Phytoextraction potential of wild type and 35S-gshI transgenic poplar trees (Populus × canescens) for environmental pollutants

This article was downloaded by: [Tamas Komives]On: 30 April 2013, At: 01:57Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House,37-41 Mortimer Street, London W1T 3JH, UK

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Phytoextraction potential of wild type and 35S-gshItransgenic poplar trees (Populus × canescens) forenvironmental pollutants herbicide paraquat, saltsodium, zinc sulfate and nitric oxide in vitroGábor Gyulai a , András Bittsánszky b , Zoltán Szabó c , Luther Waters Jr. d , Gábor Gullner b ,Györgyi Kampfl e , György Heltai e & Tamás Kőmíves b

a Institute of Genetics and Biotechnology , Szent István University , 2103 , Gödöllő , Hungaryb Plant Protection Institute, Agricultural Research Centre, Hungarian Academy of Sciences ,Budapest , 1022 , Hungaryc Agricultural Biotechnology Center , 2100 , Gödöllő , Hungaryd Department of Horticulture, College of Agriculture , Auburn University , Alabama , 36849 ,USAe Department of Chemistry and Biochemistry , Szent István University , 2103 , Gödöllő ,HungaryAccepted author version posted online: 29 Apr 2013.

To cite this article: Gábor Gyulai , András Bittsánszky , Zoltán Szabó , Luther Waters Jr. , Gábor Gullner , Györgyi Kampfl ,György Heltai & Tamás Kőmíves (2013): Phytoextraction potential of wild type and 35S-gshI transgenic poplar trees (Populus ×canescens) for environmental pollutants herbicide paraquat, salt sodium, zinc sulfate and nitric oxide in vitro , InternationalJournal of Phytoremediation, DOI:10.1080/15226514.2013.783553

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Phytoextraction potential of wild type and 35S-gshI transgenic poplar trees (Populus ×

canescens) for environmental pollutants herbicide paraquat, salt sodium, zinc sulfate and

nitric oxide in vitro

Gábor Gyulai1*, András Bittsánszky2, Zoltán Szabó3, Luther Waters Jr.4, Gábor Gullner2,

Györgyi Kampfl5, György Heltai5 and Tamás Kőmíves2

1Institute of Genetics and Biotechnology, Szent István University, 2103 Gödöllő, Hungary

2Plant Protection Institute, Agricultural Research Centre, Hungarian Academy of Sciences,

Budapest 1022, Hungary

3Agricultural Biotechnology Center, 2100 Gödöllő, Hungary

4Department of Horticulture, College of Agriculture, Auburn University, Alabama 36849, USA

5Department of Chemistry and Biochemistry, Szent István University, 2103 Gödöllő, Hungary

*Correspondence: [email protected]

E-mail addresses: [email protected]; [email protected];

[email protected]; [email protected]; [email protected];

[email protected]; [email protected]

Abstract

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Phytoextraction potentials of two transgenic (TR) poplar (Populus × canescens) clones TRggs11

and TRlgl6 were compared with that of wild-type (WT) following exposure to paraquat, zinc

sulfate, common salt and nitric oxide (NO), using a leaf-disc system incubated for 21 days on

EDTA-containing nutritive WPM media in vitro. Glutathione (GSH) contents of leaf discs of

TRlgl6 and TRggs11 showed increments to 296 % and 190%, respectively, compared with WT.

NO exposure led to a twofold GSH content in TRlgl6, which was coupled with a significantly

increased sulfate uptake when exposed to 10-3 M ZnSO4. The highest mineral contents of Na, Zn,

Mn, Cu and Mo was observed in the TRggs11 clone. Salt-induced activity of catalase enzyme

increased in both TR clones significantly compared with WT under NaCl (0.75% and 1.5%)

exposure. The in silico sequence analyses of gsh1 genes revealed that P. x canadensis and Salix

sachalinensis show the closeset sequence similarity to that of P. x canescens, which predicted an

active GSH production with high phytoextraction potentials of these species with indication for

their use where P. x canescens can not be grown.

Keywords: Phytoextraction, environmental pollutants, gene sequence analyses

1. Introduction

Poplars are frequently used for treating manure of swine houses in Hungary (Europe), and when

conditions for nitrification and denitrification are suboptimal, volatile nitrogen oxides (NO and

N2O) can be released (Kim et al., 2007). Our study aimed to evaluate the response of poplar to

NO gas. Soils around Gödöllő (Hungary) have naturally high levels of Zn content (53.3±2.3 mg

kg-1 DW soil; Gyulai et al., 2012b), and due to the overuse of paraquat in agriculture

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(Bittsánszky et al. 2009), experiments were designed to study zinc and paraquat tolerance of

poplars. Primary and secondary soil salinity is also a major environmental hazard not only in

Hungary but in the USA and Australia (Csillag et al., 1993; Metternicht and Zinck, 2003), which

is a challenge to study plant salt tolerance (Yamaguchi and Blumwald, 2005; Binh et al., 1992).

Numerous plant species are able to tolerate toxic substances and heavy metals from polluted soils

and air, such as the annual species Thlaspi caerulescens, a known nickel (Ni) and zinc (Zn)

hyperaccumulator plant; Brassica juncea, a Pb accumulator (Gleba et al., 1999); and the arsenic

(As) hyperaccumulator Pteris vittata (Nagarajan and Ebbs, 2010). Metallocrops such as oat

(Avena sativa), barley (Hordeum vulgare) and Indian mustard (B. juncea) also tend to take up

high level of Cu, Cd and Zn in hydroponics (Ebbs and Kochian, 1998). Unlike these annual

plants, given their high growth rate and perennial life history, woody poplars of both wild type

(WT) and transgenic (TR) were found to be optimal for the elimination of environmental

contaminants through phytoextraction (Peuke and Rennenberg, 2005; Bittsánszky et al., 2005;

Gyulai et al., 2012a).

Poplar trees, used in our study, were transformed to overexpress a bacterial gene encoding

gamma-glutamylcysteine synthetase (EC 6.3.2.2), which is the rate-limiting regulatory enzyme

in the biosynthesis of the ubiquitous tripeptide thiol compound glutathione (GSH) (Peuke and

Rennenberg, 2005). The overexpressed protein was directed either in the cytosol (TRggs11 line)

(Arisi et al., 1997) or in the chloroplasts (TRlgl6 line) (Noctor et al.,1998).

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The 35S-gshI TR poplars were previously investigated in vitro (Gullner et al., 2001; Gyulai et

al., 2005; Bittsánszky et al., 2006) and in situ (Peuke and Rennenberg, 2005; Peuke et al., 2012).

The gshI transgene incorporation (Gyulai et al., 2005) and gene expression levels (Bittsánszky et

al., 2006) were characterized with the capability for further gene activation by treatment with the

DNA-demethylating agent 5,6-dihydro-5'-azacytidine hydrochloride (DHAC) (Gyulai et al.,

2012a).

In the study presented GSH and cysteine (Cys) content, mineral uptake capacity, and the activity

of stress indicator enzyme catalaze (CAT) was measured in poplar leaf tissue exposed to

environmental aerial contaminants nitric oxide (NO), zinc sulfate (ZnSO4), common salt (NaCl),

and the herbicide paraquat (PQ) in aseptic leaf disc cultures in vitro. In silico sequence analyses

of plant gsh1 genes were carried out to identify plant species with high sequences similarities to

those of poplar.

2. Methods

2.1. Plant materials

A clone of the untransformed (INRA-717-1-B4) natural hybrid poplar P. × canescens (P.

tremula × P. alba), and two genetically transformed lines were used that overexpress the

bacterial (E. coli) gshI gene (gamma glutamylcysteine synthetase; EC 6.3.2.2) (NCBI: X03954;

Watanabe et al., 1986). Unlike TRggs11 (35s-gshI), transgene cassette of TRlgl6 (35S-gshI-

rbcS) carried an additional targeting sequence (32 to 202 of the 206 bp stretch; NCBI M25614)

of transit peptide (RBCS) gene rbcS (RuBPCase SSU: small subunit of RuBPCase, ribulose-1,5-

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bisphosphate carboxylase, EC 4.1.1.39), which facilitates the translocation of cytosolically

synthesized RBCS-GSH complex into chloroplasts (Leplé et al., 1992; Arisi et al., 1997; Noctor

et al., 1998).

Poplars were micropropagated and maintained in aseptic shoot cultures from nodal segments

following the methods of Gyulai et al. (2005) and Koprivova et al. (2002). Briefly, shoot

segments (0.5 cm) were placed on woody plant medium (WPM) (Lloyd and McCown, 1980)

supplemented with benzyl adenine (BA; 0.5 mg-1 L) and α-naphthaleneacetic acid (NAA; 0.2

mg-1 L), then incubated for 21 days under a 16 h/8 h (light/dark) photoperiod (3000 lux, 4.05

µmol/photons/m2/s). Auxiliary shoots that developed were excised, transferred to hormone-free

WPM media to induce rooting and incubated for an additional 21 days. Leaves of aseptic shoots

were sampled and used for leaf-disc cultures in accordance with the method of Gyulai et al.

(2012a).

The woody plant medium (WPM) (Duchefa Biochemie NL; #M0220) included the following

nutritive elements: NH4NO3 (400.0 mg L-1), H3BO3 (6.2 mg L-1), CaCl2 (72.5 mg L-1), Ca(NO3)2

x 4H2O (386.0 mg L-1), CuSO4 x 5H2O (0.25 mg L-1), EDTA x 2H2O (37.3 mg L-1), FeSO4

x7H2O (27.85 mg L-1), MgSO4 (180.7 mg L-1), MnSO4 x H2O (22.3 mg L-1), Na2MoO4 x 2H2O

(0.25 mg L-1), KH2PO4 (170.0 mg L-1), K2SO4 (990.0 mg L-1) and ZnSO4 x 7H2O (8.6 mg L-1).

2.2. Nitric oxide exposure

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Aseptic WPM medium was supplemented with a concentration series (10-7 to 10-4 M) of NaNP

(Na-nitroferricyanide III; Na2Fe(CN)5NO × 2H2O) to release NO following the method of

Floryszak-Wieczorek et al. (2006). Gas samples (250 µl) were collected with gas-tight Hamilton

syringes from the headspace of each culture vessel (1 L volume each) and analyzed with an

ANTEK 7050 NO-analyser using a chemiluminescent detector. The NO content was calculated

by one-point linear calibration using external NO gas for calibration. The peak areas, which were

proportional to the measured gas concentrations, were calculated with an HP 3396 Integrator and

Microsoft Excel software in accordance with Kampfl et al. (2007).

2.3. Paraquat (PQ) exposure

Aseptic WPM medium, solidified with 0.8 g L-1 agar, was supplemented with a concentration

series (10-8, 5 × 10-8, 10-7 and 5 × 10-7 M) of the herbicide paraquat (methyl viologen; 1,1'-

dimethyl-4.4'-bipyridinium dichloride) combined with and without 10-7 M NaNP. Leaf discs (8

mm diameter; three leaf discs per petri dishes of 6 cm, in triplicates) were laid onto the surface of

media and incubated for 21 days under a 16 h/8 h (light/dark) photoperiod (3000 lux, 4.05 µmol

photons/m2/s).

2.4. ZnSO4 exposure

Leaf discs were excised and placed on aseptic EDTA-containing (37.25 mg-1 L) WPM agar

media supplemented with a concentration series of ZnSO4 (10-2 M to 10-4 M), and incubated for

21 days, according to the standard aseptic culture methods, under a 16 h/8 h (light/dark)

photoperiod (3000 lux, 4.05 µmol photons/m2/s) following the method of Gyulai et al. (2005,

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2012a). Untreated basal WPM medium contained 29.9068 µM Zn2+ (equal to 1.955 mg-1 L Zn2+)

and 14.36 meq-1 L SO42- (George et al., 1987).

2.5. Bioconcentration factor

Bioconcentration factor (BCF) ratios were calculated by dividing the molar concentrations of the

elements measured in leaf discs with the element concentrations supplied in the WPM medium

according to Mackay (1982).

2.6. NaCl exposure

NaCl was added to WPM agar media at the concentrations of 0.75%, 1.5%, 2.25%, and 3.0%

prior to autoclaving the media. Leaf discs were incubated for 21 days under a 16 h/8 h

(light/dark) photoperiod (3000 lux, equal to 4.05 µmol photons/m2/s). Basal WPM medium

contained 4.59 mg-1 L Na supplied as the Fe-chelating agent Na2EDTA × 2H2O (37.25 mg-1 L)

(George et al., 1987).

2.7. Inductively coupled plasma emission spectrometry

The Na, Zn, Mn, Cu and Mo elemental content of poplar leaf discs was determined by ICP

(Inductively Coupled Plasma emission spectrometry) following the methods of Zarcinas et al.

(1987). Leaf discs were oven-dried at 70 ºC overnight, and 0.5 mg leaf tissue of each treatment

was transferred to digestion vessels in triplicates, digested with a mixture of 5 ml HNO3 and 2 ml

H2O2 using a MILESTONE 200 MEGA microwave oven. The microwave digestion program

was as follows: 6 min at 250 W; 6 min at 400 W; 6 min at 650 W; 6 min at 250 W closed by

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cooling for 5 min to room temperature. The digested solution was filtered through Macherey-

Nagel G 1/4 filter-paper, diluted to 25 ml, and was analyzed with Jobin Yvon 24 sequent ICP

Optical Emission Spectrometer. The operation parameters were as follows: incident power, 1

kW; outer argon flow 12 L min-1; intermediate argon flow 0.2 L min-1; inner argon flow 1.1 L

min-1; with nebulizer Meinhardt type nebulizer, and sample uptake rate 1 mL min-1 according to

Heltai et al. (2000).

2.8. HPLC analysis

Total GSH contents were measured using 0.1 g fresh leaf tissue of leaf discs after grinding in

liquid nitrogen. The content of cysteine (Cys), an amino acid precursor of GSH, was also

determined by reverse-phase HPLC with spectrofluorometric detection after derivatisation with

monobromobimane according to the methods of Strohm et al. (1995).

2.9. Catalase (CAT) activity

CAT activity was measured in extracts of 0.2 g fresh leaf tissue of leaf discs after grinding in

liquid nitrogen following the method of Aebi (1984).

2.10. Statistical analysis

At least three independent parallel measurements were carried out in each experiment.

Differences between mean values were assessed with Student's t-test at a significance level of P

= 0.05.

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2.11. Multiple sequence alignments

Nucleotide sequences of gsh1 genes were downloaded from the National Center for

Biotechnology Information (NCBI) databases (Altschul et al., 1997). Multiple sequence

alignments were applied in silico with the software programs BioEdit Sequence Alignment

Editor (North Carolina State University, USA) (Hall, 1999), MULTALIN (Combet et al., 2000),

CLUSTAL W (Thompson et al., 1994), and FastPCR (Kalendar et al., 2009). For BLAST

analysis the NCBI databases were used (Altschul et al., 1997). Hierarchical cluster analysis with

the Maximum Likelihood (ML) method (Hillis et al., 1994) was used to construct dendrograms

from the alignment sequences using computer program MEGA4 (Tamura et al., 2007).

3. Results

3.1. Paraquat exposure

A concentration of 4.0 × 10-6 M paraquat led to bleaching the leaf discs (Figure 1) of each of the

poplar clones. A concentration of 4.0 × 10-7 M paraquat caused chloroplast sublethality with a

mixture of bleached and green spots on the leaf discs (Figure 1). No bleaching effect of paraquat

was observed in the dark-incubated leaf discs (Figure 1).

3.2. GSH contents

The GSH content in both transgenic lines significantly increased by 190% (331.1±13 nmol GSH

g-1 FW for TRggs11) and 296% (504.8±22 nmol GSH g-1 FW for TRlgl6), respectively,

compared with that of WT (174.3±11 nmol GSH g-1 FW) (Figure 2).

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Under paraquat treatment, the GSH content of TRggs11 clone showed a concentration-dependent

increment with increasing concentration of the herbicide and peaked at 5 × 10-7 M paraquat

(1932.1±70 nmol GSH g-1 FW) (Figure 2).

3.3. Nitric oxide exposure

The light/dark-dependent NO release from NaNP did not show significant differences between

light and dark incubation, with the highest release of NO (300±16 ng-1 L) at the 3rd day of the

dark incubated vessels (Figure 3).

Dry weight (DW) of leaf discs of the TRggs11 clone tripled at a concentration of 10-7 M NaNP,

but NaNP had no effect on the DW of TRlgl6 and WT (Figure 4).

Nitric oxide-induced GSH overproduction at 10-7 M NaNP in the TRlgl6 clone (from 504.8±22

to 728.6±29 nmol GSH g-1 FW) and in the WT (from 174.3±11 to 272.3±29 nmol GSH g-1 FW)

(Figure 5a). The NO had no effect on the GSH content of TRggs11 clone, however the GSH

precursor amino acid Cys showed the highest level (27.7±1.1 nmol GSH g-1 FW) in this clone

without NO treatment (Figure 5b).

NaNP (10-7 M) combined with PQ (10-7 M) generated the highest level of GSH production

(1093.0±45 nmol GSH g-1 FW) in the TRggs11 clone (Figure 5c). The WT also showed PQ-

stimulated NO-responsiveness (791.0±30 nmol GSH g-1 FW) at a lower (5 × 10-7 M)

concentration of PQ combined with 10-7 M NaNP (Figure 5c).

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3.4. Zn2+ and SO42- uptake of poplars

Significantly higher S uptake capacity was observed by the TRlgl6 clone at 10-3 M ZnSO4

concentration (1171±48 nmol S g-1 DW) compared with that of TRggs11 (906±37 nmol S g-1

DW) and the WT (889±38 nmol S g-1 DW) (Figure 6).

When molar concentrations were calculated, the content of Zn and S in the leaf discs showed not

the expected 1:1 ratio with the highest level of Zn exclusions in Trlgl6 at 10-3 ZnSO4 (Figure 6).

Consequently, BCFZn of leaf discs showed low and concentration-independent values in each

clones, whereas BCFS values peaked at 10-4 M ZnSO4 in all clones with the highest value for

TRlgl6 (Figure 6).

3.5. Total content of Zn, Na, Mn, Cu and Mo

The total contents (mg kg-1 DW) of Zn, Na, Mn, Mo and Cu measured in the leaf discs incubated

on basal WPM medium were the highest in the TRggs11 clone (3.3±0.1 g kg-1 DW leaf tissue)

(Figure 7a). This clone contained also the highest contents of Zn (0.82±0.03 g kg-1 DW leaf

tissue) (Figure 7a). TRlgl6 showed the highest Na content (2.49±0.9 g kg-1 DW leaf tissue). The

highest BCFCu value was observed in the WT (Figure 7b) with significantly higher Cu (48±1.9

mg kg-1 DW) content than in the two TR clones (Figure 7a).

3.6. Salt-induced CAT activity

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The enzyme activity of CAT increased with the increasing concentration of NaCl (0.75% and

1.5%) (Figure 8) in all clones studied. However, significantly higher CAT activity was observed

in both transgenic lines compared with WT with an extreme level in the TRggs11 clone

(911.3±35 µmol H2O2 g-1 FW min-1) at 0.75% NaCl (Figure 8).

3.7. Multiple sequence alignment and phylogenetic analysis

The analysis revealed an evolutionarily conserved gsh1 cladogram (Figure 9), which strictly

followed the systematic order of plant families, and indicated that P. x canadensis and Salix

sachalinensis of Salicaceae family are genetically the closest to P. x canescens analyzed at this

gene (Figure 9).

4. Discussion

Genetic transformations provide unique plant materials for modeling plant responses to

environmental contaminations (Creissen et al., 1996; Czakó et al., 2006). Due to high growth

rate and water uptake capacity poplars became the main trees for not only the industrial (e.g.

paper production; Park et al., 2004) but also for environmental (e.g. phytoremediation) uses

(Peuke and Rennenberg, 2005; Bittsánszky et al., 2009). Transgenic poplar clones, used in our

study, were developed by genetic transformation of the bacterial (Escherichia coli) gene gshI

that encodes gamma-glutamylcysteine synthetase (Watanabe et al., 1986). It resulted in a dual

expression of poplar gsh1 and bacterial gshI genes with the endproduct of GSH, which

accumulated either in the cytosol by TRggs11 (Arisi et al., 1997) or in the chloroplasts by

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TRlgl6 (Noctor et al., 1998) with increasing GSH production (Leplé et al., 1992). As plant

phytoextraction capacities show a direct correlation with their GSH content, these trees play a

potential role in phytoremediation and phytoextraction (Noctor et al., 1995; Gullner et al., 2001).

Owing to the presence of the sulfhydril (SH) group of Cys residue in the tripeptide GSH,

GSH is a reducing and electron donor agent. When GSH is oxidized, a disulfide bond is formed

between two GSH molecules and GSH is converted to the oxidized form of glutathione disulfide

(GSSG) with the release of an electron. GSSG can be reduced back to GSH by glutathione

reductase (GR) (EC 1.6.4.2) using NADPH as an electron donor. The ratio between reduced

GSH and oxidized GSSG is a sensitive indicator of cellular toxicity (Pompella et al., 2003). GHS

combined with another reductants dithiothreitol can further enhance reduction more than GSH

alone like in the case of arsenate used in vitro (Nagarajan and Ebbs, 2010).

Paraquat, as an effective electron acceptor, primarily affects the photosynthetic electron transport

chain (PETC) located in the chloroplasts (Will et al., 2001). For paraquat treatment in our study,

a reduced sucrose concentration (1%) was supplied to the agar media to stimulate the activity of

PETCs, which are switched off by higher sucrose concentrations (2-3%) (Medgyesi et al., 1986;

Lehoczki et al., 1992). Similar to our results, transgenic poplars did not show enhanced tolerance

to photo-oxidative stress caused by paraquat in a study of Will et al. (2001). The non bleaching

effect observed in the dark-incubated leaf discs (Figure 1) confirmed the mode of action of

paraquat, which primarily targets the PETC of chloroplasts without effecting the other organelle

electron transport chain METC (mitochondrial electron transport chain) (Gyulai et al., 2005;

Bittsánszky et al., 2006).

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As a result of the dual expression of the poplar gene gsh1 and the prokaryotic transgene gshI, the

GSH content in both transgenic lines significantly increased compared with the GSH content of

WT (Figure 2). Due to the different transgene cassette of TRlgl6 (Noctor et al., 1998), the

chloroplast-directed GSH production was higher in TRlgl6 (296%) clone then in TRggs11

(190%) compared to WT (100%) (Figure 2). Nevertheless, under paraquat treatment (5 x 10-7

M), as a result of the paraquat-response plant defense mechanism, the GSH content of TRggs11

clone showed extreme high level (Figure 2). Similar to this GSH overproduction, the expression

level of the gshI gene (Bittsánszky et al., 2006) and the enzyme activity of glutathione S-

transferase (Gullner et al., 2001) also showed significant increment under paraquat stress in the

transgenic poplars studied here.

NO is a gaseous, water- and lipid-soluble reactive aerial nitrogen species known to act as a

biological messenger in cell signaling. In mammals, NO is released by the activity of nitric oxide

synthase (NOS, EC 1.14.13.39) (1433 amino acids), which catalyses the formation of citrulline

from L-arginine, and acts on the cardiovascular system as a vasodilator in mammals (Bian and

Murad, 2003). In plants, the role of NO was first studied more than a decade ago (Delledonne et

al., 1998; Durner et al., 1998) indicating that there are at least two main enzymatic routes for NO

production: the NOS-dependent L-arginine pathway, similar to animals, and the nitrite-

dependent pathway. The NOS enzymes, mapped first onto Arabidopsis chromosome 3

(Salanoubat et al., 2000), are functionally well characterized (Ötvös et al., 2005; Grün et al.,

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2006). The nitrite-dependent pathway mainly involves nitrate reductase (NR) (EC 1.7.1.3), which

catalyses the reduction of nitrate to NO (Yamasaki et al., 2001).

NaNP, which releases NO when illuminated by a high light intensity (35 mol

photons/m2/s at 20°C for 15 h), release 5–15 µM photolytic NO (Floryszak-Wieczorek et al.,

2006). In the present study the lower level of NO release might probably due to the nutritive

media used and the relatively low light intensity applied for leaf discs grow (Figure 3).

NaNP-treatment tripled the dry weight of leaf discs of the TRggs11 at a concentration of

10-7 M (Figure 4). In a study of Wang et al. (2005), NaNP, applied exogenously up to 500 µmol

L-1, also elevated the metabolism of poplar leaves by increasing the photosynthesis rate,

photochemical efficiency of PSII, and the water content.

NO-treatment increased the GSH production of TRlgl6 and WT (Figure 5a). Although,

NO decreased the GSH content of TRggs11 clone, and the content of the GSH precursor amino

acid Cys, which showed the highest level in this clone without NO-treatment (Figure 5b). These

contradictory results might be caused by the two different transgene cassettes in TRggs11 and

TRlgl6 (Noctor et al., 1998).

Nitric oxide, released in the presence of an electron acceptor such as paraquat, binds to

thiol groups of GSH, Cys and sulphur-containing proteins, resulting in S-nitrosylated derivatives,

which can interact with cellular proteins, especially the stress-responsive signaling proteins

(Grün et al., 2006). This NO-responsive GSH production was observed in the TRggs11 clone,

which produced the highest level of GSH at 10-7 M paraquat combined with 10-7 M NaNP

(Figure 5c).

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Zn homeostasis, in poplar, is maintained primarily in roots and leaves, and not in the transporting

organs of shoots and stems (Adams et al., 2011). Growth of poplars at early developmental

stages was found highly sensitive to environmental Zn, but was relatively resilient in the adult

trees (Adams et al., 2011). Unlike poplars, metallocrops like oat, barley and Indian mustard took

up high level of Zn in vivo (Ebbs and Kochian, 1998).

In our study, Zn uptake in all clones showed negative correlations with the decreasing

concentrations of ZnSO4 from 10-2 M (Figure 6). The molar concentrations of Zn and S in the

leaf discs showed not the expected 1:1 ratio, coupled with high levels of Zn exclusions, which

result indicates a separate and strictly regulated plant uptake of cationic (Zn2+) and anionic (SO42-

) moieties (Figure 6). However, TRggs11 showed a tripled Zn uptake capacity compared with

TRlgl6 and WT (Figure 7a) with the highest BCFZn value (Figure 7b).

Significantly high S uptake observed by the TRlgl6 clone at 10-3 M ZnSO4 concentration

(Figure 6), supplied sulfur for the high level of GSH synthesis with a further increment under

NO treatment (Figure 5a). Leafy cut clones of TRggs11, also showed a 1.6-fold sulfate uptake

capacity compared with WT (Herschbach et al., 2000). For a further study of Zn- and S uptake of

poplar a split-root technique (Waters et al., 1980; Marsh et al., 1985) would provide additional

information.

Plant mineral uptake, in general, relies on S-rich peptides such as GSH, metallothioneins, metal

transporter proteins and phytochelatins (Rennenberg and Brunold, 1994; Rennenberg and

Herschbach, 1995; Roosens et al., 2005). Total content of the elements measured in the poplar

leaf discs was the highest in the TRggs11 clone (Figure 7a). TRlgl6 showed the highest Na

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content, which was a 133% increment compared to an in situ data of Teuber et al. (2008). The

highest BCFCu value was observed in the WT (Figure 7b). All these patterns of minerals uptake

were effected by the chelating agent EDTA supplied to the aseptic media and the plant hormones

applied, which increased the whole plant metabolisms in the leaf-disc. Without Fe-EDTA

(Haberlandt, 1902; White, 1943) and hormones (Murashige and Skoog, 1962; Gyulai et al.,

1992) plants can not grow in vitro due to chlorosis. In pot experiments (Ebbs and Kochian,

1998), using a Zn-contaminated soil, the addition of EDTA to the soil significantly increased Zn

accumulation by B. juncea but not by oat or barley. Nevertheless, barley accumulated >2 mg of

Zn plant-1, it was 2−4 times more Zn than what was observed in Indian mustard in the presence

of EDTA (Ebbs and Kochian, 1998).

As the result of the high level of Na content of each of the poplar clones (Figure 7a), CAT

activity, as a stress indicator (McClung, 1997), was measured in the leaf discs exposed to NaCl

(Figure 8). CAT scavenges H2O2, the reaction product of superoxide dismutase, which is also

generated during the processes of mitochondrial electron transport and β-oxidation of fatty acids

during the photorespiratory oxidation (McClung, 1997). In animals a single CAT isoform is

encoded by a single gene. In contrast, CAT in plants is present as multiple isoforms encoded by

three genes of a multigene super family (Yang and Poovaiah, 2002).

Due to the plant oxidative stress response mechanisms (McClung, 1997; Yang and

Poovaiah, 2002), the activity of CAT in our study increased with the increasing concentration of

NaCl (Figure 8). Significantly higher CAT activity was observed in both transgenic lines

compared with WT with an extreme level in the TRggs11 clone at 0.75% NaCl (Figure 8). This

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result indicates that the presence of gshI transgene also improves effectively the plant oxidative

stress response capacity by the overproduction of GSH (Figure 2 and 5a). In poplar, a salt-

induced accumulation of GSH was also observed in poplar leaves as a result of enhanced

photorespiration (Herschbach et al., 2010).

Multiple sequence alignments and hierarchical cluster analyses (Moylan et al., 2004; Gyulai et

al., 2012a; Alzohary et al., 2012, 2013) of nucleotide sequences for gsh1 genes predicted

intensive GSH production for P. x canadensis and Salix sachalinensis similar to poplar, with

potential use for broadening the tools of phytoremediation (Figure 9),

5. Conclusions

To conclude, due to the aseptic in vitro conditions, the results presented gave insight into the

absolute genetic potential of WT and TR poplars exposed to common salt, zinc sulfate, the

herbicide paraquat and nitric oxide. The study of the EDTA-stimulated mineral uptake potentials

in vitro also excluded the environmental cross reactions, and indicated that WT and the two 35S-

gshI transgenic poplars might be useful for different phytoextraction purposes in situ: WT poplar

for Cu uptake; TRggs11 for uptake of Zn, Mn and Mo; and TRlgl6 for uptake of sulfate and Na.

Comparative in silico DNA sequence analyses of plant gsh1 genes indicated further species of

the Salicaceae family with a putatively high GSH production capacity and their possible uses for

phytoextraction under conditions where P. x canescens cannot be grown. Our results may

contribute to an integrated approach of soil remediation and the development and

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implementation of new bioremediation technologies that make use of plants to remove toxic

substances from contaminated sites.

Acknowledgements

Poplar clones were kindly provided by Dr. C.H. Foyer (Newcastle-upon-Tyne, UK) and Dr. H.

Rennenberg (Freiburg University, Germany). The project was supported in part by grants of the

Hungarian Scientific Research Fund OTKA-72926. OTKA-K77641, OTKA-K72926 and

OTKA-PD-75169. All authors wish to thank the Editor-in-Chief and the two anonymous

reviewers for their valuable suggestions.

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Figure Captions

Figure 1. Effects of light and dark incubation and sucrose concentration on bleaching of

poplar leaf discs by paraquat (PQ) (4 x 10-7 and 4 x 10-6 M). Leaf discs of wild-type (WT) and

the 35S-gshI transgenic poplar (Populus × canescens) lines TRggs11 and TRlgl6 were treated for

21 days on aseptic WPM media.

Figure 2. Glutathione (GSH) production in response to paraquat (PQ) exposure in poplar

leaf discs. GSH content was measured in leaf discs of wild-type (WT) and 35S-gshI transgenic

poplar (Populus × canescens) lines TRggs11 and TRlgl6 after treatment with 10-8 to 5 × 10-7 M

paraquat (PQ) for 21 days on aseptic WPM media. Mean values ± standard deviation are

indicated (n = 3).

Figure 3. Time course of nitric oxide [NO] release from NaNP (10-7 M) supplied in aseptic

WPM nutritive media and incubated under either a 16 h/8 h (light/dark) photoperiod or in

the dark. Mean values ± standard deviation are indicated (n = 3).

Figure 4. Concentration-dependent effect of NaNP on the leaf disc dry weight (DW). Leaf

discs of wild-type (WT) and 35S-gshI transgenic poplar (Populus × canescens) lines TRggs11

and TRlgl6 were treated with 10-7 to 10-4 M NaNP for 21 days added to aseptic WPM media.

Mean values ± standard deviation (n = 3) are indicated.

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Figure 5. Glutathione (GSH) and cysteine (Cys) contents in leaf discs in response to [NO]

released from NaNP and paraquat exposure. (a) Glutathione (GSH) content and (b) cysteine

(Cys) content in leaf discs of wild-type (WT) and 35S-gshI transgenic poplar (Populus ×

canescens) lines TRggs11 and TRlgl6 exposed to 10-7 M NaNP for 21 days. (c) Effect of

combined treatment of NaNP (10-7 M) and paraquat (PQ) (10-8 to 5 × 10-7 M) on GSH

production after 21 days treatment on aseptic WPM medium. Mean values ± standard deviation

are indicated (n = 3).

Figure 6. Zinc (Zn) and sulfur (S) contents, and bioconcentration factor (BCF) ratios for

Zn and S. Leaf discs of wild-type (WT) and 35S-gshI transgenic poplar (Populus × canescens)

lines TRggs11 and TRlgl6 were exposed to a concentration series of ZnSO4 (10-2 to 10-4 M) for

21 days on aseptic WPM medium. Basal WPM medium contained 1.955 mg L-1 Zn2+ and 14.36

meq L-1 SO42-. Mean values ± standard deviation indicated (n = 3).

Figure 7. Mineral uptake capacity and bioconcentration factor (BCF) ratios of poplar leaf

discs. (b) Mineral uptake capacity of leaf discs of wild-type (WT) and 35S-gshI transgenic

poplar (Populus × canescens) lines TRggs11 and TRlgl6. Mean values ± standard deviation (n =

5) are indicated. (b) BCF ratios of Na, Zn, Mn, Cu, and Mo, and Basal WPM nutritive media

contained Na (4.59 mg L-1), Zn (1.955 mg L-1), Mn (7.248 mg L-1), Mo (0.099 mg L-1) and Cu

(0.063 mg L-1).

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Figure 8. Catalase activity of leaf discs of wild-type (WT) and 35S-gshI transgenic poplar

(Populus × canescens) lines TRggs11 and TRlgl6 exposed to different concentrations of

NaCl for 21 days on aseptic WPM media. Mean values ± standard deviation (n = 3) are

indicated.

Figure 9. Bootstrap consensus ML (Maximum Likelihood) (Hillis et al., 1994) dendrogram

(Tamura et al., 2004) derived from nucleotide sequences of plant gsh1 genes with outgroup

of prokaryotic (E. coli) trangene gshI (Watanabe et al., 1986). GeneBank accession numbers

are specified for each accession. Bootstrap support values from 1000 replicates are provided

below each node. The scale bar (0.1) represents relative genetic distances with the numbers of

nucleotide substitutions per site of sequences. Plant families are indicated.

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Fig. 2.

Light

Dark

Sucrose 1.0 % Sucrose 2.0 %

Not Treated Not Treated 4.0 x 10-7 M PQ 4.0 x 10-6 M PQ 4.0 x 10-7 M 4.0 x 10-6 M PQ

TRggs11

TRlgl6

WT

TRggs11

TRlgl6

WT

264.5174.1

298.1504.8 687.9504.8 328.6

579.3

380.1354.5270.8

331.2

625.3946.5

1932.1

0

400

800

1200

1600

2000

Not Treated 10-8 M PQ 5 x 10-8 M PQ 10-7 M PQ 5 x 10-7 M PQ

nmol

GSH

x g

-1 F

W le

af t

issu

e WTTRlgI6TRggs11

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Fig. 3.

250

032

204

11497

0

149

195

0

225

35

0

41

300

217

00

236

191

220233

5982

102

188

63

33

0

50

100

150

200

250

300

350

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27Days

[NO

] rel

ease

(ng

L-1)

LightDark

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Fig. 4.

0.51.3

3.83.03.0

3.9

8.1

10.2

0.4

2.33.9

3.03.30.61.8

0

2

4

6

8

10

12

Not Treated 10-7 M 10-6 M 10-5 M 10-4 M NaNP [NO]

Leaf

dis

cs (m

g D

W)

WTTRggs11TRlgl6

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Fig. 5a.

Fig. 5b.

174.1

331.2

504.8

272.3 245.1

728.6

0

100

200

300

400

500

600

700

800

WT TRggs11 TRlgI6

Clones (Populus x canescens )

nmol

GSH

g-1

FW

leaf

tis

sue Not Treated

NaNP [NO] 10-7 M

17.6

27.7

18.1

27.2

10.8

23.6

0

5

10

15

20

25

30

35

WT TRggs11 TRlgI6Clones (Populus x canescens )

nmol

Cys

g-1

FW

leaf

tis

sue

Not TreatedNaNP [NO] 10-7 M

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Fig. 5c.

Fig. 6.

397

1985

491

15140

410

1327

410

281374

11711165

906

255

889

340 36

0.612 2.81.30.8220.68

3 413

3

106

88

117

0

200

400

600

800

1000

1200

1400

10-2 10-3 10-4 10-2 10-3 10-4 10-2 10-3 10-4

WT TR (ggs11) TR (lgl6)

ZnSO4 (mol)

- nm

ol Z

n an

d S

g-1

DW

leaf

dis

c -

0

20

40

60

80

100

120

140

- BC

F -

Zn (nmol)S (nmol)BCF-ZnBCF-S

791.0

263.0324.0277.0

429.0432.0

689.0

1093.0

589.0

608.0514.0

425.0

0

200

400

600

800

1000

1200

10-8 M PQ and 10-7 M NaNP

5x10-8 M PQ and 10-7 M NaNP

10-7 M PQ and 10-7 M NaNP

5x10-7 M PQ and10-7 M NaNP

nmol

GSH

x g

-1 F

W l

eaf

tissu

e

WTTRggs11TRlgl6

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Fig. 7a.

Na; 2489.1

Na; 1888.3Na; 1702.2

Zn; 820.6

Zn; 241.6

Zn; 291.6

Mn; 240.4Mn; 324.1

Mn; 199.1

Mo; 26.2

Mo; 37.7 Mo; 24.5

Cu; 48.4

Cu; 10.8 Cu; 17.4

0

500

1000

1500

2000

2500

3000

3500

WT TRggs11 TRlgl6Clones (Populus x canescens )

mg

kg-1 D

W le

aves

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Fig. 7b.

Fig. 8.

542.3411.4

Na; 370.8

123.6

419.7

Zn; 149.1

33.2Mn; 27.5 44.7

247.0381.2

Mo; 265.0

275.4171.3

Cu; 767.6

0

150

300

450

600

750

900

WT TRggs11 TRlgl6Clones (Populus x canescens )

BCF

239.

1

703.

2

535.

2

333.

4

911.

3

357.

1

382.

6

451.

8 621.

9

65.1

42.3

24.363

.1

23.6 87

.5

0

150

300

450

600

750

900

1050

WT TRggs11 TRlgI6Clones (Populus x canescens )

µmol

H2O

2 g-1 F

W mi

n-1

Not Treated0.75 % NaCl1.5 % NaCl2.25 % NaCl3.0 % NaCl

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HQ658456 Populus x canadensis (gsh1)

XM002297999 Populus trichocarpa

HQ228352 Salix sachalinensis

Salicaceae

AF017983 Lycopersicon esculentum

DQ444219 Nicotiana tabacum (cpDNA-gsh1)Solanaceae

Fagaceae DQ787258 Fagus sylvatica

AF128453 Glycine max

AF128454 Phaseolus vulgaris

AY204516 Lotus corniculatus

AF128455 Pisum sativum

AF041340 Medicago truncatula

Fabaceae

Y10848 Brassica juncea

NM118439 Arabidopsis thaliana

Z29490 A thaliana (cpDNA-gsh1)

Cruciferae

DICOTs

AJ302783 Zea mays

AJ508916 Oryza s japonica

AY864064 Triticum aestivum

MONOCOTs - Poaceae

MONOCOTs - Liliaceae AF401621 Allium cepa

PROKARYOTEs X03954 E coli (gshI)

99

98

89

100

77

93

77

65

94

77

90

41

58

45

21

45

0.1

Fig. 9.

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Documents

Phytoextraction potential of wild type and 35S-gshI transgenic poplar trees (Populus × canescens) for environmental pollutants