Plant Physiological Adaptations to the Massive
Foreign Protein Synthesis Occurring in
Recombinant Chloroplasts[W]
Julia Bally, Marie Nadai, Maxime Vitel, Anne Rolland, Raphael Dumain, and Manuel Dubald*
Bayer CropScience, Bioscience, F–69263 Lyon cedex 09, France (J.B., M.N., M.V., A.R., R.D., M.D.); and Centre
National de la Recherche Scientifique-Bayer CropScience Joint Laboratory, UMR 5240, F–69263 Lyon cedex 09,
France (J.B.)
Genetically engineered chloroplasts have an extraordinary capacity to accumulate recombinant proteins. We have investigated
in tobacco (Nicotiana tabacum) the possible consequences of such additional products on several parameters of plant
development and composition. Plastid transformants were analyzed that express abundantly either bacterial enzymes, alkaline
phosphatase (PhoA-S and PhoA-L) and 4-hydroxyphenyl pyruvate dioxygenase (HPPD), or a green fluorescent protein (GFP).
In leaves, the HPPD and GFP recombinant proteins are the major polypeptides and accumulate to higher levels than Rubisco.
Nevertheless, these engineered metabolic sinks do not cause a measurable difference in growth rate or photosynthetic
parameters. The total amino acid content of transgenic leaves is also not significantly affected, showing that plant cells have a
limited protein biosynthetic capacity. Recombinant products are made at the expense of resident proteins. Rubisco, which
constitutes the major leaf amino acid store, is the most clearly and strongly down-regulated plant protein. This reduction is
even more dramatic under conditions of limited nitrogen supply, whereas recombinant proteins accumulate to even higher
relative levels. These changes are regulated posttranscriptionally since transcript levels of resident plastid genes are not
affected. Our results show that plants are able to produce massive amounts of recombinant proteins in chloroplasts without
profound metabolic perturbation and that Rubisco, acting as a nitrogen buffer, is a key player in maintaining homeostasis and
limiting pleiotropic effects.
The genetic modification of the plastid genome was
achieved in higher plants more than 15 years ago (Svab
et al., 1990; Svab and Maliga, 1993). This recombinant
technology presents distinctive features that are very
attractive from a biotechnological perspective. The
most attractive of these features is the potential for
extremely high expression of the transgene in plastid
transformants, up to 70% of total soluble proteins (tsp)
in leaves for an antibacterial lysin (Oey et al., 2009). A
variety of pharmaceutical proteins have also been
produced at very high levels in transgenic chloroplasts
(Daniell et al., 2004; Daniell, 2006). The other characteristics of the technology have been extensively
reviewed recently (Maliga, 2004; Bock, 2007; Verma
and Daniell, 2007; Dubald et al., 2008) and concern (1)
the targeted insertion of the transgenes, (2) the possibility to engineer more easily complex pathways using
polycistronic vectors, (3) the apparent absence of epigenetic regulation, and (4) the natural confinement of
* Corresponding author; e-mail manuel.dubald1@bayercropscience.
com.
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy
described in the Instructions for Authors (www.plantphysiol.org) is:
Manuel Dubald ([email protected]).
[W]
The online version of this article contains Web-only data.
www.plantphysiol.org/cgi/doi/10.1104/pp.109.139816
1474
the transgenes as a result of the almost exclusive
maternal inheritance of these organelles.
Chloroplasts have an extraordinary capacity to synthesize and accumulate foreign proteins. Curiously,
very little attention has been devoted to evaluate and
analyze the consequences on the plant physiology of
this significant metabolic burden. In most reports,
which include an insecticidal toxin expressed at 46%
tsp (De Cosa et al., 2001), or a GFP expressed at 38%
tsp (Yabuta et al., 2008), no obvious phenotypic defect,
such as growth retardation, has been observed in
plastid transformants. When phenotypic modifications
were noted, these were directly linked to the specific
properties of the expressed transgenes (Tregoning et al.,
2003; Magee et al., 2004; Ruiz and Daniell, 2005;
Chakrabarti et al., 2006; Hasunuma et al., 2008; Tissot
et al., 2008). Only in the case of lysin was the hyperexpression of the recombinant protein reported to limit
plant development by exhausting the protein synthetic
capacity of chloroplasts (Oey et al., 2009). A number of
issues are therefore still very unclear: (1) are the recombinant proteins produced on top of the resident
proteins, meaning that plants naturally have the capacity to make significantly more proteins, at least if a sink
is provided? Otherwise, (2) are they produced at the
expense of all, or of only some, resident proteins,
implying that these resident proteins are normally
synthesized in excess? And (3), how are resources
allocated between resident and recombinant proteins
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How Plants Cope with Massive Recombinant Protein Synthesis
when the cell budget is reduced, in particular when
there is a limitation in nitrogen supply?
Plastid transformants expressing recombinant proteins at a high level provide a unique material to
address these fundamental questions. To draw generic
conclusions, we have for the first time, to our knowledge, studied in parallel transgenic tobacco (Nicotiana
tabacum) lines expressing recombinant proteins of completely different nature: (1) a hydroxyphenyl pyruvate
dioxygenase (HPPD) from Pseudomonas (Dufourmantel
et al., 2007), which participates in plants in the synthesis
of plastoquinones and is the target of various herbicides (Matringe et al., 2005); (2) an alkaline phosphatase
from Escherichia coli (Bally et al., 2008) with no known
substrate in chloroplasts, targeted to the thylakoids
(PhoA-L) or expressed at a lower level in the stroma
(PhoA-S); and (3) a GFP, with no enzymatic function,
accumulating strongly in the stroma. We have started
investigating the impact that massive transgene expression in chloroplasts may have on plant development, photosynthesis, leaf proteome, chloroplast
transcriptome, and amino acid composition.
RESULTS
Chloroplast Expression Vectors and Transgenic
Tobacco Lines
Tobacco plants of generation T1 obtained by selfing
and corresponding to four different vectors (Fig. 1)
were selected for analysis. These vectors target the
transgenes encoding alkaline phosphatase (PhoA-S or
PhoA-L), HPPD, or GFP to the same integration site
between the Rubisco large subunit (rbcL) and acetylCoA carboxylase subunit D (accD) chloroplast genes.
The coding regions are under the control of the strong
tobacco plastid PSII subunit D1 (psbA) gene promoter,
including its complete 5# untranslated region, except
for GFP. The latter is expressed from a dicistronic
cassette driven by the corn (Zea mays) 16SrDNA plastid
promoter (Prrn) and the ribosome binding site region
from phage lambda gene 10 (Ye et al., 2001). The
generation of homoplasmic tobacco lines and their
analysis at the DNA and protein levels has already
been documented for HPPD (Dufourmantel et al.,
2007) and PhoA (Bally et al., 2008). Concerning
pCLT554, GFP was recoded to better fit the tobacco
plastid codon usage, and we selected for analysis one
T0 transformant that displayed under UV a uniform and
strong GFP signal in leaves, localized in the chloroplasts. The T1 progeny of this transformant uniformly
expressed both antibiotic resistance and GFP fluorescence (Supplemental Fig. S1) as expected for a homoplasmic tobacco plastid transformant.
Normal Growth with Diminished Rubisco Levels in
Hyperexpressing Lines
The growth of the transgenic lines (T1 generation)
was carefully followed in the greenhouse, and no
Figure 1. Recombinant plastome genetic maps. A, Targeted tobacco
plastid genome region. B, Transformation vectors. LHRR and RHRR are
the left and right plastid recombination regions, respectively, present in
all transforming vectors. RBS, Ribosome binding site; g10L, phage
lambda gene 10; aadA, spectinomycin resistance gene. All promoters
and terminators were derived from the tobacco plastid genome except
in pCLT554 (Zm, corn).
phenotypic difference was observed compared to
wild-type tobacco during the vegetative phase (Fig.
2, A–C). Accordingly, no variation of the maximum
photochemical efficiency of PSII in the dark-adapted
state (Fv/Fm; 0.73–0.76) or of the effective quantum
yield (Fig. 2D) was measured by fluorometry on leaves
of 3-month-old plants. Expression of the recombinant
proteins in mature leaves was analyzed by SDS-PAGE
and Coomassie Brilliant Blue staining (Fig. 3A). Rubisco large (LSU) and small (SSU) subunits are the
prevalent polypeptides in wild-type tobacco. In contrast, the HPPD and GFP recombinant proteins are
expressed at an extraordinary high level and represent
in the transgenic leaves the major protein. We have
scanned the gel and quantified by densitometry the
four recombinant proteins, LSU, and SSU. The HPPD
and GFP proteins are expressed at a level close to that
of LSU in wild-type tobacco (Fig. 3B) and represent
30% to 40% tsp. When the LSU and SSU amounts are
plotted against the level of recombinant protein, there
is a clear negative correlation (Fig. 3B). Over this range
of values, the LSU to SSU ratio remains always close to
3 to 4, which matches the ratio of their respective Mrs
and is in accordance to their equal stoichiometric
contribution in Rubisco. This shows that the expression of the nucleus-encoded SSU remains tightly coupled to the expression of the plastid-encoded LSU,
suggesting that the plastid-to-nucleus retrograde signaling pathways (Nott et al., 2006) are not perturbed.
The level of many other proteins is visibly constant,
suggesting that the down-regulation of Rubisco is
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Bally et al.
Figure 2. Phenotype of transgenic lines. Comparison of T1 generation growth 2 weeks (A), 5 weeks
(B), and 3 months (C) after sowing for the wild
type (WT; 1) and for recombinant lines expressing
PhoA-S (2), PhoA-L (3), GFP (4), and HPPD (5). D,
Comparison of effective PSII photochemical
quantum yields of 3-month-old plants. Error bars
represent the SD calculated for each category from
two leaves with three independent measurements
per leaf.
specific. At the flowering stage, a specific difference
was observed in flowers of plants expressing HPPD,
which systematically lacked the usual pink pigmentation due to anthocyanins (Supplemental Fig. S2A). The
plastid transgenes encoding HPPD, GFP, and PhoA-L
are strongly expressed in the chromoplasts of tobacco
corolla (Supplemental Fig. S2B), albeit at a lower level
than in leaf chloroplasts.
Plastid Resident Transcript Levels Are Not Affected in
Transgenic Lines
High-level transgene expression could negatively
impact the plastid resident transcriptome and be responsible for the depressed level of Rubisco. A semiquantitative reverse transcription (RT)-PCR analysis
was carried out on total RNA extracted from mature
leaves at the same stage during the day. Sets of specific
primers, amplifying unique fragments, were selected to
follow in parallel the recombinant RNAs and nine
resident transcripts synthesized either by the plastidencoded polymerase (PEP) and/or by the nuclearencoded polymerase. The results (Supplemental Fig.
S3) show that the recombinant RNAs are particularly
abundant and that there is no drastic change in the level
of any of the analyzed plastid resident transcripts.
There is no variation in the amount of rbcL transcripts
despite the large variation reported above at the protein
level (Fig. 3). This is consistent with the fact that
translation initiation is considered as the limiting step
in chloroplast protein synthesis, and there is generally a
poor correlation between transcript and corresponding
protein levels (Eberhard et al., 2002). It is also noteworthy that (1) there is no up-regulation of accD transcripts,
a gene poorly transcribed by nuclear-encoded polymerase that flanks the strongly PEP-transcribed transgenes at their 3# ends; and (2) there is no variation at the
RNA level for psbA despite the use of its promoter and
5# untranslated region to drive the expression of recombinant PhoA and HPPD.
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How Plants Cope with Massive Recombinant Protein Synthesis
is observed for plants treated with 0.2 mM ammonium
nitrate. Whatever the nitrogen supply, even at the
suboptimal 2 mM concentration, there is no difference
in growth between transgenic lines or versus wildtype tobacco.
Tobacco Leaf Protein Amino Acid Composition under
Various Nitrogen Regimes
Figure 3. Protein profiles of leaves after SDS-PAGE separation. A,
Coomassie Brilliant Blue staining of protein extracts (20 mg) from wildtype tobacco (1) and from plants expressing HPPD (2), PhoA-S (3),
PhoA-L (4), and GFP (5). The position of the recombinant proteins is
indicated in the respective lanes. B, Correlation between Rubisco (LSU
in blue; SSU in red) and recombinant proteins expressed in arbitrary
units (AU). WT, Wild type.
Limiting Nitrogen Supply Does Not Unveil Any
Growth Penalty
Under normal growing conditions, we observed no
difference in the growth and development of tobacco
plants expressing extremely high levels of GFP or
HPPD, despite their depressed level of Rubisco. We
decided to investigate this situation in conditions of
limited nitrogen and to analyze how resources are then
allocated between resident and recombinant proteins.
Three-week-old wild-type and transgenic seedlings
from generation T1, sown in soil, were transplanted on
vermiculite and drenched every 2 to 3 d with a
nutritive solution containing ammonium nitrate as
the sole source of nitrogen. Four different concentrations of ammonium nitrate were provided, from 0 to 20
mM, which is the concentration used in standard plant
tissue culture media (Murashige and Skoog, 1962), and
the experiment was followed over 5 weeks before
leaves were harvested for analysis. Five days after the
start of the experiment, a positive effect on growth is
already visible on all lines at the highest nitrogen dose
(Fig. 4A-1). After 5 weeks (Fig. 4A-2), plants submitted
to complete nitrogen deprivation have not grown at all
and have become chlorotic. Very limited development
After 5 weeks, proteins were extracted from leaves
of plants shown on Figure 4A-2 and separated by SDSPAGE (Fig. 4B). At 20 mM ammonium nitrate, providing nitrogen in excess, the pattern is very similar to
that of plants grown in soil (Fig. 3). Remarkably, when
the supply of nitrogen is limited (below 2 mM), the
amount of Rubisco (LSU and SSU) is very strongly
reduced, whereas in all cases, the recombinant proteins accumulate at least at the same level as under
optimal conditions. PhoA-L, expressed at a much
lower level than HPPD or GFP, becomes the major
leaf polypeptide, exceeding largely LSU at 2 mM and
below. Even PhoA-S, which is normally completely
masked by LSU, becomes clearly visible under those
conditions (Fig. 4B).
One question that arises is whether the recombinant
proteins are synthesized in addition to the resident
proteins or if they are produced at their expense.
Leaves were harvested and the total amino acid content (free + protein-bound) was determined for each
line and at each nitrogen level. The total amino acid
content is positively correlated to the supply in ammonium nitrate, and whatever the level of nitrogen,
transgenic plants that express recombinant proteins
clearly don’t have an increased total content in amino
acids (Supplemental Fig. S4A). The amino acid composition of transplastomic and wild-type leaves from
plants grown under different conditions of nitrogen
supply was then compared (Supplemental Fig. S4B),
showing that most amino acids remain quantitatively
stable, the strongest variation being noted for Lys,
which increases in proportion with the level of nitrogen.
DISCUSSION
Our objective was to investigate how plants cope
with the massive expression of an alien gene, a situation frequently encountered with plastid transformants. Indeed, two of the transgenic tobacco lines that
we have analyzed express recombinant proteins
(HPPD and GFP) in chloroplasts at spectacular high
levels, above those of Rubisco, which is normally the
most abundant leaf protein, representing up to 60%
of the soluble proteins in C3 plants (Spreitzer and
Salvucci, 2002; Hirel and Gallais, 2006). Rubisco has
a dual role, first in carbon fixation, and second as a
potential dynamic nitrogen store, since it is present in
excess versus the amount needed to fulfill the photosynthetic requirements (Irving and Robinson, 2006;
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Bally et al.
Figure 4. Effect of nitrogen supply on growth and proteome. A, Tobacco seedlings of the wild type and of the four different
transgenic lines 5 d (1) or 5 weeks (2) after treatment with nutritive solutions containing 0 to 20 mM ammonium nitrate as unique
nitrogen source. B, SDS-PAGE and Coomassie Brilliant Blue staining of leaf proteins (20 mg) harvested after 5 weeks of treatment.
WT, Wild type.
Feller et al., 2008). The nitrogen store function of
Rubisco has been essentially documented during leaf
senescence, when proteins are remobilized and translocated for seed filling (Murchie et al., 2002; Houtz and
Portis, 2003). Hyperexpressing plastid transformants
provide an interesting model for studying protein
homeostasis, Rubisco turnover, and the plant cell
nitrogen budget. We have found that total amino
acid content of leaves overexpressing HPPD or GFP
is unchanged versus the wild type, showing that the
synthesis of the recombinant proteins occurs at the
expense of the resident proteins. Our data show that
this concerns particularly Rubisco, the major nitrogen
and amino acid pool in plant cells. The reduced level
of this enzyme could be the result of a down-regulated
synthesis or of a higher turnover. The latter hypothesis
is more likely, based on the literature related to enhanced and programmed Rubisco degradation during
senescence or under environmental stress (Houtz and
Portis, 2003; Hirel and Gallais, 2006; Feller et al., 2008).
The same compensation between Rubisco and the
recombinant protein(s) also occurs very clearly for
the high-level transient leaf expression system, based
on viral replicons (Marillonnet et al., 2005). The buffering role of Rubisco also occurs on wild-type plants
under conditions of limited nitrogen (Fig. 4). Interestingly, a recent large-scale proteomic analysis performed on nuclear transgenic rice (Oryza sativa),
expressing a human therapeutic protein in the endosperm, has also reported a decrease in endogenous
storage proteins (Luo et al., 2009).
A scenario can also be envisaged at the RNA level,
where the specific down-regulation of Rubisco would
result from the presence of the tobacco rbcL terminator
in the transgenes cassettes (Fig. 1). High-level expression of the transgenes could compete for RNA binding
factors essential for the stability or translation of the
rbcL transcript. This has been reported for the resident
tobacco chloroplast clpP gene when transgenes incorporated the 5# untranslated region of this gene (Kuroda
and Maliga, 2002). Nevertheless, this scenario is not
compatible with our finding that the steady-state
transcript levels of recombinant PhoA-S or PhoA-L
are similar to those of GFP or HPPD (Supplemental
Fig. S3) but do not strongly impact the level of Rubisco
(Fig. 3).
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How Plants Cope with Massive Recombinant Protein Synthesis
We also investigated whether there is some obvious
alteration of the chloroplast transcriptome in tobacco
plastid transformants, as a consequence of high-level
transgene expression or of competition for transcription or messenger stabilizing factors. The transcript
levels of nine plastid resident genes transcribed either
by the plastid-encoded and/or nuclear-encoded polymerase (for review, see Hess and Börner, 1999) have
been followed by quantitative RT-PCR. To our knowledge, this type of analysis has not been reported yet in
the literature for any higher-plant plastid transformant. We have found that the transgenes driven by
either the psbA (PhoA-S, PhoA-L, and HPPD) or fulllength Prrn (GFP) have a very high mRNA steady
state, only two to four times lower than the resident
psbA mRNA. No drastic or general modification has
been observed for any of the nine analyzed resident
transcripts.
We have followed the development of transgenic
lines expressing PhoA-S, PhoA-L, GFP, and HPPD and
compared it to wild-type tobacco. No visible difference
in leaf pigmentation, growth rate, flowering time, and
PSII quantum yields has been noted, despite the
massive accumulation of HPPD or GFP, accompanied
by a sharp drop of Rubisco in those lines. The absence
of penalty on plant development of such a reduction is
surprising but in line with results from past experiments using antisense RNA to down-regulate the rbcS
gene. These studies have shown that a visible phenotype is only observed in very severely impaired lines
(Quick et al., 1991; Stitt et al., 1991; Hudson et al.,
1992). Our results contrast with the recent report
mentioning an exhaustion of the protein synthetic
capacity of the chloroplast and a strongly impaired
growth in tobacco lines expressing massively a phage
lytic protein (Oey et al., 2009). This could be the consequence of this lysin’s extraordinary expression level
(70% tsp). Nevertheless, this general statement was
extrapolated only from the observed down-regulation
of Rubisco and could also result from a toxic effect of
the recombinant protein. A global exhaustion of the
chloroplast protein synthetic capacity should also
negatively impact the level of the PEP, and therefore
the level of transcripts produced by PEP, at least if the
amount of this enzyme limits transcription under
normal conditions. This was not observed in this study
for the rbcL transcript though (Oey et al., 2009). Our
results show that at least up to a recombinant protein
expression level of 30% to 40% tsp there is no penalty
for plant growth in our greenhouse conditions. Up to
that expression level also, there is no impact on PSII
quantum yield measurements, showing that the biosynthesis of the various plastid-encoded subunits of this
photosystem is not affected as well as the rest of the
electron transport chain and the major Calvin processes.
The quantitative drop of Rubisco is possibly compensated for by a higher specific activity that is dependent on its catalytic chaperone, Rubisco activase
(Parry et al., 2003; Portis, 2003). Differences in growth
could possibly be detected under other environmental
conditions where the photosynthetic activity of Rubisco could be limiting, such as higher light or temperature stress, since a reduced growth rate has for
instance been reported for antisense tobacco lines
having ,40% of normal Rubisco level (Jiang and
Rodermerl, 1995).
It could also be anticipated that under conditions of
limited nitrogen supply, the cost of the massive synthesis of a recombinant protein would become apparent and differences in growth observed. This was not
the case in our conditions over a 5-week period,
whatever the supply in ammonium nitrate (from
0–20 mM). Growth of tobacco seedlings was affected
in proportion to the severity of the nitrogen deficiency
but independent of the genetic background. This illustrates the extraordinary metabolic plasticity of
plant cells that can adapt under various conditions to
the additional important sink represented by the recombinant proteins. At the molecular level, after 5
weeks, the major modification that was observed in
the protein profile is a further specific reduction in the
amount of Rubisco allowing visualization on Coomassie blue-stained one-dimensional gels with recombinant PhoA-S and PhoA-L (50 kD), which are normally
masked by the major 53-kD large subunit of Rubisco.
PhoA-L even becomes the most abundant leaf protein
when the nitrogen supply is limited, and the levels of
GFP and HPPD then clearly exceed 50% of the leaf
soluble proteins. The proteolytic degradation machinery of plant cells is known to regulate the chloroplast
protein composition and Rubisco turnover (Feller
et al., 2008) and seems therefore less able to deal
with any of the foreign proteins (HPPD, Pho-A, or
GFP). As a consequence, these products accumulate to
higher relative levels. This finding has potential interest for molecular farming applications. A nitrogen
starving step applied on transgenic plant material
before protein extraction could increase the relative
proportion of the recombinant protein of interest and
therefore facilitate its purification.
The total amino acid composition (free and proteinbound) of leaves of plantlets grown hydroponically at
different nitrogen levels has been measured. The total
leaf amino acid content per fresh weight drops
strongly when ammonium nitrate is limiting, but there
is no difference between transgenic lines or versus the
wild type. Also, despite the fact that under nitrogen
deficiency the recombinant proteins often become by
far the predominant leaf protein (HPPD and GFP), the
global amino acid composition remains rather constant whatever the growing conditions. The reason for
this remarkable stability is probably due to the fact
that the amino acid composition of the expressed
recombinant proteins is not strikingly different from
those of Rubisco or wild-type leaves (Supplemental
Fig. S4C). This may in turn also explain why these
recombinant proteins can be expressed at such a high
level by plant cells.
The massive expression of a transgene in chloroplasts surprisingly has a rather low impact on plant
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Bally et al.
development and physiology. The recombinant proteins are synthesized at the expense of resident proteins, and Rubisco works in this respect as the major
source of nitrogen and of protein homeostasis. Interestingly, foreign proteins proportionally accumulate at
even higher levels when the nitrogen budget is limited, escaping somehow from the programmed plant
degradation machinery. We have detected only limited
changes on the one-dimensional protein profile of
leaves, the effective and maximum quantum yields
of PSII, the amino acid composition, and the transcript
level of plastid resident genes. This article provides a
set of data on how plants cope globally with high-level
transgene expression in leaves. We are currently proceeding to a more detailed leaf proteomic analysis to
determine if proteins other than Rubisco are affected
by high-level recombinant protein expression. The
consequences on other parts of the plant metabolism
under different growing conditions also merit further
investigation.
tions and was reverse transcribed using random hexamer primers and the
Thermoscript RT-PCR system (Invitrogen). The amplification of the different
target cDNAs was performed simultaneously with a LightCycler (Roche)
using the LightCycler FastStart DNA MasterPlus SYBR Green I kit (Roche
Applied Science). Each sample was run in triplicate starting with 5 ng of
cDNA, and very little variation was observed between repetitions. The
amplification conditions were the following: 10 min activation step at 95°C
for one cycle, 10 s of denaturation at 95°C, 5 s of annealing of primers at 60°C,
and 10 s of elongation at 72°C, for 45 cycles. The primer sets used in the
experiments are listed in Supplemental Figure S3B. Data analysis was
performed using the comparative Cp method described by Livak and
Schmittgen (2001). The most abundant plastid transcript according to our
analysis (16SrRNA) was chosen as reference.
Hydroponic Nitrogen Supply
Transplastomic tobacco and wild-type seeds were sown in soil in the
greenhouse at a temperature of 25°C with a daily lighting of 16 h. After 3
weeks, the seedlings were washed with water, transferred to trays filled with
vermiculite, and then received by drenching every 2 to 3 d a nutrient solution
containing microelements, iron, and vitamins (nicotinic acid, pyridoxine, and
thiamine) according to Murashige and Skoog (1962) and macroelements
according to Heller (1953) but without sodium nitrate. Different concentrations of ammonium nitrate were then added to this basal solution.
Protein Content and Amino Acid Composition
MATERIALS AND METHODS
Transformation Vectors and Plant Material
Transformation vectors pCLT515, pCLT516, and pCLT111 and the analysis
of the tobacco (Nicotiana tabacum) transgenic lines (cv PBD6) expressing PhoA
and HPPD are described by Bally et al. (2008) and Dufourmantel et al. (2007),
respectively. Transgenic tobacco lines (cv PBD6) expressing GFP (vector
pCLT554) were selected according to the procedure of Svab and Maliga
(1993) on Murashige and Skoog (1962) medium supplemented with 2 mg/L
4-benzylaminopurine and 0.05 mg/L 1-naphthaleneacetic acid. Briefly, the
abaxial side of leaves from in vitro plants measuring 3 to 5 cm were bombarded
with DNA-coated gold particles using a particle gun built in the laboratory
according to the model described by Finer et al. (1992). After 2 d, the treated
leaves were then cut into squares of on average 1 cm length and the selection
of the transformants performed with 500 mg/L of spectinomycine hydrochloride. Explants were subcultured on fresh selection medium every 10 d.
After 4 to 6 weeks, green calli or plantlets appearing on the bleached explants
were isolated and transferred to hormone-free medium for regeneration and
rooting before transfer to the greenhouse. In the greenhouse, natural light was
supplemented 16 h per day by sodium lamps providing 110 mE×m22×s21.
Protein Extraction and SDS-PAGE
Total soluble proteins were extracted from leaf material ground in liquid
nitrogen using as extraction buffer (50 mM Tris-HCl, 100 mM NaCl, and 1 mM
dithiothreitol, pH 8) supplemented with protease inhibitor cocktail tablets
(Roche Transnichon Diagnostics). Protein quantification was performed
according to Bradford (1976) using the Protein Assay Reagent kit from BioRad. Samples were combined with Laemmli (1970) buffer supplemented with
10% (v/v) b-mercaptoethanol and boiled for 5 min before separation by SDSPAGE (12%).
Measurements of Photosynthetic Parameters
The effective (Fm# 2 F)/Fm# and maximum Fv/Fm photochemical quantum
yields of PSII (Kitajima and Butler, 1975; Genty et al., 1989) were measured
with a portable PAM-2500 fluorometer (Walz) on leaves from 3-month-old
tobacco plants grown under greenhouse conditions.
Comparison of Plastid Transcript Levels by
Quantitative RT-PCR
Total RNA from each tobacco line was isolated from mature leaves using
the RNeasy Plant Mini kit (Qiagen) according to the manufacturer’s instruc-
Total amino acid composition of leaves from transgenic and wild-type
tobacco was analyzed using HPLC by the Amino Acids Analysis technical
platform of the UMR 0203 INRA/INSA (Lyon, France). Two independent
measurements were made for each line from two different samples giving
almost identical results. One set of data is presented.
Sequence data of the transforming vectors can be found in the GenBank
database under accession numbers pCLT515 (DQ882176), pCLT516
(DQ882177), pCLT111 (CQ830291), and pCLT554 (EU870886).
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S1. UV detection of GFP expression in tobacco
seedlings.
Supplemental Figure S2. Phenotype and protein profiles in flowers.
Supplemental Figure S3. RT-qPCR analysis of chloroplast transcripts.
Supplemental Figure S4. Effect of nitrogen supply on amino acid content
and composition.
ACKNOWLEDGMENT
The Ph.D. dissertation of Julia Bally was supported in part by the French
Ministry of Industry.
Received April 10, 2009; accepted May 14, 2009; published May 20, 2009.
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