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Journal of Biotechnology 145 (2010) 9–16

Contents lists available at ScienceDirect

Journal of Biotechnology

journa l homepage: www.e lsev ier .com/ locate / jb io tec

icistronic binary vector system—A versatile tool for gene expression studiesn cell cultures and plants

ahid Ali a, Heinz Martin Schumachera, Elke Heine-Dobbernacka, Antar El-Bannaa,auzia Yusuf Hafeezc, Hans-Jörg Jacobsenb, Heiko Kieseckera,∗

German Collection of Microorganisms and Cell Cultures DSMZ GmbH, Inhoffenstr. 7b, D-38124 Braunschweig, GermanyAbt. Pflanzenbiotechnologie, Leibniz University of Hannover, Herrenhäuserstr. 2, 30419 Hannover, GermanyDepartment of Biosciences, COMSATS Institute of Information Technology, Chak, Shahzad campus, Islamabad, Pakistan

r t i c l e i n f o

rticle history:eceived 5 June 2009eceived in revised form 28 August 2009ccepted 4 October 2009

eywords:grobacterium tumefaciens

a b s t r a c t

Dicistronic binary vector constructs based on pGreenII vectors for Agrobacterium mediated gene transferalleviate the translational expression monitoring of a target gene in plants. The functionality of the trans-formation vectors was proven by marker gene constructs containing a mannopine synthase promoter(p-MAS) fused to a beta-glucuronidase (gus) gene followed by an internal ribosome entry site and a fireflyluciferase (luc) gene. The cap-dependent translation of a physically independent target protein can bemonitored by the cap-independently co-translated luciferase, because both mRNAs are located on thesame strand. Among three different IRES elements, the tobamo IRES element showed highest activity

icistronicRESalt toleranceell cultureisum sativum

in transient expression. As a proof of principle for physiological studies the gus gene was replaced bya sodium antiporter gene (Atnhx1). Comparative studies with Atnhx1 transgenic luc expressing tobaccocell cultures and pea plants (Pisum sativum L.) showed improved salt tolerance in relation to their wildtype counterparts grown under corresponding conditions. A coincidence of the luc gene expression andincreased sodium chloride tolerance is demonstrated by measurement of luminescence and cell growth.

. Introduction

In eukaryotic cells binding of the ribosomal subunit to the captructure of mRNA is mediated by a set of initiation factors in the so-alled cap-dependent manner of translation (Browning, 2004). Anlternative structural element of the mRNA, called internal ribo-ome entry site (IRES) can replace the cap structure leading to aap-independent manner of translation (Kieft et al., 2001; Terenint al., 2005). Internal initiation of translation (cap-independentanner) has been demonstrated in viruses (Dorner et al., 1984),

n animal cells (Sonenberg, 1987; Pelletier and Sonenberg, 1988)s well as in plant cells (Dinkova et al., 2005). The potential ofoliovirus IRES element has been utilized for the development oficistronic vectors for correlated marker gene expression in stoi-hiometric ratios in mammalian cells (Dirks et al., 1993). In tobaccoeaves transient expression studies with dicistronic constructs,

hich contained a 148 bp 5 prime untranslated leader sequence ofcrucifer tobamo virus (TMV) subgenome (Dorokhov et al., 1994)

evealed its IRES activity. For Encephalomyocarditis virus (EMCV)RES element, cross kingdom activity was shown by Urwin et al.

∗ Corresponding author. Tel.: +49 531 2616 148; fax: +49 531 2616 418.E-mail address: hki@dsmz.de (H. Kiesecker).

168-1656/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.jbiotec.2009.10.002

© 2009 Elsevier B.V. All rights reserved.

(2000) and for Rhopalosiphum padi virus (RhPV) by Groppelli et al.(2007). In direct comparison of EMCV and TMV IRES elements intobacco protoplasts, HeLA cells and yeast, the TMV IRES elementshowed even higher activity across kingdoms (Dorokhov et al.,2002).

Although IRES elements are used in mammalian cells in variousbasic research applications dealing with coordinated expression ofat least two cistrons (Rees et al., 1996), the application in plants forthe purposes of basic research and biotechnology is rare. The TMV148 bp IRES element was successfully utilized in a binary gene trapvector. In this vector a promoterless TMV 148 bp IRES luciferasefusion construct was cloned proximal to the right border sequence(Yamamoto et al., 2003). This study presents another applicationof IRES elements in plants for functional genomics. Dicistronicconstructs achieve the successful coordinated co-expression of aphysically independent target protein, providing a physiologicaltrait, along with a reporter protein.

For subcellular localization, protein fusions between a targetand a reporter gene have often been used, but the possibility of

using fusion proteins is often limited because the functionality ofthe target protein may be affected by the fused reporter gene (Dixitet al., 2006; An et al., 2007). In conventional transformation vec-tors, selectable marker and target genes are driven by differentpromoters. Therefore, transcriptional or post-transcriptional gene

10 Z. Ali et al. / Journal of Biotechnology 145 (2010) 9–16

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ig. 1. Vector T-DNA cassette. (A) Restriction map showing the relevant single sitesKpnI/XmaI). (B) Dicistronic vector pGII0229MASnhx1/luc where Atnhx1 gene was c

ilencing can occur independently (Richter et al., 2006). It is wellnown that the integration of multiple copies of the T-DNA into thelant genome is frequently associated with gene silencing (Tang etl., 2006). In transgenic cell suspensions, occasionally consisting ofheterogeneous mixture of cell populations representing indepen-ent transformation events, there is a danger of unperceived drift

nto falling target gene expression. A method for non-invasive mon-toring of target gene transcription in a transgenic cell populationherefore would facilitate physiological studies. In this study for theonstruction of dicistronic transformation vectors three differentRES elements have been compared using the gus gene as the firstnd firefly luciferase as the second cistron. As a proof of principalor functional studies a Na+/H+ antiporter gene, Atnhx1 (Accessionumber: AF056190) from Arabidopsis thaliana was used as targetnd firefly luciferase as reporter gene. The potential of a sodiumntiporter to provide increased salt tolerance was demonstrated inild type Beta vulgaris suspension cultures (Blumwald and Poole,

987) and confirmed in transgenic plants (Apse et al., 1999; Darleyt al., 2000; Chen et al., 2008).

. Materials and methods

.1. Constructs

The basic pGII0229MAS-luc vector (not shown) harbors a lucene under the control of a mannopine synthase promoter (p-MAS)Langridge et al., 1989; Fox et al., 1992) and in divergent orienta-ion a bar gene under the control of a nopaline synthase promoterP-NOS) next to the left border. The XmaI/HindIII fragment of thentire gus gene (Escherichia coli, UidA Accession No. AY292368.1)as cloned under the control of p-MAS simultaneously with theindIII/NotI fragment of the IRES element into the XmaI/NotIigested basic vector (Fig. 1A). Three IRES elements were used:MV cp148 IRES element (Skulachev et al., 1999) amplified from they367 vector (Accession No. AB086436), putative ZM-IRES elementDinkova et al., 2005) amplified from Zea mays genomic DNA andolio-IRES element subcloned from a mammalian dicistronic vec-or system (Dirks et al., 1993) into the pGII0229MASgus/luc vector

ystem via the compatible HindIII/NotI restriction sites.

cp148HindIII: 5′-CAGAAGCTTCGATTCGGTTGCAGCATTTAAAG-3′

cp148NotI: 5′-TTCGCGGCCGCTTTCTTCTTTCAAATTAAACGAATCAGG-3′

e dicistronic vector pGII0229MASgus/luc 8807 bp: 533 bp MAS-Promoter fragmentvia XmaI and HindIII.

ZM-IRES HindIII 5′-CAGAAGCTTGTAGACTCCCGGCGAACACTCC-3′

ZM-IRES NotI 5′-AGGCGGCCGCTGCTTCTCGGTCCTCAGTC-3′

The resulting vector pGII0229MASgus/luc (Fig. 1A) exhibits thegus gene as a first cistron and the luc gene as a second cistron,linked by the IRES element. In this vector the first cistron can bereplaced using the XmaI/HindIII restriction sites. Atnhx1gene wascloned (Fig. 1B) using the following primer set:

nhx1XmaI(f) 5′-ATTCCCGGGATGTTGGATTCTCTAGTGTCGAAACTG-3′

nhx1HindIII(r) 5′-AATAAGCTTCAAGCCTTACTAAGATCAGGAGGG-3′

2.2. Vector functionality – Agrobacterium infiltration assay(English et al., 1997)

Nicotiana benthamiana plants were grown in a green house at24 ◦C, 16/8 h light/dark period in 0.3 l plastic flower pots. Agrobac-terium tumefaciens strain EHA 105 harboring a specific binaryplasmid was grown overnight (25 ml LB medium in a 100 ml flask)to an O.D. (600 nm) of 0.9–1.0 and centrifuged at 5000 rpm for10 min at 4 ◦C. The supernatant was discarded, the pellet resus-pended in infiltration medium MMA (MS salt 4.6 g l−1, sucrose20 g l−1, NAA 0.5 mg l−1, MES 1.95 g l−1, Acetosyringon 100 �M, pH5.7), adjusted to an O.D. (600 nm) of 0.9–1 and incubated for 2 hat room temperature. At 4–6 leaf stage of N. benthamiana plantsthe Agrobacterium suspension was injected through the epidermison the abaxial side of the leaves with a syringe without needle.The tip of the syringe was pressed against the undersurface of theleaf while at the same time injecting the bacterial suspension intothe apoplastic spaces. The plants were kept under moderate waterdeficit in order to alleviate the impending leaf infiltration, wateredthereafter and incubated at 19 ◦C in dark for 72 h.

2.3. Protein isolation and analysis

N. benthamiana leaves infiltrated with respective

plasmid constructs: 0229MAS-luc, 0229MAS-gus-cp148IRES-luc (0229MASgus/luc), 0229MAS-nhx1-cp148IRES-luc(0229MASnhx1/luc), 0229MAS-gus-ZM-IRES-luc (ZM-IRES) and0229MAS-gus-PolioIRES-luc (Polio-IRES) were imaged by FUJI LAS3000 after application of a luciferine potassium salt solution in

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ater (1 mM). Leaves showing highest luc activity were selectednd were grinded in liquid N2. Total RNA and proteins wereimultaneously extracted from the same samples (100 mg fresheight) with TriFast Gold (Peqlab, Erlangen, Germany) according

o the manufacturer’s instructions. The protein pellet was redis-olved in 5 M urea, 50 mM DTT buffer. Following centrifugationt 10,000 × g for 10 min, protein concentration was determinedsing 2D Quant kit (GE Healthcare, Muenchen, Germany) in thelear supernatant. A volume containing 30–150 �g of protein wasixed with loading buffer (0.025 M Tris–HCl, 4% SDS and 20%lycerin) in a 1:1 ratio, incubated at 95 ◦C for 10 min and proteinslectrophoretically separated by 12% SDS-polyacrylamide gels, andlotted onto a PVDF membrane (Millipore Corporation, Bedford,A, USA). For immunodetection, the membrane was blocked with

% skimmed milk in PBST buffer (0.14 M NaCl, 8.1 mM Na2HPO4,.7 mM KCl, 1.47 mM KH2PO4 and 0.05% TWEEN) for 1 h followedy incubation with 1:1000 diluted rabbit anti-luciferase polyclonalntibodies (MBL, Nagoya, Japan) in PBST for 2 h. The membraneas washed 3 times with PBST buffer and incubated for 45 minith goat anti-rabbit antibody (Sigma, St. Louis, USA) at a 1:20,000ilution in PBST. Afterwards the membrane was washed with PBSTtimes and incubated in substrate buffer (0.1 M Tris–base, 5 mMgCl2 and 100 mM NaCl) for 10 min. Visualization of the 61 kDa

uciferase band was performed by incubating the membraneor development in 15 ml substrate buffer containing 99 �l ofBT (stock 18.8 mg/ml in 67% DMSO) and 102 �l of BCIP (stock.4 mg/ml in 67% DMSO) for 30–60 min.

.4. RNA isolation

Total RNA was isolated simultaneously with the proteins fromhe same samples using TriFast Gold (Peqlab) according to the man-facturer’s instructions. After DNase digest the 260/280 ratio of allamples was between 1.95 and 2.1. Prior to the cDNA synthesis theNA was tested for DNA contamination via PCR by using primersgainst the 18S rRNA gene. For cDNA synthesis 200 ng total RNAs template for the cDNA synthesis the random hexamer primersnd the Revert Aid, First Strand cDNA Synthesis Kit (Fermentas, St.eon-Rot, Germany) was used.

.5. Semiquantitative PCR

For the relative quantification of the mono- and di-cistronicRNA by PCR primers against the luc sequence (Luc f: AAGC-

ATGAAACGATATGG/Luc r: GGAACAACACTTAAAATCG) were used.mplification from the cDNA was performed with Green Taq poly-erase (Fermentas), using the following PCR program: 95 ◦C, 5 min

3× (94 ◦C, 1 min; 60–1 ◦C per cycle, 72 ◦C, 1 min)], [29× (94 ◦C,min; 58 ◦C, 72 ◦C, 1 min)]; [{3×*}4× (94 ◦C, 1 min; 58 ◦C, 72 ◦C,min)]. As internal standard primers against the 18S rRNA gene

18S f: CCGACAGAAGGGACAAGA/18S r TACCTGGTTGATCCTGCC)ere used. 18S PCR amplification was done by the followingrogram: 95 ◦C, 5 min [11× (94 ◦C, 1 min; 58 ◦C, 72 ◦C, 1 min)];{3×*}4× (94 ◦C, 1 min; 58 ◦C, 72 ◦C, 1 min)].

*After each 4 cycles one set of PCR samples were taken out ofhe cycler and incubated for 10 min at 72 ◦C in order to completell PCR reactions.

.6. Stable transformation of tobacco plants

Transgenic tobacco plants were recovered as described by

orsch et al. (1985) with modifications. Leaf discs taken from initro grown tobacco plants (SR1) were incubated for max. 30 min inhe suspension of overnight grown A. tumefaciens culture harboringhe respective binary vector construct. The discs were blotted drynd placed upside down on culture plates with medium containing

ology 145 (2010) 9–16 11

MS salts including B5 vitamins (Duchefa, Haarlem, Netherlands)(sucrose 30 g l−1, BAP 1.0 �g ml−1, NAA 0.1 �g ml−1, and agar 0.8%).After 3 days of co-culture, the leaf discs were washed and trans-ferred to the same medium containing the antibiotic ticarcillin(100 mg l−1) and were cultured under dim light conditions at 16/8 hlight/dark photoperiod at 23 ◦C. After 3 weeks of growth, thecallus was subjected to selective medium containing 5 mg l−1 phos-phinothricine (ppt). Regenerated plants were cultivated in vitrounder sterile conditions in 500 ml glass jars containing hormoneand sugar free MS medium at 23 ◦C and 16/8 h light/dark photope-riod.

2.7. Initiation of callus and suspension cultures

Callus induction from wild type (WT) N. tabacum cv. SR1 andfrom transgenic T0 plants (line 06-74) was carried out on 4Xmedium (Gamborgs B5 salts including vitamins 3.16 g l−1, 2,4-D2.0 mg l−1, NAA 0.5 mg l−1, IAA 0.5 mg l−1, kinetin 0.2 mg l−1, NZ-Amine 2 g l−1, sucrose 20 g l−1, agar 8 g l−1 and for selection 5 mg l−1

ppt post-autoclaving). Callus cultures were subcultured every 4weeks. Suspension cultures were established from WT and trans-genic (T0, 06-74) callus material by transferring upper soft callusmaterial into sterile 100 ml flask. 20 ml of 4X liquid medium wasadded and the flask was incubated at 23 ◦C on a rotary shaker at aspeed of 100 rotations per minute (rpm). Disintegration of calluswas observed every 2–3 days and further 5–10 ml 4X medium wasadded as required until the callus was broken into small pieces.When a homogeneous suspension culture has developed (about2 weeks), it was transferred to a 300 ml Erlenmeyer flask and4X medium was added up to 100 ml. The cells were maintainedin 300 ml Erlenmeyer flasks containing 100 ml suspension undercontinuous light at 23 ◦C while shaking (100 rpm). Routine subcul-turing was done on a weekly basis by adding 50 ml fresh 4X liquidmedium to 50 ml suspension.

2.8. Southern blot analysis

For Southern blot analysis, 30 �g of genomic DNA isolatedfrom WT and transgenic tobacco cell suspensions (06-74), fromtransgenic pea (line 06-72-6) and WT pea plants and the plasmidpGII0229MASnhx1/luc as positive control were digested overnightwith Fast Digest enzyme XbaI (Fermentas), electrophoretically sep-arated on a 0.8% (w/v) agarose gel and subsequently transferredto a Hybond N+-nylon membrane (GE Healthcare) by the capillarytransfer method. To achieve a strong signal two dig labeled PCRfragments (801 bp and 808 bp) were amplified in order to covera total of 1609 bp of the Atnhx1 gene and two dig labeled PCRfragments (711 bp and 837 bp) were amplified in order to covera total of 1548 bp (140 bp of IRES element and 1408 bp of luc genetogether). For DIG labeling a Roche Digoxigenin PCR probe synthe-ses kit (1093657) (Roche, Mannheim, Germany) was used accordingto the manufacturer’s instruction. Hybridization was carried outfollowing standard procedures (Sambrook and Russell, 2001).

The sequences of the PCR primers used for Atnhx1 and luc probeare listed below:

nhx1 801(f) 5′-GAAGCAAGGCTATTTATCCATTTCTG-3′

nhx1 801(r) 5′-TGTGCAAGAAATAATAGTCCCAACAG-3′

nhx1 808 (f) 5′-GACATTGGAACCTTTGACTTGG-3′

nhx1 808 (r) 5′-CGGCCCTTGTAAACTTGTTGTA-3′

luc 711 (f) 5′-CGATTCGGTTGCAGCATT-3′

luc 711 (r) 5′-CGATCAAAGGACTCTGGTACAA-3′

luc 837 (f) 5′-CCTTCCGCATAGAACTGCCT-3′

luc 837 (r) 5′-TCCAAAACAACAACGGCG-3′

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A DIG luminescence detection kit (Roche, 1 363 514) and a FujiAS 3000 imager were used for Southern Blot visualization.

.9. Cell culture growth test in 24 well plates

Comparative growth test of transgenic and WT tobacco cellsas performed in 24 well titer plates (Greiner, Solingen, Ger-any) containing normal 4X solid medium and 4X solid medium

upplemented with 50 mM, 100 mM, 150 mM and 200 mM NaCl,espectively. Each well of the plate was filled with 0.9 ml medium.ell suspensions (WT and transgenic) for test inoculation wererown for 1 week on a rotary shaker and harvested by filteringff the medium through a sterile Nylon net (pore size 100 �m).00 ± 5 mg of cell mass was evenly distributed on the surface ofhe solid medium in each well under sterile conditions. Treat-

ents were made in 4 replicates. After inoculation, the plates werencubated for 3 weeks under normal growth conditions. Duringncubation cells grew and formed a callus like structure. For har-esting callus material was removed carefully from each well withspatula and fresh/dry weight was recorded using an analytical

alance.

.10. Luminescence imaging

To observe transient or stable luc expression qualitatively, trans-enic leaves were washed with detergent, rinsed with water andried on a filter paper. D-luciferin potassium salt (Cat. Nr. bc219,ynchem, Felsberg, Germany) was dissolved in water (1 mM) andprayed on the leaves. In vitro callus material and pea seeds wereprayed with filter sterilized luciferin solution without detergentretreatment. The luminescence images were taken with a Fuji LAS000 imager and processed with the Aida® quantification software.he luminescence intensity was visualized by a colour shift fromellow (lower intensity) to red (high intensity).

.11. Quantitative luciferase assay

For the quantitative luciferase activity determination, photonmission as relative light units (RLU) was measured in a luminome-er (Lumat L B 9501 by Berthold, Bad Wildbad, Germany), using aromega luciferase assay kit (Cat. No. E 1500). 100 mg of leaf/callusaterial was quickly frozen in liquid nitrogen and ground in a

re-cooled mortar, resuspended in 1× Luciferase Cell Culture Lysiseagent (CCLR – 25 mM Tris-phosphate pH 7.8, 2 mM DTT, 2 mM,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid, 10% glycerol,% Triton® X-100) provided with the Promega kit and incubatedt 4 ◦C for 1 h. The debris was removed by centrifugation and theupernatant (cell lysate) was transferred to a new tube. 20 �l of theell lysate was mixed with 100 �l of Luciferase Assay Reagent (LAR)irectly prior to measurement.

.12. Fluorimetric 4-methylumbelliferyl-b-d-glucuronide (MUG)ssay

20 �l of the cell lysate (extraction as described before usingromega luciferase kit) was added to 100 �l of MUG-buffer50 mM NaPO4, pH 7.0, 10 mM dithiothreitol (DTT), 1 mM Na2EDTA,.1% sodium lauryl sarcosine, 0.1% Triton® X-100, 1 mM MUG

-methylumbelliferyl-b-d-glucuronide). Kinetic of glucuronidasectivity was assayed in semiquantitative way in three replicatesy measuring emission at 455 nm at an excitation of 365 nm wave-

ength in a TECAN Genios (Tecan, Crailsheim, Germany) device andnalyzed by Magellan® software.

ology 145 (2010) 9–16

2.13. Statistical analysis

The box whisker plots were generated by SigmaPlot® 9.0 soft-ware in which the boxes indicate the range between 25% and the75% percentile as well as the median. The whiskers mark the 5% and95% percentile and the dots show the outliers. Statistical data anal-ysis was made with SigmaStat® 3.1 in which all pairwise multiplecomparison procedures were made according to the Holm-Sidakmethod: Overall significance level = 0.05. Power of the performedtests:alpha = 0.050:1.000.

2.14. Pea transformation

Agrobacterium mediated pea transformation was done accord-ing to the method described by Richter et al. (2006) with somemodifications. Pea seeds (cultivar Sponsor) were surface steril-ized in 70% ethanol (EtOH) (v/v) for 1 min followed by 6% sodiumhypochlorite (NaOCl) for 5–10 min, with agitation. Seeds werewashed for 5–6 times with sterile de-ionized water and soaked overnight in water. Sterilized seeds were split open, cotyledons wereremoved, radical tips were cut and the remaining embryonic axeswere sliced longitudinally 3–5 times with a razor blade (dippedinto desired Agrobacterium culture). The sliced embryos were inoc-ulated with Agrobacterium suspension according to Schroeder etal. (1993). Explants were blotted dry for 3–4 min on sterile filterpaper and plated on B5hT co-cultivation medium for 3 days inthe dark at 23 ◦C. After co-cultivation, explants (white and whitegreenish colour) were washed 3–4 times in sterile distilled watersupplemented with 300 mg/l Ticarcillin. The explants were blotteddry on sterile filter paper and cultured on shoot regeneration MSTmedium for 10 days under dim light conditions, then transferred toMST medium for another 10 days in light, 16/8 h light/dark period.Thereafter, the explants were subcultured on selection medium P2and the healthy green shoots were subcultured every 3 week onP2 medium with increased concentrations of ppt: 2.5 mg/l, 5 mg/l,7.5 mg/l, and 10 mg/l. After the fourth subculture under selectiveconditions, from luciferase expressing explants surviving shootswere propagated clone-wise. Elongating shoots were grafted tonon-transgenic root stock. Transgenic plants were grown in greenhouse at 24 ◦C, 16/8 h light/dark period in 2 l plastic flower pots.Fertilizer was provided with irrigation water. Subsequent genera-tions T1, T2 and T3 were products of self-pollination. For B5hT, MSTand P2 media recipe see Richter et al. (2006).

2.15. Root morphological study under NaCl stress

Comparative root growth studies under salt stress for WT andtransgenic pea T3 line (06-72-6) were conducted in pots containingvermiculite. Pots were irrigated two times with half strength MSO(MS salt including vitamin 2.2 g/l + MES 0.125 g/l and no sugar) sup-plemented 100 mM NaCl water 1 week prior to seed setting, furtherirrigation was performed only with MSO medium. The plants weregrown at 20 ◦C and a 16/8 h light/dark period in growth room.

3. Results

3.1. Transient expression assay for testing the vector functionality

The transcription of the monocistronic construct and thedicistronic constructs containing the different IRES elements was

tested by semiquantitative RT-PCR of the luc transcripts in relationto the 18S transcript. As shown in Fig. 2 there is no signifi-cant difference on transcriptional level among the quantity ofthe three transcripts containing the different IRES elements andthe monocistronic luciferase transcript. However, on translational

Z. Ali et al. / Journal of Biotechn

Fig. 2. Semiquantitative RT-PCR measurement of luc transcript in relation to 18Stranscript in N. benthamiana leaves infiltrated by Agrobacterium suspension harbor-ing the respective construct. (A) 0229MAS-luc, (B) 0229MASgus/luc, (C) ZM-IRES,(D) Polio-IRES, and (E) WT.

Fig. 3. Western blot analysis of firefly luciferase proteins transiently expressedfrom mono- and di-cistronic transcripts. M: Marker SM 0671 (Fermentas); (1)00I

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229MAS-luc, 30 �g total protein; (2) 0229MASgus/luc, 150 �g total protein; (3)229MASnhx1/luc, 150 �g total protein; (4) ZM-IRES, 150 �g total protein; (5) Polio-RES, 150 �g total protein; (6) WT, 150 �g total protein.

evel strongest luciferase band was observed in western blot anal-sis by monocistronic construct (0229MAS-luc). The dicistronic229MASgus/luc and 0229MASnhx1/luc constructs led to consider-ble though different luciferase expression rates, whereas the ZM-

nd Polio-IRES driven translation resulted in significantly loweruciferase expression as it is reflected by the faint bands (Fig. 3).o examine the different translation efficiencies of the three IRESlements in a more sensitive way, simultaneous enzymatic assays

ig. 4. (A) Transient �-glucuronidase activity and corresponding IRES mediated luciferaseontaining the different IRES elements are indicated. The horizontal line in the boxes reprey IRES elements in N. benthamiana leaves. From these leaves quantitative values (shown

n light intensity. Picture was made by Fuji LAS 3000 imager after application of a solutiogure legend, the reader is referred to the web version of the article.)

ology 145 (2010) 9–16 13

of gus and luciferase activity were performed from the same proteinsamples. Although gus activity was similar for all three constructs,the TMV IRES cp148 element showed significantly higher luc activ-ity (Fig. 4).

3.2. Stable expression assay for coincidence of luc activity andsalt tolerance

Suspension culture established from the transgenic T0 plant (06-74) were used to proof a coincidence of luc activity and the expectedAtnhx1 mediated salt tolerance. The presence of the transgenes insuspension culture was confirmed via PCR using specific primers(Fig. 5A). Furthermore Southern blot analysis of the transgenictobacco cell culture showed three bands after restriction digest ofthe genomic DNA with the restriction enzyme XbaI (Fig. 5B), whichcleaves once in the T-DNA between the promoter and the Atnhx1gene. The result shows that the Atnhx1 gene is integrated in at leastthree different locations of the cultured cells genome. Comparativegrowth tests were performed with the WT and the transgenic cellcultures in 24 well titer plates. Fig. 6 shows the growth of WT andtransgenic cell culture and luc expression in corresponding trans-genic cell cultures after 3 weeks of cultivation. Being exposed to saltconcentrations from 50 mM to 200 mM NaCl, fresh and dry weightaccumulation of both cell cultures decreased, but for transgeniccells the decrease was much less pronounced. As it can be seen inthe box whisker plots in Fig. 6(A and B) there is a statistically sig-nificant difference (P ≤ 0.001) among all treatments tested for thetransgenic cell line. In the concentration range from 50 mM and100 mM salt treatment the transgenic and the WT cell line show apronounced difference (P ≤ 0.001) in dry weight accumulation. Inparallel the luminescence of the transgenic cells was measured inanother set of growth tests in 24 well titer plates. The higher salttolerance of the transgenic cells measured at 50 mM and 100 mMsalt challenge coincides with luc activity. However, from colour dif-ferences in the images of the 24 well titer plates it can be seenthat the total luc activity is not evenly distributed within each well(Fig. 6D). For comparison of possible cell selection effects we grewcell cultures on media supplemented either with ppt as the onlyselective agent, with NaCl or with both ppt + NaCl. Non-transgeniccells died in all cases. An increase of luciferase activity was observedby increasing sodium concentrations (Fig. 6C and D).

3.3. Detection of IRES mediated luc activity in transgenic pea

In order to study whether luc activity, based on the functional-ity of the TMV IRES element, can be measured also in differentiated

activity. Each measurement was done in three replicates. Corresponding constructssents the median of the measured values. (B) Monitoring of luc expression, mediated

in A) were recorded. The colour shifts from yellow to red represents the increasen of luciferin potassium salt. (For interpretation of the references to colour in this

14 Z. Ali et al. / Journal of Biotechnology 145 (2010) 9–16

Fig. 5. (A) PCR confirming the presence of Atnhx1 and luc gene in transgenic tobacco cell culture. Lanes 2–5 show integration of Atnhx1 gene using 808 bp Atnhx1 specificprimers, Lanes 6–9 show integration of luc gene using 837 bp luc specific primers. Lane 1: 100 bp DNA marker, Lanes 2 and 6: transgenic suspension cells, Lanes 3 and 7:plasmid DNA (+ control), Lanes 4 and 8: wild type suspension cells, Lanes 5 and 9: water control. (B) Southern blot analysis of the cell suspension. Lane 1: Dig Marker III(Roche: 1218603), Lane 2: 30 �g of genomic DNA of cell suspension 06-74 digested with XbaI, Lane 3: Vector 0229MASnhx1/luc digested with XbaI.

Fig. 6. Growth rates of transgenic versus WT cells on solid medium. (A) shows the increase of fresh weight on media supplemented with different salt concentrations over agrowth period of 3 weeks starting from an initial inoculum cell mass (indicated by red line), (B) the corresponding dry weight measurement over a growth period of 3 weeks,(C) the total luciferase activity (LAU – linear arbitrary units) in the wells of a 24 well titer plate quantified by a Fuji LAS 3000 Imager and Aida® quantification software (thed nificant), (D) Luciferase activities imaging in titer plate cultures. The colour shifts fromy d luminescent light with Imager Fuji LAS 3000. (a) WT cells, (b) Atnhx1 transgenic cells( s (06-74) on 50 mM NaCl supplemented medium, (d) Atnhx1 transgenic cells (06-74) on1 r in this figure legend, the reader is referred to the web version of the article.)

pfglrhtoawTp

ifferences between 50 mM and 100 mM NaCl supplementation are statistically sigellow to red represents the increase in light intensity. Images taken with emitteline 06-74) on 0 mM NaCl supplemented medium, (c) Atnhx1NHX1 transgenic cell00 mM NaCl supplemented medium. (For interpretation of the references to colou

lants and in species other than tobacco, we performed pea trans-ormation. The first ten recovered ppt selected T0 shoots wererafted on non-transgenic root stocks and positively tested foruciferase activity. Progeny was raised from two out of ten T0 plantsepresenting the lines 06-72-1 and 06-72-6. The line showing theigher luciferase expression (06-72-6) was used to obtain plants ofhe T3 generation to perform further analysis. The transgenic nature

f pea plants was confirmed by PCR (not shown) and by luciferasessay (Fig. 8A). Among all plants raised no phenotypic aberrationsere observed. A single copy integration of the 0229MASnhx1/luc

-DNA was confirmed by Southern blot analysis in T3 generationlants (Fig. 7). In all generations luc activity was detected with vary-

Fig. 7. Southern blot analysis of the transgenic pea line (06-72-6). Lane 1: Dig MarkerIII (Roche:528552). Lane 2: empty. Lane 3: genomic DNA of transgenic pea. Lane 4:WT pea. Lane 5: Vector 0229MASnhx1/luc. Lanes 2–5 hybridized with Atnhx1 probe.Lane 6: transgenic pea. Lane 7: WT pea. Lane 8: Vector 0229MASnhx1/luc. Lanes 6–8hybridized with luc probe.

Z. Ali et al. / Journal of Biotechnology 145 (2010) 9–16 15

Fig. 8. (A) Luciferase activity measured with protein extracts isolated from leaves of wild type (WT) and individual transgenic plants in subsequent generations starting fromT0. In T1 and T2 two independent lines were marked by white columns (line 06-72-1, four plants) and grey columns (line 06-72-6, three plants). The luciferase expression dataof line 06-72-6 in T3 generation are shown. (B) The upper image shows opened pods of transgenic T0 and wild type plant. The lower picture shows the chemiluminescenceof the three transgenic seeds of T1 generation (line 06-72-6), whose luciferase activity in the leaves is illustrated in (A).

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Fig. 9. Root growth of transgenic versus wild type pea plants under salt challen

ng luminescence intensities (Fig. 8A). By spraying with luciferinolution luminescence can be observed in transgenic pea seedsFig. 8B).

.4. Root morphogenesis under salt stress conditions

Only slight difference in the shoot morphology could bebserved (not shown) but the root stocks of the transgenic plantsere significantly more branched and stronger (Fig. 9).

. Discussion

Hennecke et al. (2001) pointed out that the arrangement ofistrons in a dicistronic setting is crucial for IRES dependent trans-ation in mammalian cells. For comparative expression studies ofhe three IRES elements the whole dicistronic expression cassetteas therefore kept constant containing the gus gene as the first and

he luc gene as the second cistron, in order to avoid any disturbanceaused by the mRNA secondary structure. Only IRES elements wereifferent in the constructs. The TMV cp148IRES element showedignificantly higher luciferase activity as compared to ZM and Polio-RES elements.

Furthermore in the present study an increase of luc expressionas observed when the gus gene was replaced by the At-nhx1 gene

eing the first cistron (Fig. 3). Although it shows that the first cistronas an influence on the expression rate of the IRES mediated trans-

ation, which agrees with the results reported by Hennecke et al.

00 mM NaCl), 4 weeks after seedling emergence. (A) Transgenic; (B) wild type.

(2001), we cannot exclude a physiological effect of the At-nhx1encoded sodium antiporter.

Dorokhov et al. (2002) demonstrated that the TMV IRES elementshowed high activity in HeLa and yeast cells. For the EMCV IRESelement Urwin et al. (2000) demonstrated cross kingdom activityas well. Based on these facts we found that the poliovirus derivedIRES element exhibited a very low activity in tobacco cells and verylow activity was also obtained for the ZM-IRES sequence, althoughit represents a plant derived cellular IRES element (Dinkova et al.,2005).

Since in our studies the TMV cp148 IRES element contain-ing construct showed the highest activity it was used for furtherexperiments, where transgenic cell cultures showed IRES mediatedluciferase expression in combination with increased salt tolerance.Early basic work on salt tolerance performed with cell cultures byBlumwald and Poole (1987) showed that the use of cell cultures forthis purpose is possible. They observed growth rates of Beta vulgarissuspension cultures between 50 mM and 200 mM sodium chloridechallenge over a period of 10 days. In the present study similarsalt concentrations but a growth period of 3 weeks was investi-gated using cell cultures. For concentrations >100 mM (150 mM and200 mM) growth of the transgenic cells as well as the ATP depen-

dent luc activity almost disappeared and no significant differenceswere observed between transgenic and WT cells, which indicatedcell death. On the other hand plants over-expressing the Atnhx1can sustain growth in soil supplied with 200 mM sodium chloride(Apse et al., 1999), which is probably due to the fact that complex

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plified mode of internal ribosome entry. Mol. Cell Biol. 25, 7879–7888.

6 Z. Ali et al. / Journal of Bi

ifferentiated tissue can better balance the negative osmotic poten-ial. The limitations should be more significant in the case of cellultures, where all cells are directly exposed to the high salt con-entrations in the medium, since also the vacuolar H+ pumps (Niut al., 1995) must shift to a higher gear in order to sustain vacuolara+ concentrations of more than 100 mM (Darley et al., 2000).

For the salt challenge experiments the previously ppt selecteduspension cells (06-74) were immobilized on salt supplementedolid medium. The image of luciferase activity (Fig. 6D) shows theneven distribution of transgene expressing cells. Our transgenicell line has been established from a T0 transgenic plant. There-ore, cell suspensions derived from T0 plants whose transformationhimeric character cannot be excluded, may represent a heteroge-eous mixture of different transformation events in the derivedell suspension. The latter observations indicate that the presentedxpression monitoring system can be extremely helpful to observehe level of the expression of a transgene providing a physiologicalrait in a plant cell culture. In this context also luciferase expressingransgenic pea plants harboring the dicistronic Atnhx/luc expres-ion cassette showed better root stock development in comparisono the wild type. He et al. (2005) reported that the differences in theresh root masses between Atnhx1 expressing cotton and wild typelants were more pronounced than those of shoot fresh massesfter salt challenge. The experiments in the present study withransgenic pea plants demonstrated that the genetic integrity ofhe sexual propagated pea plants does not necessarily lead to lowariability in the transgene expression in the progenies (Fig. 8A) asas been shown earlier also by Richter et al. (2006) in transgenicea. Especially in this case a simple monitoring system suitable forigh throughput analysis can be helpful for correlating transgenexpression with a physiological trait.

cknowledgements

We express our gratitude to Dr. Yoshiharu Y. Yamamoto (RIKENRS, Japan) for the provision of vector yy376 harboring the respec-ive Tobamo virus IRES element. The first author is thankful toerman Academic Exchange Service (DAAD) and Prof. Dr. Erkotackebrandt (Director DSMZ) for providing scholarship to conducthis study. We are also thankful to Dr. Brian J. Tindall (DSMZ) forhe language improvement of the manuscript. Provision of greenouse facilities by Dr. Stephen Winter (JKI Braunschweig) is alsocknowledged.

ppendix A. Supplementary data

Supplementary data associated with this article can be found, inhe online version, at doi:10.1016/j.jbiotec.2009.10.002.

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