determine the proportion of
green fluorescing cells under the microscope (n = 400).
At 2 h following the delivery of 5 μM CPNs to the
culture medium, 35% of protoplasts showed green
fluorescence, while 60-75% of protoplasts treated with 10-25
μM CPNs showed fluorescence. At 24 h, the proportion
of green fluorescent protoplasts increased to 50%
following treatment with 5 μM CPNs and 79-90% following
treatment with 10-25 μM CPNs (Figure 1A, B).
Importantly, untreated samples did not fluoresce green (Figure
1C, D). Optical sections obtained by laser-powered
confocal microscopy confirming internal localization of
CPNs (data not shown). Fluorescence was mainly
cytosolic, and did not appear to be nuclear (Figure 1A, B).
Figure 1 CPN-treated BY-2 cells and protoplasts. Panels show
bright field and fluorescence images. (A,B) BY-2 protoplasts were
incubated for 2 h with 10 μM CPNs or (C, D) left untreated. (E, F)
Chains of attached BY-2 cells treated with 10 μM CPNs for 2 h.
Green fluorescence is greatest at the cross walls suggesting that
CPNs attach to the cell walls and do not penetrate the interior.
(G, H) Images of untreated BY-2 cells at 2 h. (I, J) Confocal images
of intact BY-2 cells treated with 10 μM CPNs at 24 h. Experiments
were repeated with similar results. Single optical section through
the center of the cell shows fluorescence along the cell wall and
does not penetrate the interior. Scale bars equal 50 μm.
We treated intact BY-2 suspension cells with 5, 10, 15,
and 20 μM CPNs (Figure 1E, F) and the plant cell wall
was a barrier to uptake. Optical sections obtained by
laser-powered confocal microscopy of CPN-treated BY-2
cells showed the fluorescence remained bound to the
cell surface even after 24 h of incubation (Figure 1I, J).
Untreated samples showed no green fluorescence
(Figure 1G, H).
Uptake of CPNs is reminiscent of endocytic pathway
In a recent study, positively charged nanogold particles
were transferred at the plasma membrane to the early
endosome and then into larger peripheral vesicles .
The role of the large peripheral vesicles and the
destination beyond these vesicles in plant cells has not been
described, although there is some speculation that these
are prevacuolar vesicles . In protoplasts, the CPNs
often occur in cytoplasmic granules and we
hypothesized that these are either aggregates of nanoparticles,
endocytic vesicles, or both. Given that CPNs have
positive charge, they might enter the endocytic pathway,
similar to the charged nanogold particles, and then be
released into either the cytoplasm or another membrane
bound compartment. Therefore we employed FM4-64,
which is a membrane-staining dye for live cell imaging,
to track endocytic vesicles budding from the plasma
membrane in CPN-treated protoplasts . Untreated
protoplasts stained with FM4-64 for 10 min, showed
uniform red fluorescence in the plasma membrane, and
bright spots where vesicles begin to form (Figure 2A,
arrowheads). Few internal vesicles appear. Following
staining for 20 min, the red fluorescence occurred in
prevacuolar and vacuolar membranes (Figure 2B).
Protoplasts were incubated with 10 μM of CPNs for 24
h followed by incubation with FM4-64 for 10 -30 min.
Green and red fluorescence co-localized in vesicles at the
cell margin and internally. There was a profusion of red
fluorescent vesicles, which was not seen in control
samples (not treated with CPNs). The exogenous application
of CPNs stimulated either the production of endosomes
by the cell or dye uptake by an alternative route
(compare Figure 2A, B, and 2D). We followed the transition of
FM4-64 dye over time. After 10 min of staining, green
and red fluorescence appeared in granules along the
plasma membrane (Figure 2C, D, arrowheads). Green
and red fluorescence then co localized in large peripheral
vesicles around 30 min later (Figure 2E-G). The larger
peripheral bodies (Figure 2E-G) resembled prevacuolar
vesicles (such as multi-vesiculate bodies). Given that
proteins taken up by the early endosome can be transported
either to the Golgi apparatus or prevacuolar vesicles, the
pattern of FM4-64 staining is expected. Figure 2 shows a
pattern of CPNs transitioning from small granules at the
cell surface to larger vesicles, argues that CPNs follow
the same uptake pathway as FM4-64. While further high
resolution experiments are needed to define the various
internal compartments, the pattern of CPN-uptake
suggests a membrane mediated route rather than diffusion
across the plasma membrane.
5-25 μM CPNs are nontoxic to BY-2 protoplasts
Reports indicate that cadmium-based nanoparticles
have the potential to be cytotoxic to mammalian cells.
The cytotoxic potential can be influenced by the
particle sizes and concentrations, distribution to different
regions of the cell, or liberation of Cd2+ from the
nanoparticle lattice [28,29]. Although CPNs are
Figure 2 CPN and FM4-64 treated BY-2 protoplasts examined
using confocal microscopy. (A) Protoplast that was treated with
medium (no CPNs) and then FM4-64 for 10 min or (B) more than
20 min. FM4-64 fluorescence is in the plasma membrane and
vesicles budding from the plasma membrane at 10 min. Following
a 20 min or longer incubation, red fluorescence is in the plasma
membrane, perinuclear membranes, and intracellular vesicles. Arrow
heads point to vesicles budding at the plasma membrane and in
the cortical region. (C, D) Green and red fluorescent images of
protoplasts treated with CPNs and then FM4-64 for 10 min. Arrow
heads point to vesicles along the plasma membrane that contain
both green and red fluorescence. There are a greater number of red
than green fluorescent vesicles. However, most green granules also
contain red fluorescence. (E, F, G) Protoplasts were treated with
CPNs and then FM4-64 for 20 min showed green and red
fluorescence in vesicles along the periphery of the cell. Repeated
experiments showed similar outcomes. Arrows point to examples
where green and red fluorescence overlap. Scale bars equal 20 μm.
polymers that do not contain Cd2+ and are distributed
in the cytoplasm, we cannot rule out the possibility of
a concentration dependent cytotoxicity. Therefore,
propidium iodide staining was employed to measure
the cytotoxic impact of various concentrations (0, 5,
10, 25, 50, 100, 250, and 500 μM) of CPNs following
treatments for 0, 2, 5, 8, 16, 24, and 48 h (Figure 3).
The average percent of viable protoplasts at each time
point was calculated for three replicate experiments.
Concentrations of 5-25 μM are non-toxic to
protoplast and do not significantly reduce their viability.
Typically, following preparation of BY-2 protoplasts
from intact cells, we noted 90-96% viable protoplasts
during the first 8 h of culture. This declines to 85% at
24 h and then 60% at 48 h (Table 1). 80-95% of BY-2
protoplasts were viable during the first 8 h of culture
following treatment with 5, 10, and 25 μM of CPNs (log
molar concentrations of -4.3 to -5.3), which is
comparable to untreated protoplasts. Protoplast viability
following CPN treatment further declined to 81-86% at 24 h
and 53-60% at 48 h. Thus, the average percent viability
is similar over time among the CPN-treated (5-25 μM)
and untreated protoplasts.
Concentrations over 50 μM (log molar concentrations of
-3.3 to 4.0) cause the proportion of viable protoplasts to
decline profoundly after 8 h (Figure 3; Table 1). The
average percentage of intact protoplasts treated with 50 μM
CPNs was 68% at 24 h and 32% at 48 h. For concentrations
of 100- 500 μM the average percent viability was 20 to 32%
at 24 h and 14 to 22% at 48 h (Figure 3; Table 1). These
data show that the concentrations of CPNs (5-25 μM) used
in the prior experiments for visualizing uptake are
essentially non-toxic to BY-2 protoplasts but excessive amounts
of polymer can be detrimental.
CPNs deliver both siGLO Red and NtCes1-A siRNAs to
Commercially available siGLO Red are fluorescently
labeled RNA duplexes which were combined with CPNs
and delivered to BY-2 protoplast culture medium to
assess protoplast transfection. Both green and red
fluorescence, which corresponds to CPNs and siGLO Red,
respectively, were seen inside BY-2 protoplasts within
2 h of delivery (Figure 4). FACS methods were
employed to: a) measure the fluorescence intensity in
each cell b) detect and count the number of
fluorescentprotoplasts in large populations (10,000 protoplasts).
A set of untreated BY-2 protoplasts without CPN
treatment were gated to represent the major non-fluorescent
population (Figure 5A). FACS demonstrated that 10 or
25 μM CPNs penetrated BY-2 protoplasts following
treatment for 2 or 24 h. Clearly, not all protoplasts
showed CPN-uptake. However comparing treatments at
2 and 24 h, there was undoubtedly an increase in
CPNuptake by protoplasts that was concentration and time
dependent (Figure 5B).
Cytometric analysis also showed that siGLO Red failed
to penetrate protoplasts in the absence of CPNs (Figure
5A). However, combining 10 μM or 25 μM CPNs with
200 nM siGLO Red, there is a significant and positive
shift in the number of events containing red
fluorescence (Figure 5A, C). The combined epifluorescence
microscopic and cytometric analyses (Figures 4 and 5)
indicate that CPNs were responsible for siGLO Red
Figure 3 Protoplast viability was determined following specific incubation with various concentrations of CPNs. Protoplasts were
cultured for various times between 0 and 48 h following treatment with the following CPN concentrations: 5, 10, 25, 50, 100, 250 and 500 μM.
The percentage of viable protoplasts was determined using propidium iodide staining at 0, 2,5,8,16,24 and 48 h. The data were expressed as the
average % viability at each time point for log molar concentration of CPNs taken from three independent experiments.
The ability of CPNs to deliver siRNAs targeting an
endogenous gene was also examined. siRNAs were
generated to the plant cellulose synthase gene, NtCesA-1,
and CPN-siRNA complexes were delivered to
protoplasts. Under suitable media conditions, BY-2
protoplasts can regenerate their cell walls during 3 d of
culture [30,31]. NtCesA-1 is a central factor in cell wall
Table 1 Effect of various concentrations of CPNs on
viability of BY-2 protoplasts at 0 and 24 h
deposition in plants and therefore we hypothesized that
knocking down NtCesA-1 expression would block cell
wall regeneration in protoplasts. Given that the flow
cytometry data shows ~ 40% uptake of siGLO Red
accompanied by CPNs, it is possible that a similar
population of protoplasts received CesA-1 siRNAs. We
employed calcofluor white M2R staining  to assess
cell wall regeneration following silencing NtCesA-1a and
propidium iodide staining to monitor viability .
Calcofluor white M2R staining conducted upon the
completion of cell wall digestion (T = 0 h) confirms that
there was no residual cell wall material remaining along
protoplast surfaces (Figure 6A). At 72 h, greater
amounts of calcofluor white M2R fluorescence was
observed at the margins of untreated protoplasts
indicating that the culture conditions were suitable for cell
wall regeneration (Figure 6C). For siRNA and CPN
treated protoplasts, calcofluor staining was significantly
reduced to a level that was barely visible. Rare, minor
patches of cellulose occurred along the plasma
membrane of some protoplasts at 72 h (Figure 6B).
Interestingly, we tested CPN-siRNA complexes formed by
Figure 4 CPNs deliver siGLO Red small RNAs to protoplasts. Bright field and epifluorescence images of protoplasts treated with: (A, B, C) 25 μM
CPNs and 200 nM siGLO Red small RNAs (red); (D, E) only 200 nM siGLO Red small RNAs; (F, G) untreated protoplasts (negative controls). (E) Image
shows no uptake of small RNAs in the absence of CPNs. (G) Image shows no green fluorescence, as expected. Experiments were repeated 2-3 times.
Scale bars equal 20 μm.
mixing for 3 h and overnight at 4°C and we noted that
the outcome of calcofluor staining was significantly
reduced using CPN-siRNA complexes that were
prepared by overnight incubation (data not shown). It is
worth speculating that the overnight incubation led to
maximal incorporation of siRNAs into complexes.
To determine the effectiveness of NtCesA-1
siRNAdelivery in blocking cell wall regeneration, we recorded
the average percentage of calcofluor positive protoplasts
relative to the total number of living protoplasts counted
(n = 400 protoplasts) in two replicate experiments.
Protoplast viability was confirmed using propidium iodide (see
below). For protoplasts that were untreated (no siRNA or
CPN) or treated only with NtCesA-1 siRNAs, 51-54%
were calcofluor positively by 72 h. Treating protoplasts
with CPN or siRNA alone had no effect on cell wall
regeneration until the time period between 48-72 h.
There was a steady increase in the proportion of
untreated or siRNA-treated protoplasts that stained
positive over time (Figure 6D). For protoplasts treated with
CPN alone, there is a slight plateau between 48 and 72 h
(19-23%) and only a few faint patches of newly
synthesized cell walls were seen in those protoplasts (Figure 6B
and 6D) . However, this is contrasted by protoplasts
treated with 10 μM CPNs and 200 nM NtCesA-1 siRNAs
which showed no change in cell wall regeneration
between 0 and 24 h followed by a slow increase in
calcofluor staining until 48 h. There appeared to be a plateau
between 48 and 72 h where cell wall regeneration did not
continue in a manner similar to untreated protoplasts
(Figure 6C). Calcofluor staining was seen in 33-38% of
protoplasts at 48 and 72 h, suggesting that CPN
treatment could hamper growth at later times.
Propidium iodide was used to determine the percent
viable protoplasts harvested at 0, 24, 48, and 72 h
(Figure 6E). Propidium iodide stains nonviable protoplasts and
cells, shows absorption/emission maximum at 536/617
nm, and was employed to measure the impact of CPNs on
cell viability. We counted populations of 100-250
protoplasts to determine the proportions that were propidium
iodide positive and/or contained CPNs. Untreated
protoplasts show a slow decline in viability from 90% at 0 h to
~ 60% at 72 h. Protoplasts that were treated with only
CPNs or only siRNAs showed comparable levels of
decline. However, protoplasts treated with both CPNs and
siRNAs showed a significant drop in viability between 24
and 72 h with ~ 35% of protoplasts remaining alive (Figure
6E). These data suggest that cellulose synthase activity is
essential for extending the lifetime of protoplast. The
effect of CPN plus siRNAs on cell viability and deposition
of cellulose on the cell surface  indicates that CPNs
were effective vehicle for siRNA delivery and targeted
downregulation of NtCesA-1expression.
Knockdown of NtCesA-1a transcript accumulation was
confirmed by semi-quantitative PCR (Figure 6F).
NtCesA-1a silenced protoplasts were harvested at 48 h
post delivery of CPNs alone and 200 nM siRNAs plus
CPNs. The messages were reduced >17% and >76%
compared with untreated control samples. Ubiquitin
mRNA served as an internal control for RNA quality
Figure 5 Presence of CPN and siGLO Red small RNAs in protoplasts. (A) Dot plots of BY-2 protoplasts cultured with medium only, 200 nM
siGLO Red, 10 μM CPNs, 10 μM CPNs + 200 nM siGLO Red, 25 μM CPNs, or 25 μM CPNs+200 nM siGLO Red. CPN fluorescence is detected with
FITC filter (x axis) and siGLO Red fluorescence (y axis) is detected with PE filter in protoplasts cultured for 24 h. Each dot represents a single event
with emissions frequency that is the combination of the fluorophores. The gated population in the lower left quadrant represents the majority of
nonfluorescent cells. The upper right quadrant represents the majority of events that contain both green and red fluorescence due to CPNs and
siGLO Red small RNAs. The upper left quadrant represent events that are positive only for siGLO Red small RNAs and the lower right quadrant
represent events that are positive for only CPNs. Highly fluorescent protoplasts are located furthest along the x- and y- axes. (B) Bar graph reports
the average of 10 replicate experiments using FACs to record the number of green fluorescent events inside protoplasts, as an indication of the
internalization of CPNs (lower right quadrant of dot plots). Samples were treated with 0, 10, or 25 μM CPNs and then incubated for 2 and 24 h.
Between 18-37% of protoplasts produce positive events via cytometric analyses. (C) Bar graph reports the average and stand deviations of 10
replicate experiments, recording the number of events reporting internalization of both CPNs and siGLO Red (upper right quadrant of the dot
plots). Between 27 and 42% of recorded events are positive for both CPNs and siGLO Red RNAs when they are co-delivered to protoplasts.
CPNs are fluorescent conjugated polymer nanoparticles
and are valuable for live plant cell imaging. Their
inherent photophysical properties include high fluorescence
quantum yield, large extinction coefficient, and efficient
optical signal transduction making them a superior
choice for biological imaging [22,23]. Furthermore, we
demonstrate that CPNs are an effective transfection
vehicle for delivery of siRNAs into plant protoplast.
Other transfection methods that are widely employed
for delivery of nucleic acids to plant protoplasts include
electroporation, polyethylene glycol, and lipofectamine
[34-36], and only electroporation and polyethylene
glycol has been used for direct delivery of siRNAs [37,38].
With respect to electroporation, the electric pulse can
cause an immediate loss of up to 40% viable protoplast
[35,39]. This much greater loss than following the use
of CPN-delivery, which causes only 5-20% loss of
viability within the first 24 h of delivery (Table 1). Unlike the
CPN delivery method, the optimal conditions for
delivery of siRNAs by electroporation require extensive
optimization  of the voltage and pulse time to ensure
high transfection rates. Therefore CPNs are attractive
Figure 6 CPN delivery of CesA-1 siRNAs suppress cell wall regeneration. (A) Protoplasts were harvested and immediately stained with
calcofluor white (T = 0 h) to verify complete digestion and elimination of cell walls. Image shows no calcofluor fluorescence. (B) Protoplasts at 72 h
treated with 10 μM CPNs and 200 nM NtCesA-1 siRNAs show few faint patches of blue fluorescence at the plasma membrane. (C) Untreated
protoplasts at 72 h show significant deposition of cellulose at the cell surface. Experiments were repeated five times. Scale bars represent 10 μm.
(D) The average percent of protoplasts from two experiments that showed calcofluor staining at 0, 24, 48, and 72 h following treatment with CPNs
and NtCesA-1 siRNAs. (E) Propidium iodide was used to determine the percent viable protoplasts at 0, 24, 48, and 72 h following treatment with
CPNs and NtCesA-1 siRNAs. Averages were determined for three replicate experiments. (F) Ethidium bromide- stained 1% agarose gels containing
semi quantitative RT-PCR products detecting NtCesA-1 or ubiquitin (Ubi) gene expression. The treatments with siRNAs and CPNs are indicated
above each panel and the numbers of PCR cycles from 30-45 are indicated below each lane. Lane “L” indicates DNA ladder at the bottom of the
gel and size (bp) markers are indicated on the left. As a control, semi-quantitative PCR shows ubiquitin gene expression.
and facile choice for efficient siRNA delivery into
protoplasts without compromising cell viability. Furthermore,
the intrinsic fluorescence enables real time detection of
CPN-uptake by protoplasts and offers the opportunity
to monitor the rate of cellular responses following
siRNA uptake in synchronously treated cells.
Flow cytometry for studying physiological events or
dye uptake in plant protoplasts has been used in recent
years. Protoplast morphology and the distribution of
light-scattering intensities can vary widely for different
species and cell cultures and this can impact the quality
of the results. We examined green and red fluorescence
(FL1-H and FL4-H) and expected to detect a minor
population of dots that would result from cell debris or
dying protoplasts. Notably the FACS results (Figure 5B)
and manual counting of CPN-uptake by protoplasts
produced somewhat different quantitative outcomes. A
maximum of 35% of protoplasts were green fluorescent
following treatment with 25 μM CPNs for 2 hrs as
measure by FACS, but under the microscope we noted
60-75% of protoplasts were green fluorescent. One
explanation is that the larger population that was sorted
by FACS led to a broader and unbiased assessment.
Another possibility is that the CPN fluorescence inside
some cells might be low and might overlap with the
autofluorescence of the gated population. Thus fewer
CPN-positive protoplasts may be detected by FACS than
by manual counting. A third explanation for the low
counts by FACS is that the method of mixing
protoplasts with CPNs may require further optimization to
ensure a broader population is exposed to CPNs.
Perhaps placing the culture on a rotary shaker at low speed
would enhance mixing of protoplasts and CPNs and
could increase the percentage of transfected protoplasts.
We also observed CPNs binding to debris in the cell
cultures. The nature of the debris is a mixture of lysed
cell constituents and cell wall materials. We know from
examining CPN-treated BY-2 cells that CPNs bind easily
to plant cell walls. Therefore, the presence of cell wall
material in the cultures likely depletes CPNs that could
transfect protoplasts. Perhaps improved handling of
protoplast preparations through further filtration or
washing could reduce cell wall debris and enhance the
percentage of transfected protoplasts. Further
experiments are needed to determine the conditions for
improved CPN internalization.
Cell wall biosynthesis is central to cell elongation,
plant growth and development and new methodologies
to modify the cellulose and lignin content could be
employed for generating genetically improved plants
. Researchers are working on agronomically
important crops to increase cellulose content and decrease
lignin content to improve forage digestibility and improve
the use of crops as biofuels for ethanol production. The
biosynthetic pathways leading to cell wall deposition
(cellulose and lignin) and assembly into a functional
wall are not well described . Significant advances
have been made in recent years with the identification
of CesA genes that encode the catalytic subunits for
cellulose synthase. In Arabidopsis thaliana, there are 10
members of the AtCESA gene family and only three
genes AtCESA1, AtCESA3 and AtCESA6 are known to
be important for primary cell wall synthesis . CesA
subunits assemble in rosettes and these rosettes produce
glucan chains. The rosettes assemble into microfibrils
along the plasma membrane . Patches of cellulose
occurring along the plasma membrane can be seen by
calcofluor staining . In our experiments, we noted
that at 72 h in untreated BY-2 protoplasts, there were
patches of cellulose which coalesced to form larger
areas of deposited primary cell wall (Figure 6C).
Chemicals such as ancymidol and isoxaben are known to
inhibit cell wall production and have been employed to
increase our knowledge of the mechanisms controlling
cellulose biosynthesis and deposition along the plasma
membrane. One drawback of using a chemical approach
to knockdown cellulose synthase is that they can have
additional impacts on other subcellular functions .
For example, isoxaben disrupts microtubule organization
as well as inhibits the synthesis of cellulose microfibrils
. Therefore, siRNA delivery targeting specific genes
in the cellulose biosynthesis pathway is preferred to
examine the role of each gene in cell wall deposition.
Recently, siRNA delivery via viral vectors (Potato virus
X, Barley stripe mosaic virus) to intact N. benthamiana
and barley plants has provided valuable evidence that
RNAi technology can be employed to knockdown
CesA1, CesA-2, and CesA-6 gene expression [25,44]. By
silencing individual members of the CesA gene family
researchers were able to determine which genes are
primary contributors to cell wall formation. In addition,
plants showed compensatory changes in polysaccharide
composition of the cell walls and this demonstrated that
an RNAi approach created further opportunities to
explore the relationships between the cellulose synthase
genes and pectin biosynthesis [25,44]. Importantly, using
viral vectors to deliver siRNAs into protoplasts may not
be advisable because the viruses themselves may impact
protoplast viability and cell wall regeneration. In
addition, viral delivery relies on electroporation or PEG
transfection methods which may also impact protoplast
viability. In this study, we employed a CPN-siRNA
delivery method that is taken up by protoplasts in a manner
that does not hamper viability and had no obvious effect
on cell wall regeneration during the first 48 h. CPN
delivery of CesA-1 siRNAs was effective for silencing
cellulose synthase in protoplasts showing that this
technology can be used to monitor the early stages of
cellulose deposition. When we compare Figure 6D and 6E,
we note that there is reduced protoplast viability along
with hampered cellulose synthesis. Protoplasts left
untreated, treated with siRNAs only (no CPNs), or
CPNs only (no siRNAs) showed comparable levels of
cell wall regeneration and viability. Only the addition of
10 μM CPNs plus 200 nM siRNAs caused a significant
loss in viability and cell wall regeneration. Calcofluor
staining showed no obvious buildup of cellulosic patches
along the plasma membrane, while untreated samples
showed greater regeneration. We also realize that there
is significant sequence similarity among tobacco CesA
genes and that the long piece of CesA used to generate
the siRNAs might directly knock down multiple CesA
genes. The correlation suggests that silencing CesA
genes reduced protoplast viability, indicating that
cellulose synthesis is an important housekeeping function.
Events that slow regeneration or hamper cell expansion
could lead to loss of viability. Complete and speedy
regeneration of the primary cell wall might be important
for protoplasts to respond to the osmotic pressure of the
medium and remain alive. Future research is needed to
understand the link between CesA gene function and
cell viability. These outcomes show that CPN delivery
methods may be valuable for such studies in protoplasts.
Animal cells take up extracellular materials by at least
four different routes: receptor-mediated endocytosis,
non-ionic diffusion, carrier mediated uptake, and
facilitated diffusion. These routes are largely unexplored in
plants, and little is known about the compartments
carrying endocytic cargo from the plant plasma membrane
. Several approaches have been used to identify
specific compartments in the endomembrane system. First is
the use of immune electron microscopy and antibodies
recognizing known markers to identify the compartment.
Second is the use of organelle reporters such as GFP
fused to organellar targeted protein domains [46-48].
Third is the use of FM4-64 fluorescent styryl dyes which
are taken up by endocytic vesicles and are used to follow
the vesicle trafficking network to Golgi and prevacuolar
vesicles [49,50]. Such tools have led researchers to
determine that the pathway from the early endosome to
prevacuolar/multivesiculate bodies in animals and plants differ.
In animals, the multivesiculate bodies send cargo to the
lysosome while in plants they deliver their cargo to the
vacuole. The plant prevacuolar vesicles can deliver cargo
to storage vacuoles as well as the lytic compartment .
The recycling endosome is a compartment that returns
cargo to the plasma membrane and is well described in
animal cells but is relatively unknown in plant cells,
although FM4-64 staining suggests plants likely have
these types of vesicles [50,27]. Recently, nanogold
particles have been used to probe the plant endocytic
pathways . Detailed electron microscopic analysis of BY-2
protoplasts treated with positively and negatively charged
nanogold particles revealed the presence of
clathrindependent and -independent pathways that lead to
degradation or recycling . In Figure 2 we report the
pattern of CPN localization in granules near the cell
surface followed by accumulation in larger vesicles, and this
coincides with the pattern of FM4-64 staining. The
FM464 staining was more pronounced in CPN-treated cells
than in nontreated cells suggesting that dye uptake was
stimulated by CPNs. Further research using confocal
microscopy will be needed to follow the true path of
CPN internalization, and to learn if this pathway involves
receptor mediated endocytosis or an alternative carrier
mediated uptake that coincidently leads to increased
internal staining by FM4-64. In addition, the possibility
of CPNs entering the prevacuolar compartment is
intriguing, given that we show in later experiments
CPNsiRNA complexes are effective for PTGS. Based on these
data, it is worth speculating that if CPNs-siRNAs
complexes are endocytosed, that they are then released into
the cytoplasm. Perhaps there are cellular conditions that
enable siRNAs to dissociate from CPNs and to become
active in the silencing pathway. Therefore the route for
CPNs to enter prevacuolar vesicles from the endosome
or cytoplasm is not made obvious by these experiments.
If CPN uptake is via the endosome, then one explanation
is that siRNAs exit the endosome while CPNs remain
and are transported to the prevacuolar compartment.
Perhaps a shift in the pH of the endocytic compartment
causes dissociation of the complex. An alternative route
is that CPN-siRNA complexes exit the endosome,
dissociate in the cytoplasm, and then CPNs enter the
prevacuolar compartment by an undefined mechanism.
In total, CPNs present a promising tool for live cell
imaging of the endocytic trafficking pathways in plants.
This study shows that we can deliver positively charged
CPNs to protoplast culture medium and because of
their intrinsic fluorescence we can visually monitor the
route of uptake. Future research will examine the
possibility of recording the trafficking of CPNs from the
plasma membrane to and within the endocytic pathway
alongside fluorophores fused to endocytic markers. This
is an advance over the use of fusion proteins which are
often transgenically expressed. One limitation to
employing transgenic plants is that the protein fusions
are synthesized within the cell and their pathway to the
plasma membrane potentially overlaps with their
pathway of recycling from the plasma membrane back into
the cell. On the other hand CPNs can be delivered into
the culture medium and their intrinsic fluorescence can
be relied on to visually monitor the trafficking of
molecules within the endocytic pathway to obtain new
information about these pathways.
CPNs represent a significant advance in technology for
the delivery of siRNAs to plant protoplasts. Other
methods of siRNA delivery include the use of viral vectors,
electroporation, and polyethylene glycol. For example,
one advantage of CPNs-siRNA complexes over the use
of viral vectors is the ability to deliver siRNAs that
knockdown expression of genes that are vital cellular
functions without concern for viral pathology affecting
experimental outcomes. With respect to electroporation
and polyethylene glycol, it is possible that CPNs may
have a lower impact on protoplasts viability, although
further experiments are needed to compare
CPNmediated transfer of siRNAs with these other methods
to determine which are less disruptive to cell functions.
Because CPNs are nontoxic to protoplasts and are easily
added to culture medium, this technology could be
adapted to high throughput applications. It would be
straightforward to synthesize siRNAs targeting various
regulatory steps in a pathway, deliver CPN-siRNA
complexes to protoplasts, and then monitor the outcomes of
suppressing housekeeping genes on cellular functions.
We also learned that CPNs could be a valuable
imaging tool for plant biology. The endocytic pathway is
not as well explored in plants as in vertebrate systems.
Live cell imaging recording trafficking of CPNs via the
endocytic pathway could yield valuable new information
about membrane transport in plants.
Synthesis of CPNs
The fabrication of CPNs was previously described 
(Figure 7). Briefly, an amine-containing poly(phenylene
ethynylene) (PPE) was synthesized by polymerizing
2,2’(2,2’-(2,5-dibromo-1,4-phenylene)bis(ethane-2,1-diyl))bis(oxy)diethanamine in a
mixed solvent of N-methyl pyrrolidone and morpholine
using palladium/copper catalysts. The PPE solution was
treated with excess amounts of glacial acetic acid
followed by dialysis (10,000 MWCO) against dH2O. Final
PPE- dH2O solution was filtered using a syringe filter
(0.45 μm) and stored at room temperature.
Compound (1) is 13,13’-(2,5-dibromo-1,4-phenylene)bis
(oxy)bis(2,5,8,11-tetraoxatridecane) and was prepared by
incubating at 80°C overnight a suspension of
4-methylbenzenesulfonate1 (20.6 mM; 5.9 g), 2,5-dibromohydroquinone (10.3
mMl; 2.76 g) and K2CO3 (103 mM; 14 g) in 30 mL of
dimethyl formamide (DMF). The mixture was
concentrated in vacuo and diluted with 50 mL of
dichloromethane. The solution was washed with three times with
20 mL dH2O, dried over Na2SO4, and evaporated in
vacuo. The crude product was purified by column
chromatography (silica gel, ethyl acetate/hexane (3:1, v/v).
Yield : 3.7 g (55%). 1H NMR(600 MHz) : δ = 7.15 (s, 1H,
Ar-H, J = 6), 4.12 (t, 4H, Ar-OCH2, J = 6), 3.87 (t, 4H,
Figure 7 Fabrication of compounds 1, 2, and 3.
OCH2, J = 6), 3.76 (t, 4H, OCH2, J = 6), 3.69-3.63 (m, 16H,
OCH2), 3.55 (t, 4H, OCH2, J = 6), 3.37 (s, 6H, CH3); 13C
NMR(150 MHz) : δ = 150.5, 119.4, 111.6, 72.1, 71.3, 70.9,
70.8, 70.72, 70.4, 69.8, 59.2.
Compound (2) is
(2,5-bis(2,5,8,11-tetraoxatridecan-13yloxy)-1,4-phenylene)bis(ethyne-2,1-diyl)bis(trimethylsilane) and was prepared by adding Compound (1) (5 g,
7.7 mM) to a shrink flask fitted with a stir bar and Pd
(PPh3)2Cl2 (0.54 g, 0.77 mM) and CuI (0.073 g, 0.39
mM). Thirty mL of a 2:1 mixture of tetrahydrofuran and
diisopropylamine was added to the reaction. Following
addition of trimethylsilylacetylene (4.4 mL, 31 mM), the
reaction mixture was heated to 60°C for 12 h. After
evaporating solvent, the crude mixture was dissolved in
methylenechloride and washed twice with 30 mL
saturated ammonium chloride, followed by drying over
anhydrous MgSO4. The solvent was evaporated to
produce dark brown oil, which was purified by column
chromatography (silica gel, ethyl acetate/hexane (4:1, v/
v)). Yield : 5 g (95%). 1H NMR(600 MHz) : δ = 6.91 (s,
1H, Ar-H), 4.12 (t, 4H, Ar-OCH2, J = 6), 3.87 (t, 4H,
OCH2, J = 6), 3.78 (t, 4H, OCH2, J = 6), 3.68-3.66 (m,
12H, OCH2), 3.64 (t, 4H, OCH2, J = 6), 3.54 (t, 4H,
OCH2, J = 6), 3.38 (s, 6H, CH3), 0.25 (s, 9H, SiMe3);
13C NMR(150 MHz) : δ = 154.0, 117.9, 114.3, 100.9,
100.4, 72.0, 71.2, 70.8, 70.7, 70.6, 69.7, 69.6, 59.0, 0.0.
Compound (3) is 13,13’-(2,5-diethynyl-1,4-phenylene)
bis(oxy)bis(2,5,8,11-tetraoxatridecane). A 100 mL
roundflask was charged with the compound (2) (2.5 g, 3.7
mM), 50 mL tetrahydrofuran, and 10 mL MeOH. Then
5 mL of 1 M KOH (aq) solution was added to the
reaction mixture and stirred for 2 h. The solvent was
evaporated and the reaction mixture was purified by column
chromatography (silica gel, ethyl acetate). Yield : 1.1 g
(90%). 1H NMR(400 MHz) : δ = 7.00 (s, 1H, Ar-H), 4.15
(t, 4H, Ar-OCH2, J = 6), 3.87 (t, 4H, OCH2, J = 6), 3.76
(dd, 4H, OCH2, J = 6), 3.73-3.63 (m, 16H, OCH2, J = 6),
3.55 (dd, 4H, OCH2, J = 6), 3.38 (s, 6H, CH3, J = 6),
3.35 (s, 2H, CH); 13C NMR(150 MHz) : δ = 154.1,
118.3, 113.6, 82.8, 79.6, 71.9, 71.1, 70.7, 70.6, 70.5, 69.6,
Preparation of siRNAs for cellulose synthases, NtCesA-1a
TRIzol® reagent was used to extract total RNA from
tobacco (N. tabacum) leaves (Invitrogen Corp, Carlsbad,
CA). cDNA of the putative cellulose synthase (AF233892),
NtCesA-1a and NtCesA-1b was prepared using Superscript
III reverse transcriptase (Life Technologies) and then a
fragment of the gene was PCR amplified (640 base pairs
(bp) using forward (5’-
AGTGTATGTGGGTACCGGATG- 3’) and reverse (5’- CCATATGGGACA
ATGCCTAC - 3’) primers. Following a 5-min denaturation at 94°
C, PCR was performed for 34 cycles of 94°C for 2 min, 94°
C for 15 s, 50°C for 30 s, and 68°C for 45 s, followed by
final 5-min extension at 68°C . The 640 bp PCR
product was purified using PCR Preps DNA Purification
system (Promega, Madison,WI) and cloned into the pGEM-T
Easy vector (Promega) according to manufacturer’s
instructions. The nucleotide sequence of this cDNA
fragment was confirmed as 100% and 98% identical for
NtCesA-1a and NtCesA-1b, respectively. To generate
sense and anti sense RNA, pGEM-T:NtCesA was
linearized using NcoI or SalI and in vitro transcription was
performed (RiboMAX, Promega) with SP6 and T7 RNA
polymerases. Transcription products were purified using
MEGAclear Kit (Ambion, Austin, TX). Sense and anti
sense RNAs were annealed in annealing buffer (100 mM,
KOAc, 4 mM MgCl2 and 60 mM HEPES- KOH, pH 7.4),
boiled for 5 min, and incubated overnight at 37°C .
The resulting double strand RNAs were precipitated using
ethanol and then dissolved in nuclease-free double
distilled (dd) H2O. siRNAs were generated by treating double
stranded NtCesA-1 with recombinant Dicer enzyme
according to the manufacturer’s instructions (Gene
Therapy Systems, San Diego, CA). The reaction was stopped by
adding the Dicer stop solution and 22 bp products were
detected using 3% agarose gel electrophoresis . The
final siRNA products were purified using RNA purification
column 1(Gene Therapy Systems) and dissolved in
BY-2 protoplast preparation
Typically, BY-2 cells are grown are subcultured from a 4
d-old liquid culture by transferring 10 mL BY-2 cells to
40 mL fresh BY-2 culture medium (Murashige and
Skoog salts pH 5.6 (Sigma-Aldrich Co, St. Louis, MO),
30 g.L-1 sucrose, 256 mg.L-1 KH2PO4, 100 mg.L-1
myoinositol, 1 mg.L-1 thiamine, and 0.2 mg.L-1
2,4-dichlorophenoxyacetic acid). Cells are grown on a rotary shaker
that is maintained in the dark at 120 rpm at 28°C.
Protoplasts were prepared from 3 d old tobacco BY-2
suspension cells using standard methods [52,53]. Ten ml
of packed BY-2 cells (sedimented by centrifugation at
100-g for 5 min) were resuspended in 100 mL of
enzyme solution (1.5% cellulase “Onozuka RS” (Yakult
Pharmaceutical Ind. Co. Ltd., Tokyo, Japan), 0.2%
macerase (Calbiochem-Novabiochem Corp., La Jolla,
CA), 0.5 M mannitol, and 3.6 mM 2-(N-morpholino)
ethanesulfonic acid (pH 5.5) ) in a 1 L flask and
incubated for 3-4 h at 28°C on a rotary shaker at 100 rpm.
Protoplasts were recovered by filtration through 41 μm
nylon mesh (Spectrum Laboratories, Inc., Rancho
Dominguez, CA), and washed twice with Protoplast Wash
Solution (0.5 M mannitol, 3.6 mM 2-(N-morpholino)
ethanesulfonic acid (pH 5.5) at 59 g for 5 min.
Protoplasts were resuspended in Protoplast Resuspension
Solution (BY-2 culture media plus 0.45 M mannitol) to
a density about 1 × 106 protoplasts mL-1. Protoplast
viability was measured using 0.1% fluorescein diacetate
prepared in 1 ml of 50 mM phosphate buffer (pH 7.4). For
siRNA delivery experiments, protoplasts were cultured
in cell wall regeneration medium.
siRNA delivery to protoplasts and intact BY-2 cells
Protoplasts (1 × 106mL-1) or intact BY-2 cells (1 ×
1) were mixed with CPNs in Protoplast Resuspension
Solution to a final concentration of 0, 5, 10, 15 and 20 μM
and added to 6-well culture plates (Corning Inc., Corning,
NY). Each well of the culture plate was lined with
Protoplast Resuspension Solution plus 1.0% agarose (pH 5.6).
Protoplasts and BY-2 cells were cultured at 28°C. Samples
were harvested at 2, 5, 10 and 24 h and the proportion of
CPN containing protoplasts and intact BY-2 cells were
determined using a haemocytometer. In addition, the
proportion of protoplasts or cells for which fluorescence was
seen to be associating with the cell wall, plasma
membrane, nucleus, and/or cytoplasm was recorded.
Protoplasts were resuspended in Protoplast
Resuspension Solution (BY-2 culture media plus 0.45 M mannitol)
to a density about 1 × 105 protoplasts.mL-1. CPNs (10 or
25 μM) were incubated with 200 nM siGLO Red for 3 h
(Thermo Fisher Scientific, Pittsburgh, PA) and the
complex was delivered to protoplasts. CPNs were incubated
with 200 nM NtCesA-1 siRNAs overnight and then
delivered to protoplasts. We found that mixing CPNs and
siGLO Red for 3 h was sufficient to demonstrate
transfection of CPN-siRNA complexes, but overnight mixing of
CPNs and NtCesA-1 siRNAs improved complexation and
improved siRNA delivery to plant protoplasts resulting in
a measurable phenotype. Protoplast cultures were
maintained in the dark at 28°C for 2 h and 24 h. Controls
included treating protoplasts with Protoplast Resuspension
Solution, 200 nM siGLO Red only, NtCesA-1 siRNAs or
CPNs only. The protoplast containing both CPNs and
siGLO Red were counted using a haemocytometer.
Propidium iodide and FM4-64 dye treatment
Propidium iodide, contained in the Plant Cell Viability
Assay Kit (Sigma-Aldrich Co), was solubilized according
to manufacturer’s instructions. FM4-64 (Invitrogen Corp)
staining was carried out to monitor CPN uptake, as
previously described . Protoplasts (1 × 105.mL-1) were
incubated with 10 μM CPNs for 24 h at 28°C. 20,000
protoplasts (which were previously treated with medium or
10 μM CPNs and incubated for 24 h at 28°C) were
incubated with 10 μM FM4-64 at room temperature for 10
min and then monitored using confocal microscopy.
Epifluorescence and confocal microscopy
A Nikon E600 (Nikon Corp., Tokyo, Japan) epifluorescence microscope with a B2A filter cube (470- to 490-nm
excitation filter), a DM505 dichroic mirror, and a BA520
barrier filter was used to monitor FDA staining following
enzymatic digestion of BY-2 cells and to study uptake of
CPNs protoplasts and intact BY-2 cells. Propidium iodide
was detected in protoplasts using a UV filter cube. siGLO
Red fluorescence (absorption/emission maximum at 557
nm/570 nm) was viewed using a Y-2E/C TX red filter
cube containing a 540- to 580-nm excitation filter, a
DM595 dichroic mirror, and a BA600-660 barrier filter.
Images were captured using the Optronics Magnafire
camera (Optronics Inc., Goleta, CA) and were edited
using Adobe Photoshop software (Adobe Systems Inc.,
San Jose, CA). Haemocytometer observations were
recorded using Microsoft Excel software.
A Leica TCS SP2 (Leica Microsystems, Bannockburn,
IL) confocal imaging system attached to a Leica DME
14 upright microscope equipped with Ar/Kr lasers were
used to study BY-2 cells treated with CPNs and FM4-64
staining protoplasts. Serial images were collected using
0.3 μm steps and 3-D images of 100 μm thick sections
Fluorescence activated cell sorting (FACS) flow-cytometry
of treated BY-2 protoplasts
A Becton Dickinson FACS Calibur flow cytometer
(Becton Dickinson, Franklin Lakes, NJ) equipped with an Ar
laser (excitation of 488 nm) was used to assess
CPNuptake by protoplasts. Protoplasts (1 × 106.mL-1) were
mixed with 10 μM and 20 μM CPNs in Protoplast
Resuspension Solution and added to 6-well culture
plates (Corning Inc., Corning, NY) containing Protoplast
Resuspension Solution plus 1.0% agarose (pH 5.7).
Protoplasts treated with buffer, or a 1:1 mixture of
untreated plus CPN-treated protoplasts were used as
controls. Protoplasts were cultured at 28°C and FACS
was performed at 2 h and 24 h of incubation.
The sorting capability of 10000 cells.s-1 and
fluorescence emission (FL1-H, FL2-H) was detected using a
530 nm and 585 nm band pass filters. The percentages
of fluorescence-emitting protoplasts were assessed as
evidence of CPNs and siGLO Red uptake by protoplasts.
Data were analyzed on a Macintosh computer equipped
with BD CellQuest Pro program (Becton Dickinson) and
were presented as two dimensional dot plots which
represent CPN fluorescence emissions on the X-axis
and siGLO Red fluorescence emissions on Y- axis. Data
was compiled using Adobe Photoshop software.
Semi-quantitative RT-PCR of silenced protoplasts
Semi-quantitative RT-PCR was utilized to monitor
NtCesA transcriptional levels following siRNA delivery.
Extraction of total RNA from BY-2 protoplasts was
carried out using SV Total RNA Isolation System
(Promega, Madison, WI). The first strand cDNA was
synthesized using SuperScript III reverse transcriptase
(Invitrogen Corp), 1 μg total RNA and oligo(dT)
primers. PCR was performed using NtCesA-1 specific
forward primer (5’-AGTGTA TGTGGGTACCGGATG-3’)
and NtCesA reverse
(5’-CCATATGGGACAATGCCTAC-3’) primer that also shares homology with
2. Forward (5’-GCCTCCGTGGTGGTG CTAAG- 3’),
and reverse (5’-TCAATCGGCACC GGCCTT G-3’)
primers were used to amplify ubiquitin (AY912494) cDNA
(261 bp) as the internal control. Following 10-min
denaturation at 95°C, PCR was performed for 30, 35, 40, 45
cycles of 95°C for 15 s and 60°C for 60 s. PCR products
were analyzed using ethidium bromide stained 1%
agarose gel. Gels were scanned using Alpha Image system
(Alpha Innotech, San Leandro, CA) and the images
were recorded. Densitometry was performed by Alpha
Ease FC software (Alpha Innotech).
CPN: conjugated polymer nanoparticles; PTGS: post-transcriptional gene
silencing; ddH2O: double distilled water; miRNA: microRNA; siRNA: small
interfering RNA; TRV: Tobacco rattle virus; T-DNA: Agrobacterium tumefaciens
based plasmid that can transform plant tissues; dsRNA: double stranded
RNA; CbLCV: Cabbage leaf curl virus; FDA: Fluorescein diacetate
We gratefully thank Dr. Changming Ye for assistance with primer design and
semi-quantitative PCR. This research was supported by ICx Technologies,
Oklahoma Center for Advancement of Science and Technology (OCAST)
ONAP08-018. JM acknowledges the Florida International University for
Faculty Startup Funds.
AS carried out plant protoplast experiments. JV and JM oversaw the analysis,
design and implementation. AN carried out CPN preparations. JV and JM
drafted the manuscript. All authors read and approved the final manuscript.
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