Bouchra Doukkali

Prospecting metal-resistant plant-growth promoting rhizobacteria for rhizoremediation of metal contaminated estuaries using Spartina densiflora

In the salt marshes of the joint estuary of Tinto and Odiel rivers (SW Spain), one of the most polluted areas by heavy metals in the world, Spartina densiflora grows on sediments with high concentrations of heavy metals. Furthermore, this species has shown to be useful for phytoremediation. The total bacterial population of the rhizosphere of S. densiflora grown in two estuaries with different levels of metal contamination was analyzed by PCR denaturing gradient gel electrophoresis. Results suggested that soil contamination influences bacterial population in a greater extent than the presence of the plant. Twenty-two different cultivable bacterial strains were isolated from the rhizosphere of S. densiflora grown in the Tinto river estuary. Seventy percent of the strains showed one or more plant growth-promoting (PGP) properties, including phosphate solubilization and siderophores or indolacetic acid production, besides a high resistance towards Cu. A bacterial consortium with PGP properties and very high multiresistance to heavy metals, composed by Aeromonas aquariorum SDT13, Pseudomonas composti SDT3, and Bacillus sp. SDT14, was selected for further experiments. This consortium was able to two-fold increase seed germination and to protect seeds against fungal contamination, suggesting that it could facilitate the establishment of the plant in polluted estuaries.

Cadmium biosorption properties of the metal‐resistant Ochrobactrum cytisi Azn6.2

The aim of this work was to establish the conditions for using Ochrobactrum cytisi Azn6.2 as a metal biosorbent. Azn6.2 is a novel strain from the legume symbiont O. cytisi that has been isolated from nodules of Medicago polymorpha plants grown on heavy metal‐polluted soils. Compared with the strain ESC1, Azn6.2 showed some biochemical differences, as well as antibiotic susceptibility, Azn6.2 was multi‐resistant to heavy metals, such as Cu, Cd and Zn, and bacterial pellets were able to biosorb high amounts of Cd and Zn. As shown by scanning electron microscopy coupled to energy dispersive X‐ray, most of Cd was attached to the cell surface. Optimal conditions for Cd biosorption were established, being 1 mM Cd ions in solution and 2 h of contact with the biosorbent at room temperature. At these conditions, maximal Cd loading capacity reached 32–34 mg/g. Cd desorption from bacterial pellets was achieved after washing with EDTA or, at higher efficiency, at pH 1.0. These results indicated that biosorption/desorption on O. cytisi Azn6.2 biomass should be a cost‐effective method for Cd recovery from contaminated solutions.

Improving legume nodulation and Cu rhizostabilization using a genetically modified rhizobia

The rhizobia–legume interaction has been proposed as an interesting and appropriate tool for rhizostabilization of soils contaminated with heavy metals. One of the main requirements to use this symbiosis is the availability of tolerant and symbiotically effective rhizobia. The aim of this work was to improve the symbiotic properties of the arsenic-resistant wild-type strain Ensifer medicae MA11 in Cu-contaminated substrates. The copAB genes from a Cu-resistant Pseudomonas fluorescens strain were expressed in E. medicae MA11 under the control of the nifH promoter. The resulting strain E. medicae MA11-copAB was able to alleviate the toxic effect of Cu in Medicago truncatula. At 300 µM Cu, root and shoot dry matter production, nitrogen content, number of nodules and photosynthetic rate were significantly reduced in plants inoculated with the wild-type strain. However, these parameters were not altered in plants inoculated with the genetically modified strain. Moreover, nodules elicited by this strain were able to accumulate twofold the Cu measured in nodules formed by the wild-type strain. In addition, the engineered E. medicae strain increased Cu accumulation in roots and decreased the content in shoots. Thus, E. medicae MA11-copAB increased the capacity of M. truncatula to rhizostabilize Cu, decreasing the translocation factor and avoiding metal entry into the food chain. The plasmid containing the nifH promoter-copAB construct could be a useful biotool for Cu rhizostabilization using legumes, since it can be transferred to different rhizobia microsymbionts of authoctonous legumes growing in Cu-contaminated soils.

Engineering copper hyperaccumulation in plants by expressing a prokaryotic copC gene.

In this work, engineering Cu-hyperaccumulation in plants was approached. First, the copC gene from Pseudomonas sp. Az13, encoding a periplasmic Cu-binding protein, was expressed in Arabidopsis thaliana driven by the CaMV35S promoter (transgenic lines 35S-copC). 35S-copC lines showed up to 5-fold increased Cu accumulation in roots (up to 2000 μg Cu. g(-1)) and shoots (up to 400 μg Cu. g(-1)), compared to untransformed plants, over the limits established for Cu-hyperaccumulators. 35S lines showed enhanced Cu sensitivity. Second, copC was engineered under the control of the cab1 (chlorophyll a/b binding protein 1) promoter, in order to drive copC expression to the shoots (transgenic lines cab1-copC). cab1-copC lines showed increased Cu translocation factors (twice that of wild-type plants) and also displayed enhanced Cu sensitivity. Finally, subcellular targeting the CopC protein to plant vacuoles was addressed by expressing a modified copC gene containing specific vacuole sorting determinants (transgenic lines 35S-copC-V). Unexpectedly, increased Cu-accumulation was not achieved-neither in roots nor in shoots-when compared to 35S-copC lines. Conversely, 35S-copC-V lines did display greatly enhanced Cu-hypersensitivity. Our results demonstrate the feasibility of obtaining Cu-hyperaccumulators by engineering a prokaryotic Cu-binding protein, but they highlight the difficulty of altering the exquisite Cu homeostasis in plants.

Improving Legume–Rhizobium Symbiosis for Copper Phytostabilization Through Genetic Manipulation of Both Symbionts

The presence of excess copper (Cu) in soils represents an environmental and health problem, due to the risk of groundwater pollution. Besides, it affects plant development and yield. Phytoremediation has consolidated as a low-cost and ecological technique for metal remediation. In this particular, legume–rhizobium symbioses have risen as an attractive biotechnological tool for metal phytostabilization. For this technique to be suitable, metal-tolerant symbionts are needed, which can be generated through genetic engineering. In this work, the genetic manipulation of both symbiotic partners for Cu phytostabilization was described. Concerning the plant, composite Medicago truncatula plants expressing the metallothionein gene mt4a from Arabidopsis thaliana in roots were generated, in an attempt to increase the plant tolerance towards Cu. Concerning the rhizobial strain, an Ensifer medicae strain was genetically engineered by expressing the copper resistance genes copAB from Pseudomonas fluorescens. Our results indicate the following: (a) the expression of mt4a in composite plants increases tolerance towards Cu and reduces oxidative stress caused by this pollutant. Lower levels of reactive oxygen species (ROS)-scavenging enzymes were found in mt4a-expressing plants; (b) the expression of mt4a in composite plants improves nodulation, whereas inoculation with the genetically modified Ensifer has a synergistic effect; and (c) The double symbiotic system enhances Cu accumulation in roots, without increasing metal translocation to shoots. We conclude that the genetically modified symbiosis is a suitable tool for Cu rhizo–phytostabilization.

Bacterial inoculants for enhanced seed germination of Spartina densiflora: Implications for restoration of metal polluted areas.

The design of effective phytoremediation programs is severely hindered by poor seed germination on metal polluted soils. The possibility that inoculation with plant growth promoting rhizobacteria (PGPR) could help overcoming this problem is hypothesized. Our aim was investigating the role of PGPR in Spartina densiflora seed germination on sediments with different physicochemical characteristics and metal pollution degrees. Gram negative Pantoea agglomerans RSO6 and RSO7, and gram positive Bacillus aryabhattai RSO25, together with the consortium of the three strains, were used for independent inoculation experiments. The presence of metals (As, Cu, Pb and Zn) in sediments reduced seed germination by 80%. Inoculation with Bacillus aryabhattai RSO25 or Pantoea agglomerans RSO6 and RSO7 enhanced up to 2.5 fold the germination rate of S. densiflora in polluted sediments regarding non-inoculated controls. Moreover, the germination process was accelerated and the germination period was extended. The consortium did not achieve further improvements in seed germination.

Modulation of Spartina densiflora plant growth and metal accumulation upon selective inoculation treatments: A comparison of gram negative and gram positive rhizobacteria

Metal contamination of estuaries is a severe environmental problem, for which phytoremediation is gaining momentum. In particular, the associations between halophytes-autochthonous rhizobacteria have proven useful for metal phytostabilization in salt marshes. In this work, three bacterial strains (gram-negative and gram-positive) were used for Spartina densiflora inoculation. All three bacteria, particularly Pantoea strains, promoted plant growth and mitigated metal stress on polluted sediments, as revealed from functionality of the photosynthetic apparatus (PSII) and maintenance of nutrient balance. Pantoea strains did not significantly affect metal accumulation in plant roots, whereas the Bacillus strain enhanced it. Metal loading to shoots depended on particular elements, although in all cases it fell below the threshold for animal consumption. Our results confirm the possibility of modulating plant growth and metal accumulation upon selective inoculation, and the suitability of halophyte-rhizobacteria interactions as biotechnological tools for metal phytostabilization in salt marshes, preventing metal transfer to the food chain.

Double genetically modified symbiotic system for improved Cu phytostabilization in legume roots

Excess copper (Cu) in soils has deleterious effects on plant growth and can pose a risk to human health. In the last decade, legume-rhizobium symbioses became attractive biotechnological tools for metal phytostabilization. For this technique being useful, metal-tolerant symbionts are required, which can be generated through genetic manipulation.In this work, a double symbiotic system was engineered for Cu phytostabilization: On the one hand, composite Medicago truncatula plants expressing the metallothionein gene mt4a from Arabidopsis thaliana in roots were obtained to improve plant Cu tolerance. On the other hand, a genetically modified Ensifer medicae strain, expressing copper resistance genes copAB from Pseudomonas fluorescens driven by a nodulation promoter, nifHp, was used for plant inoculation. Our results indicated that expression of mt4a in composite plants ameliorated plant growth and nodulation and enhanced Cu tolerance. Lower levels of ROS-scavenging enzymes and of thiobarbituric acid reactive substances (TBARS), such as malondialdehyde (a marker of lipid peroxidation), suggested reduced oxidative stress. Furthermore, inoculation with the genetically modified Ensifer further improved root Cu accumulation without altering metal loading to shoots, leading to diminished values of metal translocation from roots to shoots. The double modified partnership is proposed as a suitable tool for Cu rhizo-phytostabilization.