Plant Physiological Adaptations to the Massive Foreign Protein Synthesis Occurring in

Plant Physiological Adaptations to the MassiveForeign Protein Synthesis Occurring inRecombinant 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 CentreNational 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 investigatedin tobacco (Nicotiana tabacum) the possible consequences of such additional products on several parameters of plantdevelopment and composition. Plastid transformants were analyzed that express abundantly either bacterial enzymes, alkalinephosphatase (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 photosyntheticparameters. The total amino acid content of transgenic leaves is also not significantly affected, showing that plant cells have alimited protein biosynthetic capacity. Recombinant products are made at the expense of resident proteins. Rubisco, whichconstitutes the major leaf amino acid store, is the most clearly and strongly down-regulated plant protein. This reduction iseven more dramatic under conditions of limited nitrogen supply, whereas recombinant proteins accumulate to even higherrelative levels. These changes are regulated posttranscriptionally since transcript levels of resident plastid genes are notaffected. Our results show that plants are able to produce massive amounts of recombinant proteins in chloroplasts withoutprofound metabolic perturbation and that Rubisco, acting as a nitrogen buffer, is a key player in maintaining homeostasis andlimiting pleiotropic effects.

The genetic modification of the plastid genome wasachieved in higher plants more than 15 years ago (Svabet al., 1990; Svab and Maliga, 1993). This recombinanttechnology presents distinctive features that are veryattractive from a biotechnological perspective. Themost attractive of these features is the potential forextremely high expression of the transgene in plastidtransformants, up to 70% of total soluble proteins (tsp)in leaves for an antibacterial lysin (Oey et al., 2009). Avariety of pharmaceutical proteins have also beenproduced at very high levels in transgenic chloroplasts(Daniell et al., 2004; Daniell, 2006). The other charac-teristics of the technology have been extensivelyreviewed recently (Maliga, 2004; Bock, 2007; Vermaand Daniell, 2007; Dubald et al., 2008) and concern (1)the targeted insertion of the transgenes, (2) the possi-bility to engineer more easily complex pathways usingpolycistronic vectors, (3) the apparent absence of epi-genetic regulation, and (4) the natural confinement of

the transgenes as a result of the almost exclusivematernal inheritance of these organelles.

Chloroplasts have an extraordinary capacity to syn-thesize and accumulate foreign proteins. Curiously,very little attention has been devoted to evaluate andanalyze the consequences on the plant physiology ofthis 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 inplastid transformants.When phenotypicmodificationswere noted, these were directly linked to the specificproperties of the expressed transgenes (Tregoning et al.,2003; Magee et al., 2004; Ruiz and Daniell, 2005;Chakrabarti et al., 2006; Hasunuma et al., 2008; Tissotet al., 2008). Only in the case of lysin was the hyper-expression of the recombinant protein reported to limitplant development by exhausting the protein syntheticcapacity of chloroplasts (Oey et al., 2009). A number ofissues are therefore still very unclear: (1) are the re-combinant proteins produced on top of the residentproteins, meaning that plants naturally have the capac-ity to make significantly more proteins, at least if a sinkis provided? Otherwise, (2) are they produced at theexpense of all, or of only some, resident proteins,implying that these resident proteins are normallysynthesized in excess? And (3), how are resourcesallocated between resident and recombinant proteins

* Corresponding author; [email protected].

The author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policydescribed 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

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when the cell budget is reduced, in particular whenthere is a limitation in nitrogen supply?Plastid transformants expressing recombinant pro-

teins at a high level provide a unique material toaddress these fundamental questions. To draw genericconclusions, we have for the first time, to our knowl-edge, studied in parallel transgenic tobacco (Nicotianatabacum) lines expressing recombinant proteins of com-pletely different nature: (1) a hydroxyphenyl pyruvatedioxygenase (HPPD) fromPseudomonas (Dufourmantelet al., 2007),whichparticipates inplants in the synthesisof plastoquinones and is the target of various herbi-cides (Matringe et al., 2005); (2) an alkaline phosphatasefrom Escherichia coli (Bally et al., 2008) with no knownsubstrate 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 startedinvestigating the impact that massive transgene ex-pression in chloroplasts may have on plant develop-ment, photosynthesis, leaf proteome, chloroplasttranscriptome, and amino acid composition.

RESULTS

Chloroplast Expression Vectors and TransgenicTobacco Lines

Tobacco plants of generation T1 obtained by selfingand corresponding to four different vectors (Fig. 1)were selected for analysis. These vectors target thetransgenes encoding alkaline phosphatase (PhoA-S orPhoA-L), HPPD, or GFP to the same integration sitebetween the Rubisco large subunit (rbcL) and acetyl-CoA carboxylase subunit D (accD) chloroplast genes.The coding regions are under the control of the strongtobacco plastid PSII subunit D1 (psbA) gene promoter,including its complete 5# untranslated region, exceptfor GFP. The latter is expressed from a dicistroniccassette driven by the corn (Zea mays) 16SrDNA plastidpromoter (Prrn) and the ribosome binding site regionfrom phage lambda gene 10 (Ye et al., 2001). Thegeneration of homoplasmic tobacco lines and theiranalysis at the DNA and protein levels has alreadybeen documented for HPPD (Dufourmantel et al.,2007) and PhoA (Bally et al., 2008). ConcerningpCLT554, GFP was recoded to better fit the tobaccoplastid codon usage, and we selected for analysis oneT0 transformant that displayed under UVa uniform andstrong GFP signal in leaves, localized in the chloro-plasts. The T1 progeny of this transformant uniformlyexpressed both antibiotic resistance and GFP fluores-cence (Supplemental Fig. S1) as expected for a homo-plasmic tobacco plastid transformant.

Normal Growth with Diminished Rubisco Levels inHyperexpressing Lines

The growth of the transgenic lines (T1 generation)was carefully followed in the greenhouse, and no

phenotypic difference was observed compared towild-type tobacco during the vegetative phase (Fig.2, A–C). Accordingly, no variation of the maximumphotochemical efficiency of PSII in the dark-adaptedstate (Fv/Fm; 0.73–0.76) or of the effective quantumyield (Fig. 2D) was measured by fluorometry on leavesof 3-month-old plants. Expression of the recombinantproteins in mature leaves was analyzed by SDS-PAGEand Coomassie Brilliant Blue staining (Fig. 3A). Ru-bisco large (LSU) and small (SSU) subunits are theprevalent polypeptides in wild-type tobacco. In con-trast, the HPPD and GFP recombinant proteins areexpressed at an extraordinary high level and representin the transgenic leaves the major protein. We havescanned the gel and quantified by densitometry thefour recombinant proteins, LSU, and SSU. The HPPDand GFP proteins are expressed at a level close to thatof LSU in wild-type tobacco (Fig. 3B) and represent30% to 40% tsp. When the LSU and SSU amounts areplotted against the level of recombinant protein, thereis a clear negative correlation (Fig. 3B). Over this rangeof values, the LSU to SSU ratio remains always close to3 to 4, which matches the ratio of their respective Mrsand is in accordance to their equal stoichiometriccontribution in Rubisco. This shows that the expres-sion of the nucleus-encoded SSU remains tightly cou-pled to the expression of the plastid-encoded LSU,suggesting that the plastid-to-nucleus retrograde sig-naling 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

Figure 1. Recombinant plastome genetic maps. A, Targeted tobaccoplastid genome region. B, Transformation vectors. LHRR and RHRR arethe left and right plastid recombination regions, respectively, present inall transforming vectors. RBS, Ribosome binding site; g10L, phagelambda gene 10; aadA, spectinomycin resistance gene. All promotersand terminators were derived from the tobacco plastid genome exceptin pCLT554 (Zm, corn).

How Plants Cope with Massive Recombinant Protein Synthesis

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specific. At the flowering stage, a specific differencewas observed in flowers of plants expressing HPPD,which systematically lacked the usual pink pigmenta-tion due to anthocyanins (Supplemental Fig. S2A). Theplastid transgenes encoding HPPD, GFP, and PhoA-Lare strongly expressed in the chromoplasts of tobaccocorolla (Supplemental Fig. S2B), albeit at a lower levelthan in leaf chloroplasts.

Plastid Resident Transcript Levels Are Not Affected in

Transgenic Lines

High-level transgene expression could negativelyimpact the plastid resident transcriptome and be re-sponsible for the depressed level of Rubisco. A semi-quantitative reverse transcription (RT)-PCR analysiswas carried out on total RNA extracted from matureleaves at the same stage during the day. Sets of specificprimers, amplifying unique fragments, were selected tofollow in parallel the recombinant RNAs and nine

resident transcripts synthesized either by the plastid-encoded polymerase (PEP) and/or by the nuclear-encoded polymerase. The results (Supplemental Fig.S3) show that the recombinant RNAs are particularlyabundant and that there is no drastic change in the levelof any of the analyzed plastid resident transcripts.There is no variation in the amount of rbcL transcriptsdespite the large variation reported above at the proteinlevel (Fig. 3). This is consistent with the fact thattranslation initiation is considered as the limiting stepin chloroplast protein synthesis, and there is generally apoor correlation between transcript and correspondingprotein levels (Eberhard et al., 2002). It is also notewor-thy that (1) there is no up-regulation of accD transcripts,a gene poorly transcribed by nuclear-encoded poly-merase that flanks the strongly PEP-transcribed trans-genes at their 3# ends; and (2) there is no variation at theRNA level for psbA despite the use of its promoter and5# untranslated region to drive the expression of re-combinant PhoA and HPPD.

Figure 2. Phenotype of transgenic lines. Compar-ison of T1 generation growth 2 weeks (A), 5 weeks(B), and 3 months (C) after sowing for the wildtype (WT; 1) and for recombinant lines expressingPhoA-S (2), PhoA-L (3), GFP (4), and HPPD (5). D,Comparison of effective PSII photochemicalquantum yields of 3-month-old plants. Error barsrepresent the SD calculated for each category fromtwo leaves with three independent measurementsper leaf.

Bally et al.

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Limiting Nitrogen Supply Does Not Unveil AnyGrowth Penalty

Under normal growing conditions, we observed nodifference in the growth and development of tobaccoplants expressing extremely high levels of GFP orHPPD, despite their depressed level of Rubisco. Wedecided to investigate this situation in conditions oflimited nitrogen and to analyze how resources are thenallocated between resident and recombinant proteins.Three-week-old wild-type and transgenic seedlingsfrom generation T1, sown in soil, were transplanted onvermiculite and drenched every 2 to 3 d with anutritive solution containing ammonium nitrate asthe sole source of nitrogen. Four different concentra-tions of ammonium nitrate were provided, from 0 to 20mM, which is the concentration used in standard planttissue culturemedia (Murashige and Skoog, 1962), andthe experiment was followed over 5 weeks beforeleaves were harvested for analysis. Five days after thestart of the experiment, a positive effect on growth isalready visible on all lines at the highest nitrogen dose(Fig. 4A-1). After 5 weeks (Fig. 4A-2), plants submittedto complete nitrogen deprivation have not grown at alland have become chlorotic. Very limited development

is observed for plants treated with 0.2 mM ammoniumnitrate. Whatever the nitrogen supply, even at thesuboptimal 2 mM concentration, there is no differencein growth between transgenic lines or versus wild-type tobacco.

Tobacco Leaf Protein Amino Acid Composition under

Various Nitrogen Regimes

After 5 weeks, proteins were extracted from leavesof plants shown on Figure 4A-2 and separated by SDS-PAGE (Fig. 4B). At 20 mM ammonium nitrate, provid-ing nitrogen in excess, the pattern is very similar tothat of plants grown in soil (Fig. 3). Remarkably, whenthe supply of nitrogen is limited (below 2 mM), theamount of Rubisco (LSU and SSU) is very stronglyreduced, whereas in all cases, the recombinant pro-teins accumulate at least at the same level as underoptimal conditions. PhoA-L, expressed at a muchlower level than HPPD or GFP, becomes the majorleaf polypeptide, exceeding largely LSU at 2 mM andbelow. Even PhoA-S, which is normally completelymasked by LSU, becomes clearly visible under thoseconditions (Fig. 4B).

One question that arises is whether the recombinantproteins are synthesized in addition to the residentproteins or if they are produced at their expense.Leaves were harvested and the total amino acid con-tent (free + protein-bound) was determined for eachline and at each nitrogen level. The total amino acidcontent is positively correlated to the supply in am-monium nitrate, and whatever the level of nitrogen,transgenic plants that express recombinant proteinsclearly don’t have an increased total content in aminoacids (Supplemental Fig. S4A). The amino acid com-position of transplastomic and wild-type leaves fromplants grown under different conditions of nitrogensupply was then compared (Supplemental Fig. S4B),showing that most amino acids remain quantitativelystable, the strongest variation being noted for Lys,which increases in proportion with the level of ni-trogen.

DISCUSSION

Our objective was to investigate how plants copewith the massive expression of an alien gene, a situ-ation frequently encountered with plastid transform-ants. Indeed, two of the transgenic tobacco lines thatwe have analyzed express recombinant proteins(HPPD and GFP) in chloroplasts at spectacular highlevels, above those of Rubisco, which is normally themost abundant leaf protein, representing up to 60%of the soluble proteins in C3 plants (Spreitzer andSalvucci, 2002; Hirel and Gallais, 2006). Rubisco hasa dual role, first in carbon fixation, and second as apotential dynamic nitrogen store, since it is present inexcess versus the amount needed to fulfill the photo-synthetic requirements (Irving and Robinson, 2006;

Figure 3. Protein profiles of leaves after SDS-PAGE separation. A,Coomassie Brilliant Blue staining of protein extracts (20 mg) from wild-type tobacco (1) and from plants expressing HPPD (2), PhoA-S (3),PhoA-L (4), and GFP (5). The position of the recombinant proteins isindicated in the respective lanes. B, Correlation between Rubisco (LSUin blue; SSU in red) and recombinant proteins expressed in arbitraryunits (AU). WT, Wild type.





Feller et al., 2008). The nitrogen store function ofRubisco has been essentially documented during leafsenescence, when proteins are remobilized and trans-located for seed filling (Murchie et al., 2002; Houtz andPortis, 2003). Hyperexpressing plastid transformantsprovide an interesting model for studying proteinhomeostasis, Rubisco turnover, and the plant cellnitrogen budget. We have found that total aminoacid content of leaves overexpressing HPPD or GFPis unchanged versus the wild type, showing that thesynthesis of the recombinant proteins occurs at theexpense of the resident proteins. Our data show thatthis concerns particularly Rubisco, the major nitrogenand amino acid pool in plant cells. The reduced levelof this enzyme could be the result of a down-regulatedsynthesis or of a higher turnover. The latter hypothesisis more likely, based on the literature related to en-hanced and programmed Rubisco degradation duringsenescence or under environmental stress (Houtz andPortis, 2003; Hirel and Gallais, 2006; Feller et al., 2008).The same compensation between Rubisco and therecombinant protein(s) also occurs very clearly forthe high-level transient leaf expression system, based

on viral replicons (Marillonnet et al., 2005). The buff-ering role of Rubisco also occurs on wild-type plantsunder conditions of limited nitrogen (Fig. 4). Interest-ingly, a recent large-scale proteomic analysis per-formed on nuclear transgenic rice (Oryza sativa),expressing a human therapeutic protein in the endo-sperm, has also reported a decrease in endogenousstorage proteins (Luo et al., 2009).

A scenario can also be envisaged at the RNA level,where the specific down-regulation of Rubisco wouldresult from the presence of the tobacco rbcL terminatorin the transgenes cassettes (Fig. 1). High-level expres-sion of the transgenes could compete for RNA bindingfactors essential for the stability or translation of therbcL transcript. This has been reported for the residenttobacco chloroplast clpP gene when transgenes incor-porated the 5# untranslated region of this gene (Kurodaand Maliga, 2002). Nevertheless, this scenario is notcompatible with our finding that the steady-statetranscript levels of recombinant PhoA-S or PhoA-Lare similar to those of GFP or HPPD (SupplementalFig. S3) but do not strongly impact the level of Rubisco(Fig. 3).

Figure 4. Effect of nitrogen supply on growth and proteome. A, Tobacco seedlings of the wild type and of the four differenttransgenic lines 5 d (1) or 5 weeks (2) after treatment with nutritive solutions containing 0 to 20 mM ammonium nitrate as uniquenitrogen source. B, SDS-PAGE and Coomassie Brilliant Blue staining of leaf proteins (20 mg) harvested after 5 weeks of treatment.WT, Wild type.

Bally et al.




We also investigated whether there is some obviousalteration of the chloroplast transcriptome in tobaccoplastid transformants, as a consequence of high-leveltransgene expression or of competition for transcrip-tion or messenger stabilizing factors. The transcriptlevels of nine plastid resident genes transcribed eitherby the plastid-encoded and/or nuclear-encoded poly-merase (for review, see Hess and Borner, 1999) havebeen followed by quantitative RT-PCR. To our knowl-edge, this type of analysis has not been reported yet inthe literature for any higher-plant plastid transform-ant. We have found that the transgenes driven byeither the psbA (PhoA-S, PhoA-L, and HPPD) or full-length Prrn (GFP) have a very high mRNA steadystate, only two to four times lower than the residentpsbA mRNA. No drastic or general modification hasbeen observed for any of the nine analyzed residenttranscripts.We have followed the development of transgenic

lines expressing PhoA-S, PhoA-L, GFP, and HPPD andcompared it to wild-type tobacco. No visible differencein leaf pigmentation, growth rate, flowering time, andPSII quantum yields has been noted, despite themassive accumulation of HPPD or GFP, accompaniedby a sharp drop of Rubisco in those lines. The absenceof penalty on plant development of such a reduction issurprising but in line with results from past experi-ments using antisense RNA to down-regulate the rbcSgene. These studies have shown that a visible pheno-type 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 reportmentioning an exhaustion of the protein syntheticcapacity of the chloroplast and a strongly impairedgrowth in tobacco lines expressing massively a phagelytic protein (Oey et al., 2009). This could be the con-sequence of this lysin’s extraordinary expression level(70% tsp). Nevertheless, this general statement wasextrapolated only from the observed down-regulationof Rubisco and could also result from a toxic effect ofthe recombinant protein. A global exhaustion of thechloroplast protein synthetic capacity should alsonegatively impact the level of the PEP, and thereforethe level of transcripts produced by PEP, at least if theamount of this enzyme limits transcription undernormal conditions. This was not observed in this studyfor the rbcL transcript though (Oey et al., 2009). Ourresults show that at least up to a recombinant proteinexpression level of 30% to 40% tsp there is no penaltyfor plant growth in our greenhouse conditions. Up tothat expression level also, there is no impact on PSIIquantum yield measurements, showing that the biosyn-thesis of the various plastid-encoded subunits of thisphotosystem is not affected as well as the rest of theelectron transport chain and the major Calvin processes.The quantitative drop of Rubisco is possibly com-

pensated for by a higher specific activity that is de-pendent on its catalytic chaperone, Rubisco activase(Parry et al., 2003; Portis, 2003). Differences in growthcould possibly be detected under other environmental

conditions where the photosynthetic activity of Ru-bisco could be limiting, such as higher light or tem-perature stress, since a reduced growth rate has forinstance been reported for antisense tobacco lineshaving ,40% of normal Rubisco level (Jiang andRodermerl, 1995).

It could also be anticipated that under conditions oflimited nitrogen supply, the cost of the massive syn-thesis of a recombinant protein would become appar-ent and differences in growth observed. This was notthe case in our conditions over a 5-week period,whatever the supply in ammonium nitrate (from0–20 mM). Growth of tobacco seedlings was affectedin proportion to the severity of the nitrogen deficiencybut independent of the genetic background. This il-lustrates the extraordinary metabolic plasticity ofplant cells that can adapt under various conditions tothe additional important sink represented by the re-combinant proteins. At the molecular level, after 5weeks, the major modification that was observed inthe protein profile is a further specific reduction in theamount of Rubisco allowing visualization on Coomas-sie blue-stained one-dimensional gels with recombi-nant PhoA-S and PhoA-L (50 kD), which are normallymasked by the major 53-kD large subunit of Rubisco.PhoA-L even becomes the most abundant leaf proteinwhen the nitrogen supply is limited, and the levels ofGFP and HPPD then clearly exceed 50% of the leafsoluble proteins. The proteolytic degradation machin-ery of plant cells is known to regulate the chloroplastprotein composition and Rubisco turnover (Felleret al., 2008) and seems therefore less able to dealwith any of the foreign proteins (HPPD, Pho-A, orGFP). As a consequence, these products accumulate tohigher relative levels. This finding has potential inter-est for molecular farming applications. A nitrogenstarving step applied on transgenic plant materialbefore protein extraction could increase the relativeproportion of the recombinant protein of interest andtherefore facilitate its purification.

The total amino acid composition (free and protein-bound) of leaves of plantlets grown hydroponically atdifferent nitrogen levels has been measured. The totalleaf amino acid content per fresh weight dropsstrongly when ammonium nitrate is limiting, but thereis no difference between transgenic lines or versus thewild type. Also, despite the fact that under nitrogendeficiency the recombinant proteins often become byfar the predominant leaf protein (HPPD and GFP), theglobal amino acid composition remains rather con-stant whatever the growing conditions. The reason forthis remarkable stability is probably due to the factthat the amino acid composition of the expressedrecombinant proteins is not strikingly different fromthose of Rubisco or wild-type leaves (SupplementalFig. S4C). This may in turn also explain why theserecombinant proteins can be expressed at such a highlevel by plant cells.

The massive expression of a transgene in chloro-plasts surprisingly has a rather low impact on plant





development and physiology. The recombinant pro-teins are synthesized at the expense of resident pro-teins, and Rubisco works in this respect as the majorsource of nitrogen and of protein homeostasis. Inter-estingly, foreign proteins proportionally accumulate ateven higher levels when the nitrogen budget is lim-ited, escaping somehow from the programmed plantdegradationmachinery. We have detected only limitedchanges on the one-dimensional protein profile ofleaves, the effective and maximum quantum yieldsof PSII, the amino acid composition, and the transcriptlevel of plastid resident genes. This article provides aset of data on how plants cope globally with high-leveltransgene expression in leaves. We are currently pro-ceeding to a more detailed leaf proteomic analysis todetermine if proteins other than Rubisco are affectedby high-level recombinant protein expression. Theconsequences on other parts of the plant metabolismunder different growing conditions also merit furtherinvestigation.

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 cmwere 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 hydro-

chloride. 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 Bio-

Rad. Samples were combined with Laemmli (1970) buffer supplemented with

10% (v/v) b-mercaptoethanol and boiled for 5 min before separation by SDS-

PAGE (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-

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�Cfor 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 concentra-

tions of ammonium nitrate were then added to this basal solution.

Protein Content and Amino Acid Composition

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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