The TFIIS and TFIIS-like genes from Medicago truncatula are involved in oxidative stress response

Gene 470 (2011) 20–30

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Gene j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / g e n e

The TFIIS and TFIIS-like genes from Medicago truncatula are involved in oxidative stress response Anca Macovei a, Alma Balestrazzi a, Massimo Confalonieri b, Armando Buttafava c, Daniela Carbonera a,⁎ a b c

Dipartimento di Genetica e Microbiologia, via Ferrata 1, 27100 Pavia, Italy C.R.A.-Centro di Ricerca per le Produzioni Foraggere e Lattiero-Casearie, viale Piacenza 29, 26900 Lodi, Italy Dipartimento di Chimica Generale, Via Taramelli 12, 27100 Pavia, Italy

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Article history: Accepted 7 September 2010 Available online 19 September 2010 Received by G. Theissen Keywords: Barrel medic Elongin A Oxidative stress QRT-PCR TFIIS TFIIS-like

a b s t r a c t The cDNA sequence coding for a novel putative TFIIS (transcription elongation factor II-S), hereby named MtTFIIS-like, was isolated from barrel medic (Medicago truncatula Gaertn.) by reverse transcriptasepolymerase chain reaction. The nucleotide sequence contains an open reading frame of 1074 bp, predicting a 40.0 kDa protein, conserved among plant species. The N-terminal region of the MtTFIIS-like protein includes a LW motif, characterized by highly conserved leucine (L) and tryptophan (W) residues, also found in the canonical TFIIS protein, elongin A (transcription elongation factor S-III) and CRSP70 (cofactor required for Sp1 activation), while a proline-rich region is present in the C-terminal domain. The expression profiles of the MtTFIIS-like gene were evaluated by quantitative real-time PCR (QRT-PCR) in barrel medic plantlets grown in vitro under oxidative stress conditions induced by copper (CuCl2 0.05, 0.1 and 0.2 mM) and polyethylene glycol (PEG6000 50, 100 and 150 g/L), respectively. Both stress agents caused ROS (reactive oxygen species) accumulation. Moreover, EPR spectra of leaves from plantlets exposed to toxic copper doses confirmed that the heavy metal is translocated from roots to the aerial parts, where it is found predominantly in the Cu2+ redox state. The MtTFIIS-like gene expression was significantly enhanced (up to 2.9-fold) in aerial parts of copper-treated plants, and in roots (up to 4.4-fold) in response to PEG treatments. The expression profiles of the MtTFIIS-like gene were compared to those of the MtTFIIS gene, encoding the canonical TFIIS protein, which was similarly up-regulated in response to both stresses. Interestingly, the MtTFIIS-like and MtTFIIS genes were significantly up-regulated (up to 3.2- and 4.3-fold, respectively) during seed imbibition, a physiological process which requires active DNA repair. Based on the reported data, the possible roles played in planta by the novel MtTFIIS-like gene are discussed. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Abiotic stresses (e.g. drought and heavy metal pollution) cause deleterious effects on plant viability by inducing accumulation of genotoxic reactive oxygen species (ROS). The response at the nuclear level, in terms of DNA damage sensing and repair, is crucial to maintain the plant genome integrity (Bray and West, 2005). Oxidative DNA lesions, resulting from ROS accumulation can block transcription elongation and this impairment can be removed through transcrip-

Abbreviations: DAB, 3,3′-Diaminobenzidine; EPR, Electron paramagnetic resonance; Mt, Medicago truncatula; NBT, Nitroblue tetrazolium; 8-oxo-dG, 7,8-Dihydro-8oxoguanine; PEG, Polyethylene glycol; QRT-PCR, Quantitative real-time polymerase chain reaction; ROS, Reactive oxygen species; SOD, Superoxide dismutase; TFIIS, Transcription elongation factor S-II. ⁎ Corresponding author. Tel.: + 39 0382 985583; fax: + 39 0382 528496. E-mail address: [email protected] (D. Carbonera). 0378-1119/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2010.09.004

tion-coupled repair (TCR), which is a part of the nucleotide excision repair (NER) system (Fouster and Mullenders, 2008). It is known that transcription of protein-coding genes by RNA polymerase II (RNAPII) starts with the assembly of a preinitiation complex at the gene promoter region and proceeds through transcription initiation, elongation and termination (Hahn, 2004). The transcription elongation factor II-S (TFIIS) was the first RNAPIIassociated factor to be purified, due to its ability to promote efficient synthesis of long transcripts in vitro (Sekimizu et al., 1976). Furthermore, it has been reported that TFIIS stimulates RNAPII to transcribe through DNA regions containing blocks induced by DNAbinding proteins and drugs (Reines and Mote, 1993). Recently, Kuraoka et al. (2007) demonstrated that TFIIS from HeLa cells enables RNAPII to bypass 7,8-dihydro-8-oxoguanine (8-oxoG) but not other types of oxidative DNA damage, thus playing a key role in the tolerance to oxidative stress. Based on this finding, a role of TFIIS in TCR has been hypothesized (Kuraoka et al., 2007). The TFIIS protein is conserved in eukaryotes while homologs are also present in archaea and in some viral genomes (Booth et al., 2000).

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The transcription elongation factor II-S contains three folding domains showing distinct features. The N-terminal domain I is composed of a four-helix bundle and includes a LW motif involved in nuclear targeting (Ling et al., 2006). Domain I is dispensable for TFIIS function in yeast while in human cells it directly interacts with the RNAPII holoenzyme (Booth et al., 2000). The central domain II which mediates the interaction of TFIIS with the largest subunit of RNAPII (Morin et al., 1996) and the C-terminal domain III required for the stimulation of RNA cleavage are both essential for the function of this protein (Awery et al., 1998). Regions with homology to domain I of TFIIS are found in both animal elongin A (Shilatifard et al., 1996) and CRSP70 (Ryu and Tjian, 1999). Elongin A, also known as transcription elongation factor S-III, increases the rate of transcription by suppressing transient pausing in the elongation complex (Yasukawa et al., 2007). CRSP70 is a subunit of the CRSP (cofactor required for Sp1 activation) complex which is required for the activity of the enhancer binding protein Sp1 (Ryu et al., 1999; Deniaud et al., 2006). In a recent work Uzureau et al. (2008) identified two trypanosome (Trypanosoma brucei) proteins with homology to TFIIS, named TbTFIIS1 and TbTFIIS2-1, respectively. These proteins show different structural features, since TbTFIIS1 contains only the canonical domains II and III while TbTFIIS2-1 contains also the domain I and a PWWP (Proline–Tryptophan–Tryptophan–Proline) domain, usually required for the binding of DNA methyltransferases to heterochromatin. Each gene appears not essential for cell viability, while the double knockdown mutant shows impaired cell growth. However, no differences in the response to oxidative stress of TFIIS single and double knock-down trypanosome cells were evidenced (Uzureau et al., 2008). As for plants, the ET1 protein, found in maize chloroplasts, has a remarkable sequence similarity to the domain III of the eukaryotic TFIIS. It has been demonstrated that ET1 plays a role in transcription elongation in chloroplasts (da Costa e Silva et al. 2004). More recently, Grasser et al. (2009) isolated and characterized the TFIIS gene from Arabidopsis thaliana. Differently from human, where three conserved TFIIS isoforms are present, one expressed ubiquitously and the others in a tissue-specific way (Labhart and Morgan, 1998), a unique TFIIS transcript was detected in Arabidopsis tissues, suggesting for a constitutive expression pattern in plants (Grasser et al., 2009). In yeast, the TFIIS gene is not required for cell survival, but its inactivation has lethal effects on mouse embryos (Ito et al., 2006). The down-regulation of TFIIS gene in Arabidopsis did not induce significant differences in the global gene expression (Grasser et al., 2009), contrarily to results reported in human cells (Hubbard et al., 2008). However, lack of TFIIS gene or reduced expression levels significantly shortened the dormant stage of fully developed Arabidopsis seeds before germination. For this reason it has been suggested that TFIIS might be involved in the regulation of seed dormancy by modulating those genes which play a key role in this process (Grasser et al., 2009). In the present work we have identified and characterized a novel gene from barrel medic (Medicago truncatula Gaertn.), named MtTFIIS-like, encoding a protein that shares some common features not only with the canonical TFIIS, but also with elongin A and CRSP70. It is worth nothing that the extensive databases currently available for molecular investigation in M. truncatula, make this plant an ideal target to explore novel gene functions in legumes (Ané et al., 2008) and to develop innovative biotechnological methods (Confalonieri et al., 2009; Scaramelli et al., 2009). Based on the role played by the canonical TFIIS in the antioxidant response in animal cells and in the attempt to elucidate the biological function of MtTFIIS-like protein, the expression profiles of both genes were evaluated and compared in barrel medic plants exposed to oxidative stress. Furthermore, in a parallel investigation, the analysis of both MtTFIIS and MtTFIIS-like gene expression was extended to the early phase of seed hydration, when DNA repair is activated in order to withstand ROS injury and preserve genome integrity.

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2. Materials and methods 2.1. Plant material and treatments The M. truncatula Gaertn., genotype R108-1, used in this study was selected by Trinh et al. (1998), due to its high embryogenic potential and genetic transformation properties. Plants were grown in greenhouse, in pots containing a mixture of peat and soil (1:2). Cultivation was performed following standard procedures. Plants were not supplied with any additional nutrients and watered when needed. Tissues were collected from plants at the vegetative and reproductive stages and stored in liquid N2. For oxidative stress treatments, plantlets were grown in vitro in sterile containers (Micropoli, Cesano Boscone, Italy) on a medium containing macrosalts and microsalts MS (Murashige and Skoog, 1962), vitamin SH (Shenk and Hildebrandt, 1972), 20 g/L sucrose and 4 g/L gerlite (Duchefa Biochemie, Haarlem, Netherlands). The plantlets were maintained in a climate chamber at 22–24 °C with a 16 h light/18 h dark cycle photoperiod and a photosynthetic photon flux of 65– 70 μmol/m2/s under a cool white fluorescence lamp. As for the heavy metal treatments, plantlets were grown in vitro in the presence of different copper concentrations (CuCl2, Sigma-Aldrich, Milan, Italy; 0, 0.05, 0.1 and 0.2 mM). Osmotic stress was induced by growing plantlets in vitro in the presence of increasing PEG6000 concentrations (0, 50, 100 and 150 g/L) (Duchefa Biochemie). The water potential of the culture medium was estimated at −0.30 MPa (0 g/L PEG6000), − 0.60 MPa (50 g/L PEG6000), − 0.66 MPa (100 g/L PEG6000) and −1.0 MPa (150 g/L PEG6000). For imbibition experiments, seeds were transferred to Petri dishes (9 cm diameter; 100 seeds per dish) with two filter papers moistened with 2.5 mL distilled water and kept in the dark at 20 °C. Seeds were collected at the indicated time intervals and stored in liquid N2. 2.2. Cloning procedure and sequence analysis RNA isolation was carried out using the NucleoSpin RNA II extraction kit (Macherey-Nagel, M-Medical S.r.l., Cornaredo, Italy), according to the manufacturer's instructions. cDNAs were obtained using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems International, Monza, Italy) according to the manufacturer's suggestions. The oligonucleotide primers Mt-F1 (5′-CCGATGGATTTGGACGATTT-3′) and Mt-R1 (5′-GTATCACCAGTGCTGCCTTCC-3′) were used to amplify the full-length MtTFIIS-like cDNA. Oligonucleotide primers were designed using Web Primer DNA Design software (http://www.yeastgenome. org/cgi-bin/web-primer). Amplification was obtained using the following PCR conditions: 94 °C 50 s, 60 °C 50 s, 72 °C 2 min (35 cycles) in a TGradient PCR apparatus (Biometra GmbH, Goettingen, Germany), using the Go HotMaster Taq DNA Polymerase (Promega S.r.l., Milan, Italy). PCR products were purified from agarose gel (Duchefa Biochemicals) using the GFX™ PCR DNA and Gel Band Purification Kit (Amersham Biosciences GmbH, Milan, Italy) and subsequently cloned into the pJET1/blunt vector, using the GeneJetTM PCR Cloning Kit (Fermentas, MMedical S.r.l., Cornaredo, Italy). The recombinant plasmid pJET-TFIIS-like was then purified using the WizardR Plus SV Minipreps DNA Purification System (Promega). Sequence analysis was performed with the ABI PRISMR BigDye™ Terminator Cycle Sequencing Ready Reaction Kit, using the ABI PRISM R310 Genetic Analyzer (Applied Biosystems). 2.3. In situ ROS detection Leaves were collected from 30-day old barrel medic plantlets grown in vitro in the presence/absence of oxidative stress conditions. The staining with nitroblue tetrazolium (NBT, Sigma-Aldrich) was used to detect superoxide radicals (O− 2 ) as described by Rao and Davis (1999). The production of hydrogen peroxide (H2O2) was detected using 3,3′diaminobenzidine (DAB, Sigma-Aldrich) as described by Thordal-

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Christensen et al. (1997). Quantification of stained areas was performed using a Biostep GmbH apparatus (Jahnsdorf, Germany) with Argus X1 3.3.0. software.

3. Results

2.4. Electron paramagnetic resonance (EPR) measurements

A bioinformatic investigation carried out in the M. truncatula database, using the A. thaliana TFIIS amino acid sequence (accession no. At2g38560) as starting point, revealed not only the presence of the canonical MtTFIIS gene, but also the occurrence of an additional sequence containing the domain I found in TFIIS, elongin A and CRSP70. This novel gene was thus named MtTFIIS-like. With the aim to isolate the full-length cDNA, RNA was extracted from barrel medic plantlets and utilized to synthesize the corresponding cDNA by RTPCR. The full-length MtTFIIS-like cDNA (1522 bp) includes a 5′untranslated region (243 bp), followed by an open reading frame (ORF, 1074 bp) and a 3′-untranslated end (205 bp). The ORF encodes a protein of 357 amino acids (aa) with a predicted molecular mass of 40.0 kDa. The MtTFIIS gene from barrel medic (accession no. AC137821), identified through bioinformatic approach, encodes a protein of 369 aa with a putative molecular mass of 40.0 kDa. The comparison between the canonical MtTFIIS and the novel MtTFIISlike amino acid sequences revealed a reduced identity (12.7%) (Fig. 1). The canonical MtTFIIS protein is composed of three structural domains: the N-terminal domain (domain I, aa 2–98), the central domain (domain II, aa 201–324) and the C-terminal domain (domain III, aa 329–367) (Supplemental Fig. S1). The domain organization of the MtTFIIS-like protein from M. truncatula is also described in Supplemental Fig. S1, and compared with those of canonical TFIIS proteins (from barrel medic and human, respectively). The schematic representation highlights the fact that, although located at different positions, domain I is a common feature of these proteins. The estimated amino acid identity of domain I in MtTFIIS-like with MtTFIIS and HsTFIIS is 24 and 32.8%, respectively. Furthermore, domain I of MtTFIIS-like shares 31.4 and 21.4% amino acid identity with the same region of the human elongin A and CRSP70. With the aim to verify the presence of the MtTFIIS-like sequence in other plant species, a bioinformatic investigation was carried out. As shown in Fig. 2, the MtTFIIS-like amino acid sequence from M. truncatula (GenBank accession no. ABE81609) was compared to the putative amino acid sequences from A. thaliana (GenBank accession no. BAB09406), Vitis vinifera (GenBank accession no. CAO43626) and Oryza sativa (GenBank accession no. ABA95889). The overall amino acid sequence identity among the analyzed plant TFIIS-like amino acid sequences is about 30%, while the identity increased up to 57% within the N-terminal domain.

EPR measurements were carried out with an X Band Bruker EMX/12 Spectrometer (Bruker BioSpin GmbH, Karlsruhe, Germany) equipped with data-acquisition system and automatic temperature-control apparatus. The analysis of the spectra was performed by computer simulation with the Bruker package SimFonia, which implements a Hamiltonian including Zeeman nuclear and electronic terms and the isotropic and anisotropic components of the hf (hyperfine) and g tensors. Spectral intensity measurements were performed by double integration using the Bruker WINEPR software. The EPR spectra of the samples in the form of fine powders were recorded at 77–130 K and at room temperature. The setting parameters were 100 kHz modulation frequency, modulation amplitude 5 G and microwave power 5 mW. The g-values were estimated using 2,2-Diphenyl-1-picrylhydrazyl radical (DPPH, Sigma-Aldrich) as a reference standard. Quantitative estimation of Cu in the samples was performed by comparison with glassy copper sulfate aqueous solutions of known concentration.

2.5. Quantitative real-time polymerase chain reaction (QRT-PCR) QRT-PCR was carried out with the Green Dye Master Mix (Rovalab, Cabru S.A.S., Arcore, Italy) in a final volume of 25 μL according to supplier's indications, and using a Rotor-Gene 6000 PCR apparatus (Corbett Robotics Pty Ltd, Brisbane, Queensland Australia). Amplification conditions were as follows: denaturation at 95 °C for 15 min, and 45 cycles of 95 °C 15 s, 55 °C 30 s and 72 °C 30 s. Quantification was carried out using the M. truncatula γ-tubulin (DFCI annotation TC130143) as reference gene. Oligonucleotide primers from the barrel medic MtTFIIS-like (DFCI annotation TC117757), MtTFIIS (DFCI annotation TC95251) and superoxide dismutase (SOD) (DFCI annotation TC119940) coding sequences were designed using the RealTime PCR Primer Design program from GenScript (https://www. genscript.com/ssl-bin/app/primer) (Supplemental Table 1). For each oligonucleotide set, a no-template water control was used. The QRTPCR results were interpreted using the LinRegPCR computer software (Ramakers et al., 2003). The logarithm of relative fluorescence unit (LogRFU) was used for graphic representation.

3.1. Isolation and sequence analysis of the barrel medic MtTFIIS-like cDNA

2.6. Bioinformatic analysis The barrel medic sequences were obtained from DFCI Medicago truncatula Gene Index Database (http://compbio.dfci.harvard.edu/tgi/). Comparison of amino acid sequences was performed using the Expasy SIB BLAST Network Service (http://www.expasy.ch/tools/blast//). The search for the nuclear localization signal was carried out using the NucPred program (http://www.sbc.su.se/~maccallr/nucpred/cgi-bin/single.cgi). The search for sumoylation sites was carried out at: http://www.abgent. com/tools/sumoplot. The Motif Scan tool (http://myhits.isb-sib.ch/cgibin/motif_scan) was used to identify the other reported motifs within the MtTFIIS-like protein sequence.

2.7. Statistical analysis Three replicated plantlets from each treatment combination were selected for tissue analysis. Results were subjected to analysis of variance (ANOVA) and the means were compared by the Tukey's test. Percentage data were transformed to arcsin√x before statistical analysis.

3.2. The MtTFIIS-like and MtTFIIS genes are constitutively expressed in barrel medic tissues The expression analysis of MtTFIIS-like and MtTFIIS genes was carried by QRT-PCR. Both genes were constitutively expressed in plants grown in greenhouse, both at the vegetative (one month old) and reproductive (two month old) growth stage. At the vegetative growth stage, no significant (P = 0.08) fluctuation of the MtTFIIS-like mRNA was detected among the plant tissues (Fig. 3). As for the reproductive growth stage, the analysis of variance revealed a significant (P b 0.0001) variation in the average level of MtTFIIS-like transcript among the tested tissues. The gene expression was outstanding in stems followed by flowers, leaves, roots and pods, while the lowest amount was observed in nodules. The MtTFIIS transcript was detected in all the tested tissues (Fig. 3). The analysis of variance revealed significant (P = 0.006) differences among plant tissues for MtTFIIS expression level: at the vegetative growth stage, young and old leaves, as well as nodules showed, on average, significant (P = 0.04) higher MtTFIIS expression levels than

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Fig. 1. Comparison between the canonical MtTFIIS (accession no. AC137821) and the novel MtTFIIS-like (accession no. ABE81609) amino acid sequences. * = conserved amino acid residues. : = conserved amino acid substitutions. • = semi-conserved amino acid substitutions.

roots, according to Tukey's test. As for the reproductive growth stage, no significant (P = 0.22) differences were detected among plant tissues for MtTFIIS gene expression. The comparison between the two genes shows a higher expression level of MtTFIIS at the vegetative stage.

3.3. ROS accumulation in barrel medic leaves exposed to oxidative stress In order to investigate the barrel medic response to oxidative stress, plantlets were exposed to copper and PEG6000 treatments. The morphology of a M. truncatula plant grown in vitro for 30 days under

Fig. 2. Comparison of the Medicago truncatula (Mt) TFIIS-like amino acid sequence with the Arabidopsis thaliana (At) (accession no. BAB09406), Vitis vinifera (Vv) (accession no. CAO43626) and Oryza sativa (Os) (accession no. ABA95889). Conserved amino acid residues are represented in bold. The TFIIS N-terminal domain (domain I) is underlined and the conserved leucine (L) and tryptophan (W) residues of the LW motif are highlighted by gray boxes.

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Fig. 3. Results from QRT-PCR analyses carried out on the same plants. Values are expressed as means ± SD of three independent replicated plants. The logarithm of relative fluorescence unit (LogRFU) was used for graphic representation. Vegetative stage (one month-old plants): n, nodules; r, roots; yl, young leaves; ml, mature leaves. Reproductive stage (two month-old plants): n, nodules; r, roots; s, stems; l, leaves; f, flowers; p, pods.

physiological condition is shown in Fig. 4A, while the exposure to 0.05, 0.1 and 0.2 mM CuCl2, resulted into evident stem shortening and reduction of the root volume (Fig. 4B–D). On the other hand, the osmotic stress treatments (50, 100, and 150 g/L PEG6000) caused reduced plant development, both in aerial parts and roots, especially when the 100 and 150 g/L doses were applied (Fig. 4E–G). Accumulation of endogenous hydrogen peroxide (H2O2) in leaves of 30-day old plantlets grown in vitro on increasing copper and PEG6000 concentrations was determined by DAB staining (Fig. 4A and D), while the level of superoxide radical (O− 2 ) was assessed by staining with nitroblue tetrazolium (NBT) (Fig. 4B and E). As shown in Fig. 4C, the percentage of DAB- or NBT-stained areas from untreated plantlets was 3.3% of the total leaf area. On the other hand, the accumulation of hydrogen peroxide and hydroxide radicals increased in parallel to copper concentrations. The lowest dose (0.05 mM CuCl2) resulted into 38 and 40% of DAB- and NBT-stained areas, respectively, while there was a significant increase with the highest concentrations,

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quantified as 52 and 61% (0.1 mM CuCl2) and as 77 and 74% (0.2 mM CuCl2). The analysis of variance revealed significant differences (P b 0.0001) among copper treatments for the percentage of DABand NBT-stained leaf areas, respectively. A similar analysis was carried out on the PEG6000-treated plantlets (Fig. 4D and E). As shown in Fig. 4F, the percentage of DAB- or NBT-stained areas from untreated plantlets was 3.3% of the total leaf area. The accumulation of hydrogen peroxide and superhydroxide radicals increased in response to PEG6000 treatments. After 30 days of treatment with PEG6000 (50 g/L, 100 g/L and 150 g/L), the percentage of NBT-stained leaf area was 35%, 78% and 81%, respectively, while the percentage of DABstained leaf area was quantified at 31% (50 g/L PEG6000), 76% (100 g/L PEG6000) and 85% (150 g/L PEG6000). The analysis of variance revealed significant differences (P b 0.0001) among PEG6000 treatments for the percentage of DAB- and NBT-stained leaf areas, respectively. In conclusion, both copper and PEG6000 treatments induced an evident ROS accumulation in barrel medic leaves.

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Fig. 4. Thirty-day old M. truncatula (genotype R108-1) plantlets grown in vitro in the absence of stress (A) and in the presence of 0.05, 0.1 and 0.2 mM CuCl2 (B–D) and in the presence of 50, 100 and 150 g/L PEG6000 (E–G).

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3.4. EPR copper measurements Accumulation of copper ions in barrel medic plantlets was evaluated using electron paramagnetic resonance (EPR) technique. The low temperature EPR spectra of dried powder leaves of 30-day old barrel medic plantlets grown in vitro in the presence (A) and absence (B) of 0.2 mM CuCl2 are shown in Fig. 5. The spectrum A is characterized by a narrow single line at g ≈ 2.004 which can be assigned to organic free radicals, a sextet of lines attributed to manganous Mn2+ ions at g ≈ 2 and clear features related to the hyperfine splitting of the copper Cu2+ ions (I = 3/2) at low field. The spectrum B shows only the Mn2+ sextet and the free radical line. The higher contribution in spectrum A, about 87%, comes from the Mn2+ ions. The subtraction of B from A gives a typical copper ion EPR pattern C contributing the remaining 13%. The organic free radicals are quantitatively negligible in respect to the metals species. The Cu2+ concentration resulting from EPR measurements is estimated on average 85 mg kg− 1. Fig. 5D shows the simulation of the difference spectrum C. The EPR parameters g1 = 2.03, g2 = 2.07, g3 = 2.24, A = 17.7 mT are consistent with an almost axially species and suggest, on the basis of Vanngard–Peisach–Blomberg plots, the presence of three or four nitrogen donor atoms coordinated to copper ion. 3.5. The MtTFIIS-like and MtTFIIS genes are up-regulated in response to oxidative stress The expression profiles of the MtTFIIS-like and MtTFIIS genes were evaluated by QRT-PCR in roots and aerial parts of barrel medic plantlets grown in vitro in the presence of increasing copper concentrations (0, 0.05, 0.1 and 0.2 mM CuCl2) (Fig. 6A). Both genes were up-regulated in response to copper treatments. In the case of MtTFIIS-like expression

level in aerial parts, the analysis of variance revealed significant (P b 0.0001) differences among treatments. The MtTFIIS-like gene was significantly (P = 0.001) up-regulated (2.1-fold) in aerial parts in response to the lower copper dose (0.05 mM CuCl2) and the average amount of MtTFIIS-like transcript increased in the aerial parts exposed to the highest copper dose (Fig. 6A). Although the average amount of MtTFIIS-like transcript resulted higher in the roots from plants treated with increasing copper concentrations compared to the untreated tissues, the analysis of variance revealed no significant (P = 0.23) differences among treatments. According to Tukey's test, the average level of MtTFIIS mRNA was significantly (P = 0.0005 and P = 0.005, respectively) enhanced in aerial parts and roots exposed to increasing copper concentrations. The up-regulation of MtTFIIS in aerial parts from plants treated with the highest copper doses (0.1 and 0.2 mM CuCl2) resulted respectively 7.1- and 6.6-fold the values measured for the untreated tissues while a less pronounced up-regulation was observed in roots in response to copper treatments (Fig. 6A). The SOD gene was used as a marker of the antioxidant response outside the nuclear compartment (Fig. 6A and B). The analysis of variance revealed significant (P b 0.0001) differences among treatments for the average level of SOD mRNA in aerial parts. The average amount of SOD mRNA detected in aerial parts in response to increasing copper concentrations was significantly (P = 0.05) higher than that observed for the untreated tissues, according to Tukey's test. The up-regulation of SOD transcript in aerial parts from plants exposed to the highest copper concentration was estimated 1.9-fold. As for the SOD gene expression in roots, Tukey's test revealed a significant (P = 0.02) increase in the average amount of the target mRNA in the roots of 0.2 mM CuCl2-treated plantlets when compared to the untreated tissues. The response of the MtTFIIS-like and MtTFIIS genes was also evaluated in barrel medic plantlets grown in vitro in the presence of

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Fig. 5. Accumulation of endogenous hydrogen peroxide (H2O2) and superoxide radicals (O− 2 ) in leaves of 30-day old M. truncatula plantlets grown in vitro under oxidative stress conditions. DAB-stained leaf tissues exposed to increasing doses of CuCl2 (A) and PEG6000 (D), respectively. NBT-stained leaf tissues exposed to increasing doses of CuCl2 (B) and PEG6000 (E), respectively. The stained leaf areas were quantified by densitometric analysis (C and F). Values are expressed as means ± SD of three independent replicated plantlets.

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Fig. 6. Results from EPR analysis carried out on dried powder leaves of 30-day old barrel medic plantlets grown in vitro in the presence (A) and absence (B) of 0.2 mM CuCl2. (C) The subtraction of B from A gives a typical copper ion EPR pattern. (D) The simulation of the difference spectrum C.

increasing PEG6000 concentrations (0, 50, 100 and 150 g/L) (Fig. 6B). Concerning the MtTFIIS-like gene expression in aerial parts, the analysis of variance revealed significant (P = 0.01) differences among treatments. At the highest PEG6000 concentration, the upregulation of MtTFIIS-like in aerial parts was estimated 1.5-fold (Fig. 6B). The analysis of variance also revealed significant (P b 0.0001) differences among treatments for the average level of MtTFIIS-like transcript in roots. The MtTFIIS-like gene was significantly (P = 0.001) up-regulated (3.7-fold) in response to the lower PEG6000 concentration (50 g/L) and the transcript level slightly increased in the roots exposed to higher PEG6000 concentrations. As far as MtTFIIS expression level in the aerial parts, the analysis of variance revealed significant (P = 0.001) differences among treatments. According to Tukey's test, the average amount of MtTFIIS mRNA detected in aerial parts in response to the increasing PEG6000 concentrations was significantly (P = 0.008) higher than that observed for the untreated tissues. At the highest PEG6000 concentrations (100 and 150 g/L), the up-regulation of MtTFIIS in aerial parts was estimated 4.3- and 5.4fold, respectively (Fig. 6B). According to Tukey's test, MtTFIIS was also significantly (P = 0.001) up-regulated (1.7- and 2.1-fold, respectively) in roots in response to the same PEG6000 concentrations. As for SOD expression level in aerial parts and roots, the analysis of variance revealed significant (P = 0.005 and P = 0.01, respectively) differences among treatments. The up-regulation of SOD transcript in aerial parts and roots from plants exposed to the highest PEG6000 concentration was estimated 1.7- and 2.2-fold, respectively (Fig. 6B). As control, the house keeping gene encoding γ-tubulin was evaluated in barrel medic plantlets grown in the presence/absence of stress agents. As shown in Fig. 7, no fluctuations were observed in the amount of γ-tubulin transcript in response to copper treatments in both

roots and aerial parts. A similar response was evidenced when the barrel medic tissues exposed to PEG were examined (Fig. 7). The occurrence of stable transcript levels of this house keeping gene further supports for the stress-dependent up-regulation of MtTFIIS and MtTFIIS-like genes.

3.6. The MtTFIIS-like and MtTFIIS genes are up-regulated during seed imbibition The expression profile of the MtTFIIS-like and MtTFIIS genes were evaluated during seed imbibition, a physiological process which requires active DNA repair to maintain the plant genome integrity (Holdsworth et al., 2008). The gain in the fresh weight during seed imbibition is represented in Fig. 7A. The first phase of imbibition is characterized by a rapid intake of water into the seeds, corresponding to the first 8 h of exposure. During the second phase, no further water intake was observed. The analysis of variance revealed significant (P b 0.0001) differences among the different sampling times following hydration for the average level of MtTFIIS-like transcript. The MtTFIISlike gene expression was up-regulated during seed hydration with a significant increase (3.2-fold) after 8 h of hydration (Fig. 7B). Subsequently, a decrease in the level of MtTFIIS-like mRNA was detected at 12 h, followed by a further enhancement occurring at 24 and 36 h. The analysis of variance also revealed significant differences (P b 0.0001) among the different sampling times following hydration for the average level of MtTFIIS transcript. According to Tukey's test, the average amount of MtTFIIS mRNA detected at 12 and 24 h was significantly (P = 0.02) higher compared to that observed at other time points (Fig. 7B). The SOD gene expression was also evaluated in barrel medic seeds during imbibition. The SOD gene was significantly

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(P = 0.01) up-regulated (2.9-fold) at 8 and 12 h after hydration, while the expression decreased after these time points (Fig. 7B). The expression profiles of the house keeping barrel medic gene encoding γ-tubulin were also evaluated during seed imbibition. As shown in Fig. 8, the amount of γ-tubulin transcript did not change during seed rehydration, thus confirming that the observed upregulation of MtTFIIS and MtTFIIS-like genes was strictly related to the activation of DNA repair processes. 4. Discussion In the present work, the MtTFIIS-like gene encoding a novel protein which shares common features with the transcription elongation factor II-S (TFIIS), the elongin A and the CRSP70 protein has been investigated in the model legume M. truncatula (Gaertn.). The MTFIISlike and MtTFIIS genes were constitutively expressed in all tissues both at the vegetative and reproductive growth stages, suggesting for their essential role in the physiology of plants. In animal cells, the TFIIS protein seems to play a protective role against damage caused by 7,8-dihydro-8-oxoguanine accumulation (Kuraoka et al., 2007). Since, in this regard, no information are available in plants, we decided to assess the possible involvement of the MtTFIIS-like and MtTFIIS genes in Medicago antioxidant response, utilizing copper and PEG as stress agents. Differently from other metals, the redox state and coordination of copper in plant tissues have been so far poorly explored. EPR was used by Orsega et al. (2003) to analyze copper translocation from soil to

leaves of Sambucus nigra L. seedlings, where the heavy metal was detected in the oxidation state Cu2+. The ligand coordination geometry of the Cu2+ ion complex was not, however, clearly defined. More recently, Mijovilovich et al. (2009) investigated the complexation of copper in leaves of Thlaspi caerulescens. A large proportion of copper was bound by sulfur ligands but additional signals in the EPR spectra indicated complexation of Cu2+ by the non proteogenic amino acid nicotinamine and the presence of a hexadentate complex was suggested. The copper concentration estimated by EPR in M. truncatula leaves is in agreement with data previously obtained by spectrometric analyses (Macovei et al., 2010), as well as the good resolved lines of the copper nucleus hyperfine coupling, and is indicative of the fact that most of the copper ions are in one predominant single species in the Cu2+ redox state. However, we cannot exclude the possibility that Cu+ ions underwent oxidation to Cu2+. In conclusion, the EPR analysis confirmed that copper is translocated from roots to the aerial parts in M. truncatula plantlets. The reported ROS accumulation in barrel medic leaves exposed to copper and PEG represents a clear evidence of the oxidative stress conditions induced by these agents. The working system hereby investigated has been used in a previous study (Macovei et al., 2010) to analyze the expression profiles of barrel medic Tdp1 (Tyrosyl-DNAphosphodiesterase) and top1 (DNA topoisomerase I) genes, known to be involved in DNA repair in animal cells (Interthal et al., 2001; Balestrazzi et al., 2010). The copper and PEG concentrations, tested in the present work, have been already demonstrated to induce the

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Rehydration time (h) Fig. 8. (A) Fresh weight increase in Medicago truncatula seeds during imbibition. (B) Expression profiles of MtTFIIS-like, MtTFIIS and SOD genes in seeds at different times following hydration obtained by QRT-PCR analysis. As control, the house keeping gene encoding γ-tubulin was also analyzed. The logarithm of relative fluorescence unit (LogRFU) was used for graphic representation. Values are expressed as means ± SD of three independent replicated plants.

antioxidant response in barrel medic plantlets grown in vitro. The latter have been also characterized in terms of chlorophyll content, proline accumulation and 8-oxo-dG levels as a parameter of oxidative DNA damage (Macovei et al., 2010). It is worth noting that the inhibition of root growth and elongation, observed in the M. truncatula plants exposed to copper and analyzed in the present study, was a common feature well evidenced in rice (O. sativa) (Xu et al., 2008), tomato (Lycopersicon aesculentum) (Mediouni et al., 2006) and white poplar (Populus alba L.) (Balestrazzi et al., 2009). When oxidative stress treatments were applied, both the MtTFIIS-like and MtTFIIS genes were significantly up-regulated in roots and aerial parts of barrel medic plantlets. It is generally acknowledged that genes involved in DNA and RNA metabolism are regulated not only with respect to physiological processes but also in response to environmental stress (Vashisht and Tuteja, 2006). As for copper, the global changes in gene expression occurring in plants exposed to this heavy metal have been extensively analyzed, revealing that copper treatments particularly affect those genes involved in defense stress response, photosynthesis and transport (Sudo et al., 2008). However, information concerning genes related to DNA and RNA metabolism are still scanty. The finding that both MtTFIIS-like and MtTFIIS genes are upregulated in response to copper well correlates with previously reported data which evidenced the presence of high levels of DNA oxidative damage in leaves of Medicago plants exposed to toxic copper doses (Macovei et al., 2010). The hypothesis of the possible involvement of MtTFIIS-like and MtTFIIS genes in the response to PEG mediated-stress was further strengthened by the observation that changes in water potential from −0.30 to −1.00 MPa were able

to significantly increase the gene expression level both in the aerial parts and roots up to 1.5- and 4.4-fold, respectively. It is known that high molecular weight PEG induces osmotic stress in plants by decreasing the water potential of nutrient solutions. The SOD gene (coding for the cytosolic Cu/Zn superoxide dismutase) was used in this study as a marker of the antioxidant response outside the nucleus, since it has been reported that this specific enzyme is induced in response to copper (Luna et al., 1994). As expected, this gene was upregulated in aerial parts of plantlets treated with increasing copper doses, while in roots no significant up-regulation was observed. This might be possibly due to the fact that the SOD gene undergoes posttranslational regulation (Rubio et al., 2001; Ruzsa and Scandalios, 2003). Moreover, a significant up-regulation characterized also the response to PEG6000 at the highest concentration. Considering the possible role hypothesized for the animal TFIIS in the transcriptioncoupled repair mechanism, both the MtTFIIS-like and MtTFIIS genes were evaluated, in terms of expression profiles, during seed imbibition, a process known to require the activation of DNA repair events (Holdsworth et al., 2008). The MtTFIIS-like gene was upregulated at 8 h following rehydration while a temporal shift of about 4 h characterized the MtTFIIS transcript accumulation. It is worth noting that both transcripts are required throughout seed imbibition, a phase which anticipates germination. This finding well correlates with the work from Grasser et al. (2009), who demonstrated that the TFIIS gene plays critical role in the regulation of seed dormancy in Arabidopsis. In plants, the response to the genotoxic effects induced by ROS involves activation of different DNA repair pathways having basic

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features in common with those found in animals (Kimura and Sakaguchi, 2006). However, several DNA repair genes are present in multiple copies in plant genomes suggesting that the presence of multiple alleles might increase tolerance to oxidative damage (Morgante et al., 2005; Balestrazzi et al., 2010). Within this context, the isolation of the MtTFIIS-like gene might be indicative of the novel DNA repair functions in plants. The present investigation highlighted both structural and functional similarities between the MtTFIIS-like protein and the canonical MtTFIIS. The finding that the Xenopus laevis, rat, mouse and human TFIIS genes encodes three distinct isoforms (TCEA1, TCEA2 and TCEA3), each with tissue-specific expression patterns, suggests for different requirements for this relevant nuclear function (Wind and Reines, 2000). TCEA1, called “general SII”, is expressed in all tissues (Umehara et al., 1995), TCEA2 is found in testis and ovaries (Xu et al., 1994) while TCEA3 is present mostly in heart, liver, skeletal muscle and kidney (Taira et al., 1998). On the other hand, Uzureau et al. (2008) reported on the identification of two TFIIS proteins with different domain architectures in T. brucei, which seem to be functionally redundant. As for the possible role of the MtTFIISlike protein, the hypothesis of an elongin-like function, suggested by the presence of the domain I in both proteins, is not totally convincing, due to the lack of the transcriptional activation domain characterizing elongin A. Since no elongin encoding genes have been so far identified in plants, further studies will be relevant to highlight the presence of elongin-like functions. However, we cannot rule out the possibility that the putative MtTFIIS-like product might be involved in the transcription elongation, with a role more similar to that played by the canonical TFIIS protein. To our knowledge, this is the first report describing a MtTFIIS-like protein in plants, not having homologous in animal databases (Macovei et al., unpublished data). As for the MtTFIIS gene, the present investigation adds new information to the results reported by Grasser et al. (2009), demonstrating, for the first time, the involvement of the TFIIS gene in the plant response to oxidative stress, in agreement with the literature currently available for the animal TFIIS gene (Kuraoka et al., 2007). Supplementary data to this article can be found online at doi:10.1016/ j.gene.2010.09.004. Acknowledgments This research was supported by Fondo di Ateneo per la Ricerca— University of Pavia. Authors would like to thank Dr. Lorenzo Concia for his helpful suggestions concerning QRT-PCR technique. We are grateful to Dr. Sergio Arcioni and Andrea Porceddu (IGV-CNR, Perugia, Italy) for providing R108-1 M. truncatula genotype. References Ané, J.M., Zhu, H., Frugoli, J., 2008. Recent advances in Medicago truncatula genomics. Int. J. Plant Genomics 1–11. Awery, D.E., et al., 1998. Yeast transcription elongation factor (TFIIS), structure and function. J. Biol. Chem. 273, 22595–22605. Balestrazzi, A., et al., 2009. Expression of the PsMTA1 gene in white poplar engineered with the MAT system is associated with heavy metal tolerance and protection against 8hydroxy-2′-deoxyguanosine mediated-DNA damage. Plant Cell Rep. 28, 1179–1192. Balestrazzi, A., et al., 2010. Response to UV-C radiation in topo I-deficient carrot cells with low ascorbate levels. J. Exp. Bot. 61, 575–585. Bray, C.M., West, C.E., 2005. DNA repair mechanisms in plants: crucial sensors and effectors for the maintenance of genome integrity. New Phytol. 168, 511–528. Booth, V., Koth, C., Edwards, A.M., Arrowsmith, C.H., 2000. Structure of a conserved domain common to the transcription factor TFIIS, elongin A, and CRSP70. J. Biol. Chem. 275, 31266–31268. Confalonieri, M., et al., 2009. Enhanced triterpene saponin biosynthesis and root modulation in transgenic barrel medic (Medicago truncatula Gaertn.) expressing a novel β-amyrin synthase (AsOXA1) gene. Plant Biotechnol. J. 7, 172–182. da Costa e Silva, O., et al., 2004. The Etched1 gene of Zea mays (L.) encodes a zinc ribbon protein that belongs to the transcriptionally active chromosome (TAC) of plastids and is similar to the transcription factor TFIIS. Plant J. 38, 923–939. Deniaud, E., Baguet, J., Mathieu, A.L., Pagès, G., Marvel, J., Leverrier, Y., 2006. Overexpression of Sp1 transcription factor induces apoptosis. Oncogene 25, 7096–7105.

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