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TRANSGENIC TOBACCO PLANTSEXPRESSING A FUNGAL LACCASE AREABLE TO REDUCE PHENOL CONTENTFROM OLIVE MILL WASTEWATERSPasquale Chiaiese a , Francesca Palomba a , Carolina Galante a

, Silvana Esposito a , Margherita-Gabriella De Biasi a & EdgardoFilippone aa Department of Soil, Plant, Environmental and Animal ProductionSciences, School of Biotechnological Sciences, University of Naples“Federico II”, Portici, ItalyAccepted author version posted online: 30 Mar 2012.Publishedonline: 07 Jun 2012.

To cite this article: Pasquale Chiaiese , Francesca Palomba , Carolina Galante , Silvana Esposito ,Margherita-Gabriella De Biasi & Edgardo Filippone (2012): TRANSGENIC TOBACCO PLANTS EXPRESSINGA FUNGAL LACCASE ARE ABLE TO REDUCE PHENOL CONTENT FROM OLIVE MILL WASTEWATERS,International Journal of Phytoremediation, 14:9, 835-844

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International Journal of Phytoremediation, 14:835–844, 2012Copyright C© Taylor & Francis Group, LLCISSN: 1522-6514 print / 1549-7879 onlineDOI: 10.1080/15226514.2011.628715

TRANSGENIC TOBACCO PLANTS EXPRESSING AFUNGAL LACCASE ARE ABLE TO REDUCE PHENOLCONTENT FROM OLIVE MILL WASTEWATERS

Pasquale Chiaiese†, Francesca Palomba†, Carolina Galante,Silvana Esposito, Margherita-Gabriella De Biasi, andEdgardo FilipponeDepartment of Soil, Plant, Environmental and Animal Production Sciences, Schoolof Biotechnological Sciences, University of Naples “Federico II”, Portici, Italy

A biotechnological approach was applied to reduce phenol content in olive mill wastewatersby transgenic tobacco plants. The cDNA laccase of poxC gene from Pleurotus ostreatus,carrying its own signal peptide for extracellular secretion, was transferred into the Nicotianatabacum genome. Transgenic tobacco plants were obtained and the recombinant enzyme wassecreted into the rhizosphere by the plant root apparatus, confirming the ability of the plantmachinery to recognize the fungal POXC peptide signal leader appropriately as secretorytag. Total laccase activity assayed by ABTS in transgenic lines increased sharply comparedto control plants. Moreover, plants cultivated in a hydroponic solution with the addition ofolive mill wastewaters were able to reduce the total phenol content up to 70%.

KEY WORDS: phytoremediation, Pleurotus ostreatus, laccase, Nicotiana tabacum, metabolicengineering, olive oil mill wastewaters

INTRODUCTION

The olive oil industry generates large amounts of by-products that are harmful to theenvironment. Olive mill wastewaters (OMWs) are effluents with a high load of phenoliccompounds. Since phenols are amphiphilic, only a fraction of them enters the oil phase: alarge proportion (>98%) is lost with the waste stream during processing (Rodis et al. 2002).Phenols are also considered responsible for the black colour of OMWs, they are phytotoxicand have antimicrobial properties (Tsioulpas et al. 2002). For these reasons, the uncontrolleddisposal of OMWs has traditionally become a great problem in the Mediterranean area dueto their polluting effects on soil and water (Piotrowska et al. 2006; Sierra et al. 2007).World olive oil production is estimated to be about 2.9 million metric tons per year ofwhich approximately 98% is from the Mediterranean (Cardinali et al. 2010).

†equal contribution of these authorsAddress correspondence to Pasquale Chiaiese, Department of Soil, Plant, Environmental and Animal Pro-

duction Sciences, School of Biotechnological Sciences, University of Naples “Federico II” Via Universita, 10080055, Portici, (Naples), Italy. E-mail: chiaiese@unina.it

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Among methods that can be applied to remediate contaminated soils, phytoremedi-ation (Pilon-Smits 2005) has two main advantages: it preserves the natural soil structure(Arvanitoyannis et al. 2007; Vangronsveld et al. 2009) and it is less expensive than otherremediation strategies (Mench et al. 2009). On the other hand, the main constraint to usingplants for remediation is the absence of highly efficient enzymes for phenol degradation.To enhance the potential of phenol degradation, biotechnological strategies can be applied,through either the over-expression of endogenous genes or the expression of transgenes(Abhilash et al. 2009). In recent years, among enzymes capable of degrading phenols,laccases have raised interest and are already used in a variety of biotechnological andenvironmental applications (Gianfreda and Rao 2004).

Laccases (EC 1.10.3.2) are enzymes belonging to the oxidoreductase family whichexert their activity on a wide variety of phenolic and non-phenolic compounds (Bailey et al.2004). An important feature of this enzyme is the ability to degrade phenols into less toxiccompounds (Majeau et al. 2010) with a concomitant reduction of molecular oxygen to water(Canas and Camarero 2010). Laccases are widely distributed in bacteria, fungi and higherplants (Alexander 2000; Duran and Esposito 2000). In nature, soil bacteria and white-rotfungi can oxidize organic pollutants, decreasing their toxicity by producing enzymes withdegradation activity. Some species of white rot-fungi, such as Pleurotus ostreatus, havebeen shown to degrade phenols in soil (Elisashvili and Kachlishvili 2009; Haritash andKaushik 2009; Giardina et al. 2010). To enhance the efficiency of xenobiotic removal byplants a biotechnological approach consists in overexpressing genes that are involved intheir degradation (James and Strand 2009).

This study aimed to assess to reduce the phenol content of OMW using tobacco plantsexpressing a fungal laccase: poxC cDNA, isolated from the basidiomycetes Pleurotusostreatus, was transferred into the Nicotiana tabacum genome. Transgenic plants werecharacterized by molecular and enzymatic assays and the ability to reduce the total phenolcontent in the OMW.

MATERIALS AND METHODS

Signal Peptide Analysis

In silico translation of the poxC cDNA sequence (Z34848) was performed using theTranseq program (www.ebi.ac.uk/emboss/transeq/). Identification of sequences resemblingplant signal peptide was carried out by http://www.cbs.dtu.dk/service/SignalP/.

Plant Genotype and In Vitro Growing Conditions

Nicotiana tabacum cv Samsun NN was propagated by stem nodes on nutrient growthmedium (PO) based on Murashige and Skoog (1962) mineral salts, with the addition ofsucrose 30 g l−1 (Duchefa) and Microagar 8 gl−1 (Duchefa) as gelificant agent.

Plant Expression Vector and Tobacco Genetic Transformation

The cDNA-clone of pox2 gene, also termed poxC, isolated from the white-rot fungusPleurotus ostreatus (Palmieri et al. 1993) was cloned into SacI-XbaI sites of the pKYLXplant binary vector driven by CaMV35S2 constitutive promoter. The expression vector

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was transferred into Agrobacterium tumefaciens strain LBA4404 (Ooms et al. 1982) byelectroporation (Biorad E. Coli pulser).

Tobacco genetic transformation was carried out as reported in Horsch et al. (1985).After co-cultivation, leaf explants were transferred onto PO medium with BAP 2.0 mgl−1, IAA 0.4 mg l−1 and kanamycin 100.0 mg l−1 added as selective agent. Every 10 daysexplants were transferred onto a selective fresh medium. After 6 weeks, regenerated shootswere transplanted into PO basal medium containing kanamycin 150.0 mg l−1.

Screening and Identification of Transgenic Tobacco Plants

PCR analysis was performed on genomic DNA extracted from tobacco leaves (Doyleand Doyle 1987) collected from regenerated shoots. PCR was carried out using twopoxC cDNA-specific primers (forward 5′-CACGGATTCTTGCTACGCTTA-3′; reverse 5′-CTTAGGACGGAACGATGCCTC-3′). The PCR program consisted of 5 min at 95◦C,followed by 30 cycles of 60 sec at 94◦C, 60 s at 60◦C, and 60 s at 72◦C. The final extensionstep was for 10 min at 72◦C.

Estimation of poxC Gene Copies Integrated in the Transgenic

Tobacco Genome

The number of poxC copies integrated into the transgenic tobacco lines was es-tablished by Southern blot. Genomic DNA digested with SacI (Promega was separatedby electrophoresis through a 0.8% (w/v) agarose gel and blotted to Hybond nylon mem-brane (Amersham Biosciences). Blots were hybridized with a probe of 1.7 kbp, previouslyobtained from SacI and XbaI digestion of the expression vector. Probe labelling was per-formed with (α-32P) dCTP by the Prime-a-Gene (Promega) labelling kit, according to themanufacturer’s instructions.

Total Leaf Protein Extraction

Leaf tissues were grinding in liquid nitrogen and homogenizing in the extractionbuffer, as reported by Nagel et al. (1992). Insoluble material was removed by centrifugationfor 15 minutes at 4◦C at 14K rpm; the supernatant was then transferred to a fresh tube.Total protein concentration was determined by the Bradford method (Bradford 1976).

Western and Zymogram Analysis

To perform immunoblot analysis 100 µg of total soluble proteins for each transgenicline were separated on 15% SDS polyacrylamide gels and electroblotted onto nitrocellulosemembrane (Amersham), following the standard protocols as reported in Sambrook et al.(1989). The heterologous laccase was detected with polyclonal antibodies raised in rabbitagainst the purified laccase from Pleurotus ostreatus. The secondary antibody was anti-rabbit IgG (Biorad).

To perform zymogram analyses 60 µg of total leaf protein for each sample were loadedonto 12.5% SDS polyacrylamide gel under native conditions as reported by Palmieri et al.(2000). After running, gel was soaked in a 0.1 M sodium citrate solution at pH 3.0 contain-ing 2 mM ABTS [2,2-azobis(3-ethylbenzothiazoline-6-sulphonic acid)] as chromogenicsubstrate for 5 min (Hatakka 1994). ABTS is a synthetic non-phenolic substrate, which

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produces a green radical cation when oxidised by laccase (Singh Arora and Kumar Sharma2010).

Enzymatic Activity of Transgenic Root Exudates

Nodes of each transgenic line were subcultured in PO liquid medium for 3 weeksin sterile conditions. Rooted plants were then transplanted into 30 ml PO liquid medium,and after 7 days aliquots of culture medium were taken under sterile conditions. Totalprotein content of culture medium was quantified by Bradford assay (Bradford 1976). Theheterologous laccase activity was evaluated by oxidation of 2mM ABTS at 420nm (ε =36.000 cm−1 M−1) in 0.1M citrate buffer (pH 3.0) at 25◦C (Piscitelli et al. 2005). Enzymaticactivity was expressed as international units (U), where U corresponds to 1 µmol of ABTSoxidised per minute. Assays were performed in triplicate.

In vitro OMW Phenolic Reduction Assay

Olive mill wastewaters (OMWs) from a local oil press was centrifuged at 10,000 rpmfor 45 min and then filtrated by a fine mesh aiming to remove solid particles. The liquidphase was filter sterilised with a 0.22 µm nitrocellulose filter under a laminar flow hood toobtain an axenic OMW solution.

Rooted tobacco transgenic lines and control plants were transferred into sterile con-tainer pots in which OMW was diluted 1:25 (v/v) to obtain a final phenol concentration of0.1 g l−1. Plants were grown for 16 days in a growing chamber at 24◦C under a 16:8 hourslight:darkness photoperiod. The concentration of total phenols was detected in accordingto Folin et al. (1927) on liquid culture aliquot every two days. Two replicates were madefor each plant and three replicates for each sample.

Statistical Analysis

All data reported were averaged at least over three separate experiments. ANOVAanalyses were performed by Systat 11.0 software. Differences were considered to be sig-nificant at P < 0.05.

RESULTS

In silico analysis was carried out for the recognition of the P. ostreatus poxC signalpeptide sequence as plant secretory tag; which results infer that the signal peptide had aprobability of about 90% to target the recombinant protein to the apoplast. Furthermore, ahigh similarity (86%) between poxC cDNA and the tobacco genome was estimated.

Stable nuclear genetic transformation was achieved by co-cultivation of tobacco leafdisks with the Agrobacterium tumefaciens strain bearing the pKYpoxC plasmid. Integrationof the poxC gene in the putative transgene plant genome was established PCR analysis asshown in Figure 1, in which the expected band of 1700 bp was detected in the putativetransgenic plants. No amplicons were observed from non-transgenic plant genomic DNA.

Twenty-five independent transgenic lines were chosen among 170 PCR positiveplants. Expression of the P. ostreatus laccase was assayed by Western blot and the re-combinant protein was the expected size compared to the purified enzyme (Figure 2A).The activity of heterologous poxC enzyme in the 25 transgenic samples was revealed by

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Figure 1 Screening of putative transgenic tobacco shoots by PCR analysis performed on genomic DNA extractedfrom leaves. In track 1–30: putative transgenic plants, C+: P. ostreatus poxC, M: marker size, C−: untransformedplant.

zymogram assay (Figure 2B). By Southern blot analysis, the number of transgene copiesintegrated into genomic tobacco plants was estimated from one to seven (Figure 3).

In order to confirm the root secretion of poxC and to evaluate laccase activity, ABTSassay was performed on liquid culture medium from 14 plants growing in a hydroponicsystem under sterile conditions. No activity was detected in untransformed plant medium,while all transgenic lines showed different levels of laccase activity from their culturemedium (Figure 4). Eight out of 14 transgenic plants showed a very low enzymatic activityand were therefore not reported in Figure 4. On the basis of the enzymatic activity, thesix plants were grouped into three classes by statistical analysis. The first and secondclass showed an enzymatic activity ranging from 1.7-to 12.6 mU mg−1 total protein. Onetransgenic plant (poxC-40) showed the highest activity (47.0 mU mg−1 total protein) forroot exudates. Therefore, these results demonstrated the secretion of the fungal laccase intothe apoplast and hence its release into the culture medium.

The ability of heterologous poxC to reduce the phenolic content of OMW by rootexudates was established for four transgenic lines in hydroponic culture in sterile conditions.The efficiency of phenolic removal was assayed by monitoring the total phenolic content ofOMWs during a 16-day experiment (Figure 5). All plants assayed were able to reduce theOMW phenolic content and a statistical reduction was observed for the poxC-40 genotypefrom the fourth day of culture. From the 14th day of culture, the other genotypes showeda considerable reduction in phenolic content compared with the control plant. At the endof the experiment, the poxC-40 plant was able to reduce the phenolic content to about74% of the initial value. Data from transgenic plants were significantly different from thecontrol. The other transgenic genotypes, on average, were able to reduce this content toabout 51% of the initial value, while the control plant showed a reduction of about 28%(Figure 5).

Figure 2 Detection and enzymatic assay of heterologous phenol oxidase in tobacco leaves. (A) Western analysison leaf total proteins of transgenic tobacco plants and wild type. Rabbit polyclonal antibodies against the purifiedlaccase from P. ostreatus were used. (B) Zymogram of phenoloxidase extracted from transgenic tobacco leavesand wild type. Tracks labelled 1 to 25 transgenic plants, C−: control plants, C+: purified laccase, M:molecularweight marker.

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Figure 3 Southern blot analysis. In track 1-25: putative transgenic plants, C+: P. ostreatus poxC, C−: untrans-formed plant.

DISCUSSION

In nature, organisms such as ligninolytic fungi are able to produce enzymes such aslaccase that are useful for environmental remediation (Christian et al. 2005; Singh Aroraand Kumar Sharma 2010). Kissi et al. (2001) demonstrated that in an airlift bioreactorPleurotus ostreatus was able to reduce the amount of phenolic compounds in OMW mainlydue to release of laccase enzyme into the medium. Among several different molecular formsof laccase secreted by P. ostreatus, POXC is the most abundant and has been molecularlywell characterized (Palmieri et al. 1993). Therefore, our final goal was to enhance catabolic

Figure 4 Enzymatic activity of transgenic root exudates using ABTS as substrate. Laccase activity of rootexudates is calculated as Enzymatic Units per milligrams of total protein. Column with different letters aresignificantly different (p < 0.05).

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TRANSGENIC TOBACCO REDUCE PHENOLS 841

Figure 5 Phenol removal by transgenic plants from olive oil mill wastewaters. Transgenic plants reduction ofphenols contents is reported as percentage. For each group column, with different letters are statistically different(p < 0.05).

activity of higher plants by manipulating their genomes towards phenolic content in acomplex matrix such as that of OMWs. In this regard, we evaluated the ability of transgenicplants to reduce OMW phenolic content by overexpressing the poxc gene in tobacco, chosenas a model species for genetic manipulation.

Ligninolytic enzymes such as laccases are not easily expressed in heterologous sys-tems in an active form, even in other fungi families. White-rot fungal laccases have beenfound to be expressed in Saccharomyces cerevisiae (Cassland and Jonsson 1999), Pichiapastoris (Jonsson et al. 1997) and Aspergillus oryzae (Yaver et al. 1996); which laccaseswere expressed at low concentrations in these fungi. In a few cases the expression of afungal laccase has been reported in plants, such as Trametes versicolor laccase I in maize(Hood et al. 2003; Bailey et al. 2004); Melanocarpus and Pycnoporus laccases in rice (deWilde et al. 2008); Coriolus versicolor and Schizophyllum commune laccases in tobacco(Sonoki et al. 2005; Hirai et al. 2008).

To pursue our goal, the secretion of the transgenic enzyme into the polluted environ-ment was an important task. Bailey et al. (2004) showed that T. versicolor laccase targetingto maize embryo cells extra-cellular compartment affected the regeneration ability, thusproducing a reduced number of transgenic plants. Moreover, the plants died prior to beingtransferred in vivo, suggesting a lethal effect of the transgenic enzyme. In our experiments,after genetic transformation of tobacco cv. Samsun NN with a construct bearing the P.ostreatus poxC cDNA with its own signal peptide, we obtained several stable transformedplants that were able to grow and to seed set.

The secretion of the POXC transgenic enzyme in the plant growth liquid mediumconfirmed the in silico analysis, hence the correct recognition of the 23 amino acid fungalsignal peptide by the plant secretory machinery. C. versicolor signal peptide has also beenshown to be recognised by the tobacco cell secretory machinery (Sonoki et al. 2005).

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However, our results demonstrated for the first time that also P. ostreatus POXC signalpeptide was correctly recognised in plants although this sequence did not prove similar tothat of C. versicolor.

When cultured in liquid medium, transgenic tobacco plants show a wide range ofextracellular phenoloxidase activity. Wang et al. (2004) obtained transgenic A. thalianaplants expressing a cotton laccase gene bearing an extracellular signal sequence leader.Root exudates exerted a phenoloxidase activity of about 6mU mg−1 of total proteins in50-fold concentrated liquid media. The best activity that we detected in tobacco liquidculture medium was eight times higher than that reported by the above authors.

In a 16-day time course experiment in which plants were grown in a diluted OMWhydro-culture system, tobacco transgenic plants were able to survive and to reduce thecontent of phenolic compound in the medium. In one case the phenolic content was reducedto 74% of its initial concentration. Since there was an average reduction of 26% in phenoliccompounds in the control plant medium, we can infer that phenol reduction is related tothe release of transgenic POXC into the culture medium. Our best result was in agreementwith that reported by Sonoki et al. (2005), whereby the efficiency to oxidize bisphenolA or pentachlorophenol of one tobacco transgenic plant secreting C. versicolor laccasewas two times higher than control plants. Similar results were obtained by Hirai et al.(2008), who demonstrated that tobacco plants transformed with a codon-usage optimizedSchizophyllum commune laccase gene were able to reduce about 1.8 fold the trichlorophenolcontent in hydroponic culture. In our experiments, the best transgenic plant reduced the totalphenolic compounds in OMW by about 2.6 fold without any gene sequence modification.Interestingly, this is the first report of total phenol reduction in OMW-added medium, whilstothers have reported the degradation activity on just one phenolic compound.

In this study we demonstrated that plants expressing a fungal laccase constitute auseful tool for phytoremediation of hazardous phenolic contaminants in a complex matrixsuch as olive mill wastewaters. This proof-of-principle could be applied to other plantspecies that have faster growth, produce more biomass, have a deep root apparatus and areadaptable to different environmental conditions.

ACKNOWLEDGMENTS

The authors wish to thank Sara Siervo for her valuable help in plant tissue culture,Rosario Nocerino for his technical assistance and Prof. Giovanni Sannia for providing thepoxC cDNA and the antibodies.

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