Expression of an exogenous 1-aminocyclopropane-1-carboxylate deaminase gene in Mesorhizobium spp. reduces the negative effects of salt stress in chickpea

Authors

  • Clarisse Brígido,

    1. Laboratório de Microbiologia do Solo, ICAAM, Instituto de Ciências Agrárias e Ambientais Mediterrânicas, Universidade de Évora, Núcleo da Mitra, Évora, Portugal
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  • Francisco X. Nascimento,

    1. Laboratório de Microbiologia do Solo, ICAAM, Instituto de Ciências Agrárias e Ambientais Mediterrânicas, Universidade de Évora, Núcleo da Mitra, Évora, Portugal
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  • Jin Duan,

    1. Department of Biology, University of Waterloo, Waterloo, ON, Canada
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  • Bernard R. Glick,

    1. Department of Biology, University of Waterloo, Waterloo, ON, Canada
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  • Solange Oliveira

    Corresponding author
    1. Laboratório de Microbiologia do Solo, ICAAM, Instituto de Ciências Agrárias e Ambientais Mediterrânicas, Universidade de Évora, Núcleo da Mitra, Évora, Portugal
    • Correspondence: Solange Oliveira, Departamento de Biologia, Universidade de Évora, Apartado 94, 7002-554 Évora, Portugal. Tel.: 351266760878;

      fax: 351266760913; e-mail: ismo@uevora.pt

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Abstract

Our goal was to study the symbiotic performance of two Mesorhizobium ciceri strains, transformed with an exogenous 1-aminocyclopropane-1-carboxylate deaminase gene (acdS), in chickpea plants under salinity stress. The EE-7 (salt-sensitive) and G-55 (salt-tolerant) M. ciceri strains were transformed with an acdS gene present on plasmid pRKACC. Salinity significantly reduced the overall growth of plants inoculated with either wild-type strains. Although the growth of plants inoculated with either salt-sensitive or salt-tolerant strain was reduced under salinity, the salt-tolerant strain showed a higher ability to nodulate chickpea under salt stress compared with the salt-sensitive strain. The shoot dry weight was significantly higher in plants inoculated with the acdS-transformed salt-sensitive strain compared with the plants inoculated with the native strain in the presence of salt. The negative effects of salt stress were also reduced in nodulation when using acdS-transformed strains in comparison with the wild-type strains. Interestingly, by expressing the exogenous acdS gene, the salt-sensitive strain was able to induce nodules in the same extent as the salt-tolerant strain. Although preliminary, these results suggest that genetic modification of a Mesorhizobium strain can improve its symbiotic performance under salt stress and indicate that ACC deaminase can play an important role in facilitating plant–rhizobium interaction under salinity conditions.

Introduction

Salinity stress is one of the most important abiotic stresses due to its impact in reducing crop yield worldwide. More than 800 million hectares of land is salt-affected (Essah et al., 2003), accounting for more than 6% of the world's total land area.

Legumes represent a very significant group of crops in agriculture, and therefore, their tolerance to salt stress is important to worldwide agricultural practice. Chickpea (Cicer arietinum L.) is one of the most important grain legume crops because it is a protein source in both human and animal diets. Furthermore, it plays a significant role in the maintenance of soil fertility, through its symbiotic association with rhizobia (Saxena & Singh, 1987). Like other legumes, chickpea is very sensitive to salinity, which affects its growth and development. On the other hand, rhizobial tolerance to salinity is important for the symbiosis, particularly if salt concentrations could have a detrimental effect on rhizobial populations as a result of direct toxicity and/or as through osmotic stress (Tate, 1995).

Despite the fact that legume plants are more sensitive to salinity than their rhizobial partners, the establishment of the symbiosis between them is highly sensitive to salt stress (Zahran, 1999). Symbiotic performance in saline soils depends on the salt tolerance of both the host and the microsymbiont (Saxena & Rewari, 1992; Zahran, 1999). The cultivated chickpea has a narrow genetic variation (Udupa et al., 1993), which makes it difficult for breeders to produce elite cultivars with durable resistance to the many major abiotic stresses. However, the use of genomic analysis of potential salt-tolerant chickpea cultivars may contribute significantly to the selection of salt-tolerant cultivars (Mantri et al., 2007; Varshney et al., 2009). Meanwhile, several strategies have been followed in an attempt to improve plant growth under stressful conditions, such as selection of high-salt-tolerant rhizobia strains and reduction in deleterious ethylene concentrations in the plant, that seem to be promising approaches.

Ethylene is produced by plants in response to several environmental stresses (Bari & Jones, 2009) and is also known for its negative role in nodulation (Ma et al., 2002; Middleton et al., 2007), as it inhibits the formation and functioning of nodules (Nandwal et al., 2007; Ding & Oldroyd, 2009). Under salinity, the ethylene levels increase in plant tissues (Abeles et al., 1992). Nandwal et al. (2007) reported a positive correlation between the levels of salinity and the amount of 1-aminocyclopropane-1-carboxylate (ACC) content (the immediate ethylene precursor) and ethylene production in chickpea nodules. From this perspective, the approach of lowering the deleterious ethylene levels in plants through the activity of ACC deaminase (encoded by the acdS gene), which converts ACC into α-ketobutyrate and ammonia, may be an alternative to help chickpea plants to overcome the negative effects caused by salt stress.

Recently, the acdS gene was detected in several Mesorhizobium species (Kaneko et al., 2000; Nascimento et al., 2012b), including chickpea mesorhizobia isolates. Although the Mesorhizobium strains harbour an acdS gene, an improvement of nodulation efficiency and bacterial competiveness was obtained in plants when they were inoculated with strains that had been genetically transformed to constitutively express an exogenous copy of the ACC deaminase gene (Conforte et al., 2010; Nascimento et al., 2012ac), suggesting that modulation of the ethylene levels in root tissues through ACC deaminase is an effective strategy to increase nodulation competitiveness of the bacterium.

Taking into account our previous results, it should be possible to transform Mesorhizobium spp. with acdS genes from free-living bacteria and thereby enhance nodulation and growth of legumes under stressful conditions. Thus, the main objectives of the work reported here were the following: (1) to evaluate the symbiotic performance of native salt-tolerant and salt-sensitive rhizobia strains in chickpea plants under salt stress; and (2) to improve the symbiotic performance of Mesorhizobium ciceri strains in chickpea plants under salinity stress by transformation with an exogenous acdS gene.

Materials and methods

Bacterial growth conditions

The bacterial strains and plasmids used in this study are listed in Table 1. Two chickpea mesorhizobia were selected based on their salt tolerance in the liquid media (Brígido et al., 2012) and their similar symbiotic effectiveness under control conditions (Alexandre et al., 2009), namely Mesorhizobium ciceri EE-7 (salt-sensitive) and Mesorhizobium ciceri G-55 (salt-tolerant). The mesorhizobia strains were grown at 28 °C in tryptone yeast (TY) medium (Beringer, 1974) or in minimal medium (Robertsen et al., 1981). The growth medium for transformed mesorhizobia strains was supplemented with tetracycline (20 μg mL−1). The E. coli DH5α and MT616 strains were grown in Luria–Bertani medium (Sambrook & Russell, 2001) at 37 °C. Appropriate antibiotics were added to the medium when necessary. For the E. coli strain containing pRKACC or pRK415, 15 μg mL−1 of tetracycline was used, while for the strain with pRK600, the medium was supplemented with 25 μg mL−1 of chloramphenicol.

Table 1. Bacterial strains and plasmids used in this study
Plasmid or BacteriaRelevant featuresReference or source
  1. Tcr, Tetracycline resistance; Cmr, Chloramphenicol resistance.

Plasmids
pRKACCThe broad-host-range plasmid pRK415 containing the acdS gene from Pseudomonas putida UW4, TcrShah et al. (1998)
pRK415Broad-host-range plasmid, TcrShah et al. (1998)
pRK600pRK2013, npt::Tn9, CmrFinan et al. (1986)
Bacteria
M. ciceri EE-7M. ciceri strain, acdS+, salt-sensitiveAlexandre et al. (2009), Brígido et al. (2012) and Nascimento et al. (2012b)
M. ciceri G-55M. ciceri strain, acdS+, salt-tolerantAlexandre et al. (2009), Brígido et al. (2012) and Nascimento et al. (2012b)
M. ciceri EE-7 (pRK415)M. ciceri EE-7 strain containing pRK415This study
M. ciceri G-55 (pRK415)M. ciceri G-55 strain containing pRK415This study
M. ciceri EE-7 (pRKACC)M. ciceri EE-7 strain containing pRKACCThis study
M. ciceri G-55 (pRKACC)M. ciceri G-55 strain containing pRKACCThis study
E. coli DH5αSupE44 Δ lacU169 (φ80lacZΔM15), hsdR17, recA1, endA1, gyrA96, thi-1, relA1Sambrook & Russell (2001)
E. coli MT616Strain containing helper plasmid pRK600Finan et al. (1986)

Triparental mating

In this study, plasmid pRKACC, containing the acdS gene from Pseudomonas putida UW4 or pRK415, were used to transform the two selected chickpea mesorhizobia strains, by triparental conjugation as described previously by Nascimento et al. (2012a).

To confirm the transfer of the plasmids to mesorhizobia cells, the tetracycline-resistant mesorhizobia colonies were picked and plasmids were extracted following the manufacturer's procedures (DNA-Spin™ Plasmid DNA Purification Kit, Intron) and visualized in a 1.0% agarose gel stained with ethidium bromide.

ACC deaminase activity assay

The native mesorhizobia cells and the respective transconjugants were tested for ACC deaminase activity following the methodology described before (Nascimento et al., 2012a). Mesorhizobium strain LMS-1 (pRKACC) was used as positive control, and Mesorhizobium sp. MAFF303099 was used as negative control (Nascimento et al., 2012a).

Chickpea seed sterilization

Chickpea seeds (cultivar Chk 3226) were surface-sterilized with 2.5% sodium hypochlorite for 30 min. After sterilization, seeds were rinsed six times in sterilized distilled water and incubated for 2 h at 28 °C. Seeds were placed in sterilized vermiculite and then incubated in the dark for 48 h at 28 °C.

Evaluation of the chickpea plants tolerance to salt

After germination, the seeds were transferred to plastic pots filled with sterile vermiculite and grown in a growth chamber, under a 16/8 h light/dark cycle and 24/18 °C day/night temperature and at a relative humidity of 65%.

To determine the salt concentration to use in the plant growth trial, the chickpea plants were tested with three different NaCl concentrations: 0.075%, 0.15% and 0.3% supplemented in the nutrient solution (Broughton & Dilworth, 1971) applied in alternate watering. Chickpea plants watered with nitrogen-free nutrient solution without supplemented NaCl were considered as negative control plants, and plants supplemented with nitrogen (140 ppm nitrogen as KNO3, in the nutrient solution) were used as positive control. Three plants per treatment were used, and the nutrient solution was applied three times a week. Two months after sowing, the chickpea plants were harvested and the nonlethal salt concentration was determined.

Plant growth assay under control and salt stress

To evaluate the symbiotic performance of the transformed and wild-type mesorhizobia strains in chickpea plants under control and salinity conditions, a plant growth assay was conducted in a growth chamber. The Mesorhizobium strains were grown in TY liquid medium (supplemented with tetracycline for transformed mesorhizobia strains) at 28 °C for 72 h. After incubation, the cell suspension was centrifuged at 10 000 g and washed twice with TY liquid medium (without antibiotic). The bacterial cultures were standardized to an OD540 nm of 0.8, and 1 mL of the bacterial suspension was used to inoculate each seed.

For control conditions, nutrient solution (Broughton & Dilworth, 1971) with and without nitrogen was used. For salinity conditions, 15 days after inoculation, the plants were subjected to salt stress using the nutrient solutions supplemented with 0.15% NaCl and applied in alternate watering. The nutrient solutions were applied three times a week. Uninoculated plants watered with a nitrogen-free nutrient solution or with a nutrient solution supplemented with nitrogen were used as negative and positive controls, respectively. After 8 weeks, the plants were harvested and several parameters were measured, such as shoot dry weight, root dry weight, number of nodules and nodule dry weight.

RT-PCR from root nodules RNA

To evaluate the acdS gene expression in symbiotic conditions, detection of the transcript in the acdS-transformed strains was performed by RT-PCR as described (Nascimento et al., 2012b). Total RNA was extracted from 8-week-old nodules using the RNeasy Mini Kit (Qiagen) according to the manufacturer's protocol. After extraction, about 3 μg of total RNA was treated with DNase I (Roche Applied Science). Conversion of total RNA to cDNA was conducted using the RETROscript Reverse Transcription for RT-PCR kit (Ambion) according to the manufacture's suggestions. Amplification of the acdS gene from the acdS-transformed strains was performed using the acdS-F and acdS-R2 primers (Nascimento et al., 2012b).

Statistical analysis

Six replicates per treatment were used for statistical analysis. These data were examined by an analysis of variance, and multiple comparisons among treatment means were made by Student–Newman–Keuls test. Statistical analyses were performed using MSTAC software, version 2.1.

Results

The two selected chickpea mesorhizobia were transformed with the plasmid pRKACC, containing the ACC deaminase gene from the plant growth-promoting bacterium, Pseudomonas putida UW4, or with the plasmid pRK415. The successful transformation of the two Mesorhizobium strains was confirmed by plasmid extraction and visualization in an agarose gel (data not shown). Expression of the exogenous acdS gene under symbiotic conditions was confirmed by RT-PCR using total RNA extracted from root nodules (Supporting Information, Fig. S1).

The ACC deaminase activity was measured in all mesorhizobia strains, both native and transformed strains. No ACC deaminase activity was detected in any of the wild-type strains or strains transformed with pRK415 in free-living conditions (Supporting Information, Table S1). On the other hand, the acdS-transformed mesorhizobia strains showed significantly different levels of ACC deaminase activity; the ACC deaminase activity of M. ciceri EE-7 (pRKACC) and M. ciceri G-55 (pRKACC) was 0.768 ± 0.036 and 0.398 ± 0.068 μmol of α-ketobutyrate mg of protein−1 per h, respectively.

The chickpea cultivar CHK3226 was tested for salt tolerance. Of the three NaCl concentration tested, only 0.3% NaCl was found to be lethal to the chickpea plants (data not shown). The other two NaCl concentrations applied to chickpea plants also showed negative effects on plants growth, however, in a lower extent. For this reason, 0.15% NaCl concentration was chosen for the evaluation of symbiotic performance of the mesorhizobia strains under salt stress.

A chickpea plant growth assay was performed to assess the symbiotic performance of the native salt-tolerant and salt-sensitive rhizobia strains as well the transformed strains, under control and salt stress conditions. Shoot dry weight (SDW), root dry weight (RDW), number of nodules (NN) and nodule dry weight (NDW) of plants inoculated with either strain showed no significant differences under control conditions (Figs 1 and 2). However, in the presence of salt, the growth of chickpea plants inoculated with each native strain was significantly inhibited compared with plants grown in control conditions (Fig. 1). The salt stress imposed on the chickpea plants led to a reduction in all analysed parameters when compared with the plants grown under control conditions (Figs 1 and 2). The symbiotic performance of the salt-sensitive strain EE-7 was affected in a greater extent when compared to the salt-tolerant strain G-55. For instance, plants inoculated with the salt-sensitive strain EE-7 showed a reduction of 54.5% in the SDW and 48.7% in the total biomass, while the plants inoculated with the salt-tolerant strain showed a decrease of 35.9% in the SDW and 33.6% in the total biomass, when compared to plants grown under control conditions (Fig. 1a and c).

Figure 1.

Results obtained from plant growth assay using chickpea plants inoculated with transformed and native mesorhizobia salt-tolerant and salt-sensitive strains under control and salinity conditions. Data correspond to the mean and standard error values of six plants after 8 weeks of inoculation. Light grey bars indicate plant under control conditions, and grey bars correspond to plants subjected to salinity conditions. (a) Shoot dry weight (g plants−1); (b) root dry weight (g plants−1) and (c) chickpea total biomass (g plants−1). Means sharing the same letters are not significantly different at ≤ 0.05 (n = 6).

Figure 2.

Results obtained from plant growth experiments using chickpea plants inoculated with acdS-transformed and no-transformed mesorhizobia salt-tolerant and salt-sensitive strains under control and salinity conditions. No nodules were obtained in uninoculated plants. Data correspond to the mean and standard error values of six plants after 8 weeks of inoculation. Light grey bars indicate plants under control conditions, and grey bars correspond to plants subjected to salinity conditions. (a) Number of nodules per plant; (b) nodule dry weight per plant. Means sharing the same letters are not significantly different at ≤ 0.05 (= 6).

No significant differences were observed between chickpea plants inoculated with native mesorhizobia strains and the ones transformed with pRK415 either under control or salt stress conditions (Table S2). Concerning the symbiotic performance of the acdS-transformed mesorhizobia strains, under salinity and with both strains, all analysed parameters from plants inoculated with the transformed mesorhizobia strain are higher compared with the plants inoculated with the native strain (Figs 1 and 2).

A significant difference between SDW of plants inoculated with strain EE-7 (pRKACC) and wild-type strain EE-7 was observed under salinity. Strain EE-7 (pRKACC) promoted an increase in SDW and chickpea total biomass of 54% and 35%, respectively, in plants under salinity stress when compared to those inoculated with the wild-type strain under the same conditions (Fig. 1a and c). An increase in the nodulation abilities of the strain EE-7 (pRKACC) was also observed, especially regarding the NN formed in chickpea plants under salinity. Strain EE-7 (pRKACC) showed an increase of 120% in the NN when compared to the NN formed by the wild-type strain EE-7, under salinity conditions). Interestingly, the NN formed by the acdS-transformed mesorhizobia strains in chickpea plants under salt stress conditions was similar to the NN formed in plants inoculated with the same strains, under control conditions (Fig. 2a).

Although in a lesser extent, the plants inoculated with the strain G-55 (pRKACC) also showed an improvement in tolerance to salinity conditions, compared with the plants inoculated with the native strain. The strain G-55 (pRKACC) resulted in a 38% and 25% increase in SDW and chickpea total biomass, respectively, when compared to the plants inoculated with the native strain G-55, under salinity (Fig. 1).

Discussion

Selection of rhizobia strains and host cultivars is required for effective symbiosis to maximize plant production under stressful conditions, such as salinity. In the present work, the effect of NaCl on chickpea plants inoculated with two specific Mesorhizobium ciceri strains was evaluated as well as the impact of the expression of an exogenous acdS gene in the improvement of the symbiotic performance of the strains.

Salinity significantly reduced the overall growth of chickpea plants when inoculated with either of the native strains. Similar depressive effects of NaCl in chickpea growth have been reported in other studies (Soussi et al., 1998; Garg & Singla, 2004; Eyidogan & Öz, 2007).

A considerable inhibition of nodulation was also detected in the present study. This notwithstanding, the native salt-tolerant mesorhizobia strain showed an increased ability to nodulate chickpea under salt stress compared with the salt-sensitive strain. These results are in agreement with the observations in other reports (Saxena & Rewari, 1992; Elsheikh & Wood, 1995; Mhadhbi et al., 2004), suggesting that the salt tolerance of the microsymbiont partner influences the symbiosis performance. However, the differences observed between plants inoculated with the salt-tolerant and the salt-sensitive Mesorhizobium strains are insufficient to preferentially use salt-tolerant rhizobia as a major approach to overcome the negative effects of salinity in plants.

Although both native mesorhizobia strains possess the acdS gene in their genome (Nascimento et al., 2012b), no ACC deaminase activity was detected in either strain in free-living conditions. Similarly, in other studies with mesorhizobia strains possessing the acdS gene, no ACC deaminase activity was detected in free-living conditions (Ma et al., 2003; Nascimento et al., 2012a). Herein, the two acdS-transformed strains showed ACC deaminase activity in free-living conditions. While the detected ACC deaminase activities of the two acdS-transformed mesorhizobia strains are low compared with the ACC deaminase activity of Pseudomonas putida UW4 (Ma et al., 2003), the ACC deaminase activities detected in this study are within the expected range of activities detected previously (Shah et al., 1998).

Both acdS-transformed mesorhizobia strains improved the chickpea plant growth under salt conditions when compared to plants inoculated with native mesorhizobia strains under the same stressful conditions. With the salt-sensitive M. ciceri EE-7 (pRKACC) an increased ability to nodulate chickpea plants under salinity was observed. Moreover, the salt-sensitive-transformed strain was able to form nodules in the same extent as the salt-tolerant wild-type M. ciceri G-55. On the other hand, the expression of an exogenous ACC deaminase in the salt-tolerant strain M. ciceri G-55 did not significantly influenced the nodulation abilities of this strain. It is possible that the different improvement obtained in the symbiotic performance of the two strains upon transformation with the acdS gene is due to the different levels of ACC deaminase activity in the two strains, with a higher ACC deaminase activity in EE-7 (pRKACC) contributing to a higher alleviation of the negative effects of ethylene.

The reduction in the negative effects of salt stress by the acdS-transformed strains was observed in all of the parameters analysed. Similar results were previously obtained with other plants (canola, tomato, cucumber and red pepper) inoculated with rhizobacteria expressing ACC deaminase and subjected to salt stress (Cheng et al., 2007; Gamalero et al., 2010; Siddikee et al., 2010, 2011). In addition, the increased symbiotic performance of acdS-transformed mesorhizobia strains under salinity is achieved by reducing the ethylene levels in plant tissues, which, in turn, lead to an increased number of nodules.

The acdS-transformed strains did not promote significant plant development under control conditions. Similar results were previously observed with cucumber plants inoculated with AcdS+ strains that showed improved plant development only under salinity stress (Gamalero et al., 2008, 2010). In contrast, several studies showed the beneficial effects on plant growth through an exogenous ACC deaminase activity displayed by acdS-transformed rhizobial strains under control conditions (Ma et al., 2004; Tittabutr et al., 2008; Nascimento et al., 2012a).

Taking into account that M. sp. MAFF303099 only expresses ACC deaminase activity in symbiotic conditions, under the transcriptional control of a NifA2-regulated promoter (Uchiumi et al., 2004; Nukui et al., 2006), and that the acdS gene of a chickpea mesorhizobia (M. ciceri UPM-Ca7T) was found to be transcribed under symbiotic conditions as well (Nascimento et al., 2012b), we can postulate a similar regulation of the endogenous acdS gene of the mesorhizobia strains used herein. The lack of a difference in symbiotic performance between native and acdS-transformed strains under control conditions may be due to the presence of the endogenous ACC deaminase activity, together with the low exogenous ACC deaminase activity whose beneficial effect on plant growth is only detectable in conditions that increase the ethylene to deleterious levels, such as salinity stress. A lower loss rate of the plasmid pRKACC under salinity stress conditions than under control conditions could also account for these results, with the salt stress acting as a de facto selective condition for the retention of the plasmid. This latter possibility is supported by the observation that tetracycline-resistant bacteria were only recovered from nodules of plants grown under salinity stress (data not shown). Accordingly, expression of the exogenous acdS gene in 8-week-old nodules was detected only in plants grown under salinity conditions (Fig. S1). These results are consistent with the observation that bacteria isolated from the rhizosphere of plants growing on the more stressful (including excessive sunlight and frequent drought) South-Facing Slope of Evolution Canyon in Israel are ten times more likely to possess ACC deaminase than were those from plants on the less stressful North-Facing Slope (Timmusk et al., 2011). In that instance, bacterial ACC deaminase is effectively being selected for by the plants growing under more stressful conditions, protecting plants and facilitating both bacterial and plant survival (Timmusk et al., 2011). In addition, an undetected low level of ACC deaminase activity under free-living conditions, which facilitates nodulation, cannot be ruled out. Rhizobia infection and nodulation causes only a very small and localized increase in plant ethylene levels, which may require only a low level of enzyme activity to prevent this from occurring (Glick, 2005). Under control conditions, the ethylene level is expected to be low in the plant tissues, so it is possible that the expression of the endogenous acdS gene may be enough to reduce those levels. Although the increase in ethylene in chickpea subjected to salinity has been reported (Nandwal et al., 2007), future work should include the measurement of ethylene levels in inoculated chickpea plants grown under control and salt stress to confirm the role of ACC deaminase enzyme in lowering the ethylene concentration under stress condition.

The preliminary results herein presented suggest that expression of an exogenous acdS gene in mesorhizobia improves the symbiotic performance of the bacteria when they were used as inoculants of chickpea plants grown under saline conditions, thus alleviating the negative effects caused by salinity. Further investigation on the use of acdS-expressing rhizobia for protection of chickpea plants grown under salinity conditions is required, namely the integration of an exogenous acdS gene into the mesorhizobia chromosome for constitutive expression.

Acknowledgements

The authors thank the funding from FEDER Funds through the Operational Programme for Competitiveness Factors - COMPETE and National Funds through FCT - Foundation for Science and Technology under the Strategic Project PEst-C/AGR/UI0115/2011, research projects FCOMP-01-0124-FEDER-007091 and PTDC/BIA-EVF/4158/2012, and from the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement n° 247669. C. Brígido acknowledges a FCT fellowship (SFRH/BD/30680/2006). B.R. Glick and J. Duan were supported by the Natural Science and Engineering Research Council of Canada. The authors thank Professor Mário de Carvalho for helping in the statistical analysis and to G. Mariano for technical assistance.

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