Chromium (Cr) pollution is due to large number of industrial processes such as chrome plating, wood preserving, pigmenting, pulp and paper, textile dyeing, tanning and leather processing. (Dhal et al. 2013). Its widespread use has converted Cr in a serious pollutant of air, soil and water (Zayed and Terry 2003). Alternative techniques for the clean-up of polluted soil and water, such as the cost-effective and less disruptive phytoremediation, have gained acceptance in recent years (Pilon-Smits 2005; Vangronsveld et al. 2009). Plant species used for phytoremediation of heavy-metal pollution may promote the growth of microorganisms, which in turn have capacity to detoxify metals efficiently by transforming them into insoluble salts or relatively nontoxic oxidation states (Kuiper et al. 2004; Belimov et al. 2005). Improvement of the beneficial associations between microorganisms and plants, particularly in the rhizosphere, is a research area of global interest.
Silene vulgaris (Moench) Garcke is a perennial facultative metallophyte species phenotypically diverse and widely distributed across its native range of Eurasia and North Africa (Marsden-Jones and Turrill 1957). It has also been introduced to other regions such as North America, South America and Australia where it colonizes disturbed habitats such as roadsides, railroad tracks and agricultural settings (Marsden-Jones and Turrill 1957). The tolerance to a diversity of metals has been described for this species (Paliouris and Hutchinson 1991; Ernst and Nelissen 2000) as well as local adaptation to metal-contaminated soils (Schat et al. 1996). The effectiveness of S. vulgaris in the revegetation of contaminated soils seemed to result in a different reduction of heavy metal toxicity on soil bacteria (Martinez Iñigo et al., 2009). Recent studies (Pradas del Real et al. 2013) showed that Cr uptake in S. vulgaris increased in the presence of Cr(VI) and a remarkable clonal variability was observed. In addition specifically differences were found in the elemental composition of root exudates in S. vulgaris genotypes due to Cr speciation in the medium (Pradas del Real et al. 2014b). These characteristics along with a wide range of adaptation and high intraspecific diversity (Sloan et al. 2012) make this species of great interest for phytoremediation purposes.
There is clear evidence that the plant-microorganisms interactions in the rhizosphere play important roles in plant growth, phytoextraction and metal accumulation as well as stress reduction in the plant (Kuiper et al. 2004; Abou-Shanab et al. 2008; Bakker et al. 2013). Heavy metals may change soil microbial biomass, activities and structure (Giller et al. 1998; Kahn and Scullion 2000; Anderson et al. 2009; Wang et al. 2010). In spite of the increasing knowledge of metal–microorganism interactions, few studies have attempted to characterize the bacterial communities in the rhizosphere of metallophytes under heavy metal stress (Wang et al. 2008; Navarro-Noya et al. 2010; Xu et al. 2012; Zhang et al. 2012). In addition, as far we know no studies have been performed to describe the structure and diversity of rhizobacterial communities under Cr stress.
Plants may actively shape rhizosphere microbial communities due to the release of large amounts of organic carbon by the roots (Bais et al. 2006; Hartmann et al. 2009). Considering that plant root exudates differ among plant species, cultivars or accessions (López-Bucio et al. 2000), it can be inferred differences in rhizosphere microbiomes of different plant species (Smalla et al. 2001; Costa et al. 2006) and at genotype level within a plant species (Micaleff et al. 2009).
Organic acids have been mentioned as playing an important role in the transport, storage and heavy metal tolerance, including Al, Cd, Cr, Fe, Ni and Zn (Hall 2002; Zeng et al. 2008). Organic acids can bind heavy metals and may therefore be deployed in response to metal toxicity. Malate and citrate organic acids are involved in root to shoot metal transport through the xylem and in vacuolar sequestration (Rauser 1999). Furthermore, citrate and oxalate have been reported to play an important role in phytoremediation of Cr-contaminated soils by enhancing Cr uptake and increasing translocation to shoots (Davies et al. 2001). Heavy metal stress commonly induces important accumulation of low molecular weight organic acids in various plant organs reinforcing the hypothesis that these molecules are involved in metal tolerant mechanisms. (Mnasri et al. 2015).
Plant-microbe interactions are considered to be important processes determining the efficiency of phytoremediation of heavy metal contaminated soils. However relatively little is known about how these interactions are influence by Cr contamination. Monitoring the changes in some molecular and biochemical parameters involved in Cr stress responses, could open potential pathways to enhance plant tolerance to Cr soil contamination. Furthermore, identification of rhizobacteria having potential for heavy metal tolerance, is of particular interest for bacteria assisted phytoremediation. In the present study two Cr-tolerant genotypes were selected attending to differences in metal uptake, exudation rates and differences on elemental composition of root exudates in response to Cr under hydroponic conditions (Pradas del Real et al. 2013; 2014b). The purpose of this study was: a) to evaluated the metal induced response on biomass production, lipid peroxidation (MDA) and root organic acid composition b) to assess and compare the effect of Cr contamination on the structure and composition of rhizosphere microbial communities by PCR-DGGE and bacterial 16S rDNA sequencing and c) to determine the effect of plant bacterial interactions over Cr uptake.
Materials and methods
Plant material and greenhouse experimental design
Silene vulgaris natural populations were collected throughout Madrid Province and, after two growing seasons, rhizome cuttings were used to propagate individual plants. Genetically uniform clones were developed and vegetatively propagated on permanent field plots at the “El Encín” agricultural experiment station (Alcalá de Henares, Spain: 40º 03´ N, 3º 19´ W). Two genotypes from two different populations were selected: genotype SV-21 (Rozas de Puerto Real) and genotype SV-38 (Valdemaqueda). A pot experiment was conducted under greenhouse conditions (humidity 50-65 %, average air temperature 21 ºC and natural light). Plastic pots were filled with 17 kg of agricultural loamy sand soil collected from the top layer (0-30 cm). Soil characteristics were analyzed according to the official soil analysis in Spain (MAPA, 1994). Basically,: pH 6.62, carbonates 0.6 %, electrical conductivity 0.65 dS m-1, total nitrogen 0.13 %, available P 55 mg kg-1 organic matter 2.67 %; sand 75.5 %, silt 10 %, clay 14.5 %; exchangeable cations Ca 1437 mg kg-1, Mg 131 mg kg-1, Na 8.15 mg kg-1, K 193 mg kg-1; metals Cr 29 mg kg-1, Pb 26 mg kg-1, Cd 0.24 mg kg-1, Cu 15 mg kg-1, Zn 37.12 mg kg-1. The experiment was performed in a randomized block design with three replicates and a 2×3 factorial arrangement with two levels of pollution: i) no pollution, ii) K2Cr2O7 to simulate a Cr(VI) spilling; and two vegetations treatments: i) S. vulgaris genotype SV-21 and ii) S. vulgaris genotype SV-38.
Cr pollution was simulated by spiking the soil with K2Cr2O7 solution (1000 mg Cr L-1) to reach a final concentration of total Cr in pots of 100 mg kg-1soil. The soil was brought to 60 % water holding capacity and maintained by addition of deionized water for three months. After soil consolidation, 10 cuttings per genotype were transplanted to each pot and maintained during two growing periods. The total amount of water applied in each irrigation cycle was based on the normal rainfall in Mediterranean conditions.
Plant analysis
At the end of the experimental period shoots and roots were collected, weighed and dried in a forced air oven for 48 h at 70 ºC. The dry weights were determined and the samples were digested by adding 0.5 ml of HNO3(65 % Suprapur®) and 0.5 ml of HClO4 (70 %, Suprapur®). Total Cr concentration was measure by Flame Atomic absorption spectrophotometer (Varian fast sequential model AA240FS). Bush branches and Leaves (DC73348GSV-1) were used as reference material. The translocation factor (TF) was calculated to determine relative translocation of Cr content (mg kg-1) from the root to the aerial part of the plant: TF= Cshoot/Croot. The ratio of metal concentration in the plant to soil was used to determine the bioconcentration factor (BF): BF = Cp/Cso, where Cp and Cso are metal concentrations in the plant (shoot and root) and in the soil, respectively. Tolerance index (TI) was defined as the ratio between plant biomass after Cr treatment and control: TI= WCr/Wcontrol.
Lipid peroxidation of shoots and roots was evaluated as malondialdehyde (MDA) by the method of Reilly and Aust (2001). Absorbance was measure at 532 nm. Absorbance at 600 nm was subtracted to this measure to eliminated the interferences of soluble sugars in the samples. Both absorbances were determined by UV-VIS light spectrophotometer (Thermo Spectronic Helios Alpha).
Organic acids were extracted from fresh root tissues as described by Arnetoli (2008). Samples (1 g) of frozen fresh weight were homogenized in 10 mL of Milli-Q water using a mortar and pestle in liquid N2; then homogenates were centrifuged for 20 min at 10,000 rpm at 4 ºC. The supernatant was stored at -20 ºC and filtered through PVC filters of 0.20 μm before analysis. Organic acids (oxalic, citric, malic, formic, lactic, acetic and succinic) were measured by Ionic Chromatography (Dionex DX 500) using a conductivity detector. Chromatographic conditions were as follows: sample loop volume, 25 L); analytical column, IonPac ICE-AS6; eluent, 0.4 mM heptafluorobutyric acid (flow rate, 1.0 mL min-1); suppressor, MicroMembrane Anion-ICE; regenerant, 5 mM tetrabutylammonium hydroxide (flow rate: 5 mL min-1); analysis time, 20 min. Organic acids were identified by comparing the retention times of the samples against retention times of the standards. Calibration curves have been performed using Merck reagents from 50 to 280 mg L-1 for oxalic acid; from 1 to 50 mg L-1 for citric, malic and acetic acids; from 1 to 30 mg L-1 for lactic and succinic and from 0.5 to 10 mg L-1 for formic acid.
Soil DNA extraction, PCR and DGGE analyses
Rhizosphere soil was collected at flowering time from each of the three replicates. From each pot three soil samples were bulked into a single sample. After the plants were uprooted and shaken, rhizosphere samples were collected from soil that remained gently adhered to plan roots (Lynch 1990) and collected into sterile Petri dishes and store at 4 ºC until DNA was extracted. Soil DNA was extracted using the UltraClean Soil DNA Isolation Kit (Mo Bio Laboratories, Inc.) according to the manufacturer´s instructions. Bacterial 16S rRNA gene fragments were amplified with the primer set 341F with a GC clamp (40-nucleotide GC-rich sequence, 5’-CCT ACG GGA GGC AGC AG-3´) and 907R (5’ CCG TCA ATT CMT TTG AGT TT-3’) specific for the domain Bacteria (Schäfer and Muyzer 2001). Amplicons
2018-9-6-1536220555
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