Targeting Acr3 from Ensifer medicae to the plasma membrane or to the tonoplast of tobacco hairy roots allows arsenic extrusion or improved accumulation. Effect of acr3 expression on the root transcriptome.

Transgenic tobacco hairy roots expressing the bacterial arsenite efflux pump Acr3 from Ensifer medicae were generated. The gene product was targeted either to the plasma membrane (ACR3 lines) or to the tonoplast by fusing the ACR3 protein to the tonoplast integral protein TIP1.1 (TIP-ACR3 lines). Roots expressing Acr3 at the tonoplast showed greater biomass than those expressing Acr3 at the plasma membrane. Furthermore, higher contents of malondialdehyde (MDA) and RNA degradation in ACR3 lines were indicative of higher oxidative stress. The determination of ROS-scavenging enzymes depicted the transient role of peroxidases in ROS detoxification, followed by the action of superoxide dismutase during both short- and medium-term exposure periods. Regarding As accumulation, ACR3 lines accumulated up to 20-30% less As, whereas TIP-ACR3 achieved a 2-fold increase in As accumulation in comparison to control hairy roots. Strategies that presumably induce As uptake, such as phosphate deprivation or dehydration followed by rehydration in the presence of As, fostered As accumulation up to 10 800 µg g-1. Finally, the effects of the heterologous expression of acr3 on the root transcriptome were assessed. Expression at the plasma membrane induced drastic changes in gene expression, with outstanding overexpression of genes related to electron transport, ATP synthesis and ATPases, suggesting that As efflux is the main detoxification mechanism in these lines. In addition, genes encoding heat shock proteins and those related to proline synthesis and drought tolerance were activated. On the other hand, TIP-ACR3 lines showed a similar gene expression profile to that of control roots, with overexpression of the glutathione and phytochelatin synthesis pathways, together with secondary metabolism pathways as the most important resistance mechanisms in TIP-ACR3, for which As allocation into the vacuole allowed better growth and stress management. Our results suggest that modulation of As accumulation can be achieved by subcellular targeting of Acr3: expression at the tonoplast enhances As accumulation in roots, whereas expression at the plasma membrane could promote As efflux. Thus, both approaches open the possibilities for developing safer crops when grown on As-polluted paddy soils, but expression at the tonoplast leads to better growth and less stressed roots, since the high energy cost of As efflux likely compromises growth in ACR3 lines.

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.

Bioaugmentation with bacteria selected from the microbiome enhances Arthrocnemum macrostachyum metal accumulation and tolerance.

A glasshouse experiment was designed to investigate the role of bacterial consortia isolated from the endosphere (CE) and rhizosphere (CR) of Arthrocnemum macrostachyum on its metal uptake capacity and tolerance in plants grown in metal polluted sediments. A. macrostachyum plants were randomly assigned to three bioaugmentation treatments (CE, CR and without inoculation) during 120days. Bioaugmentation with both bacterial consortia enhanced A. macrostachyum capacity to accumulate ions in its roots, while shoot ions concentration only increased with CE treatment. Furthermore bioaugmentation ameliorated the phytotoxicity levels, which was reflected in an increment of plant growth of 59 and 113% for shoots and 52 and 98% for roots with CE and CR treatments, respectively. This effect was supported by bacteria beneficial effect on photochemical apparatus and the modulation of its oxidative stress machinery. These findings indicated that bacteria selected from the microbiome can be claimed to improve A. macrostachyum metal remediation efficiency.

Removal of copper from aqueous solutions by rhizofiltration using genetically modified hairy roots expressing a bacterial Cu-binding protein.

The aim of this work was to develop a biotechnological tool to hyperaccumulate high copper (Cu) concentrations from wastewaters. Transgenic tobacco hairy roots were obtained by expressing, either the wild-type version of the gene copC from Pseudomonas fluorescens in the cytoplasm of plant cells (CuHR), or a modified version targeted to the vacuole (CuHR-V). Control hairy roots transformed with the empty vector (HR) were also generated. The roots were incubated in the presence of solutions containing Cu (from 1 to 50 mM). At 5 mM external copper, transgenic hairy roots accumulated twice the amount of copper accumulated by control hairy roots. However, at 50 mM Cu, accumulation in both transgenic and control roots reached similar values. Maximum Cu accumulation achieved by transgenic hairy roots was 45,000 µg g-1 at 50 mM external Cu. Despite the high Cu accumulation, transgenic hairy roots, particularly CuHR-V, showed less toxicity symptoms, in correlation with lower activity of several antioxidant enzymes and lower malondialdehyde (MDA) levels. Moreover, CuHR-V roots displayed low values of the oxidative stress index (OSI) - a global parameter proposed for oxidative stress - indicating that targeting CopC to the vacuole could alleviate the oxidative stress caused by Cu. Our results suggest that expressing copC in transgenic hairy roots is a suitable strategy to obtain Cu-hyperaccumulator hairy roots with less toxicity stress symptoms. ABBREVIATIONS: APX: ascorbate peroxidase; ATSDR: Agency for Toxic Substances and Disease Registry (U.S.); BCF: bioconcentration factor; CuHR: copper-hairy roots; EDTA: ethylenediamine tetracetic acid; EPA: Environmental Protection Agency (U.S.); GSH: glutathione; HM: heavy metals; HR: control hairy roots; ICP-OES: Inductively Coupled Plasma/Optical Emission Spectrometry; MDA: malondialdehyde; NBT: nitroblue tetrazolium; OD: optical density; OSI: oxidative stress index; PCR: polymerase chain reaction; PVP: polyvynilpirrolidone; PX: peroxidase; ROS: reactive oxygen species; SOD: superoxide dismutase.

Screening beneficial rhizobacteria from Spartina maritima for phytoremediation of metal polluted salt marshes: comparison of gram-positive and gram-negative strains.

The aim of our work was the isolation and characterization of bacteria from the rhizosphere of Spartina maritima in the metal contaminated Odiel estuary (Huelva, SW Spain). From 25 strains, 84 % were identified as gram-positive, particularly Staphylococcus and Bacillus. Gram-negative bacteria were represented by Pantoea and Salmonella. Salt and heavy metal tolerance, metal bioabsorption, plant growth promoting (PGP) properties, and biofilm formation were investigated in the bacterial collection. Despite the higher abundance of gram-positive bacteria, gram-negative isolates displayed higher tolerance toward metal(loid)s (As, Cu, Zn, and Pb) and greater metal biosorption, as deduced from ICP-OES and SEM-EDX analyses. Besides, they exhibited better PGP properties, which were retained in the presence of metals and the ability to form biofilms. Gram-negative strains Pantoea agglomerans RSO6 and RSO7, together with gram-positive Bacillus aryabhattai RSO25, were selected for a bacterial consortium aimed to inoculate S. maritima plants in metal polluted estuaries for phytoremediation purposes.

Microcystin-tolerant Rhizobium protects plants and improves nitrogen assimilation in Vicia faba irrigated with microcystin-containing waters.

Irrigation of crops with microcystins (MCs)-containing waters-due to cyanobacterial blooms-affects plant productivity and could be a way for these potent toxins entering the food chain. This study was performed to establish whether MC-tolerant rhizobia could benefit growth, nodulation, and nitrogen metabolism of faba bean plants irrigated with MC-containing waters. For that, three different rhizobial strains-with different sensitivity toward MCs-were used: RhOF96 (most MC-sensitive strain), RhOF125 (most MC-tolerant strain), or Vicz1.1 (reference strain). As a control, plants grown without rhizobia and fertilized by NH4NO3 were included in the study. MC exposure decreased roots (30-37 %) and shoots (up to 15 %) dry weights in un-inoculated plants, whereas inoculation with rhizobia protects plants toward the toxic effects of MCs. Nodulation and nitrogen content were significantly impaired by MCs, with the exception of plants inoculated with the most tolerant strain RhOF125. In order to deep into the effect of inoculation on nitrogen metabolism, the nitrogen assimilatory enzymes (glutamine synthetase (GS) and glutamate synthase (GOGAT)) were investigated: Fertilized plants showed decreased levels (15-30 %) of these enzymes, both in shoots and roots. By contrast, inoculated plants retained the levels of these enzymes in shoots and roots, as well as the levels of NADH-GOGAT activity in nodules. We conclude that the microcystin-tolerant Rhizobium protects faba bean plants and improves nitrogen assimilation when grown in the presence of MCs.

Unraveling the effect of arsenic on the model Medicago-Ensifer interaction: a transcriptomic meta-analysis.

The genetic regulation underlying the effect of arsenic (As(III)) on the model symbiosis Medicago-Ensifer was investigated using a combination of physiological (split-roots), microscopy and genetic (microarrays, qRT-PCR and composite plants) tools. Nodulation was very sensitive to As(III) (median inhibitory dose (ID50) = 20 µM). The effect on root elongation and on nodulation was local (nonsystemic). A battery of stress (salt, drought, heat shock, metals, etc.)-related genes were induced. Glutathione played a pivotal role in tolerance/detoxification, together with secondary metabolites ((iso)flavonoids and phenylpropanoids). However, antioxidant enzymes were not activated. Concerning the symbiotic interaction, molecular evidence suggesting that rhizobia alleviate As stress is for the first time provided. Chalcone synthase (which is involved in the first step of the legume-rhizobia cross-talk) was strongly enhanced, suggesting that the plants are biased to establish symbiotic interactions under As(III) stress. In contrast, 13 subsequent nodulation genes (involved in nodulation factors (Nod factors) perception, infection, thread initiation and progression, and nodule morphogenesis) were repressed. Overexpression of the ethylene responsive factor ERN in composite plants reduced root stress and partially restored nodulation, whereas overexpression of the early nodulin ENOD12 enhanced nodulation both in the presence and, particularly, in the absence of As, without affecting root elongation. Several transcription factors were identified, which could be additional targets for genetic engineering aiming to improve nodulation and/or alleviate root stress induced by this toxic.

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.

Endophytic Cultivable Bacteria of the Metal Bioaccumulator Spartina maritima Improve Plant Growth but Not Metal Uptake in Polluted Marshes Soils.

Endophytic bacterial population was isolated from Spartina maritima tissues, a heavy metal bioaccumulator cordgrass growing in the estuaries of Tinto, Odiel, and Piedras River (south west Spain), one of the most polluted areas in the world. Strains were identified and ability to tolerate salt and heavy metals along with plant growth promoting and enzymatic properties were analyzed. A high proportion of these bacteria were resistant toward one or several heavy metals and metalloids including As, Cu, and Zn, the most abundant in plant tissues and soil. These strains also exhibited multiple enzymatic properties as amylase, cellulase, chitinase, protease and lipase, as well as plant growth promoting properties, including nitrogen fixation, phosphates solubilization, and production of indole-3-acetic acid (IAA), siderophores and 1-aminocyclopropane-1-carboxylate (ACC) deaminase. The best performing strains (Micrococcus yunnanensis SMJ12, Vibrio sagamiensis SMJ18, and Salinicola peritrichatus SMJ30) were selected and tested as a consortium by inoculating S. maritima wild plantlets in greenhouse conditions along with wild polluted soil. After 30 days, bacterial inoculation improved plant photosynthetic traits and favored intrinsic water use efficiency. However, far from stimulating plant metal uptake, endophytic inoculation lessened metal accumulation in above and belowground tissues. These results suggest that inoculation of S. maritima with indigenous metal-resistant endophytes could mean a useful approach in order to accelerate both adaption and growth of this indigenous cordgrass in polluted estuaries in restorative operations, but may not be suitable for rhizoaccumulation purposes.

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.

Paenibacillus prosopidis sp. nov., isolated from the nodules of Prosopis farcta.

A bacterial strain, designated PW21(T), was isolated from root nodules of Prosopis farcta in Tunisia. Phylogenetic analysis based on 16S rRNA gene sequences placed the isolate into the genus Paenibacillus, with its closest relatives being Paenibacillus glycanilyticus DS-1(T) and Paenibacillus castaneae Ch-32(T) with identity values of 96.9 %. DNA-DNA hybridization measurements showed values of less than 25 % with respect to these two species. The isolate was a Gram-variable, motile and sporulating rod. Catalase activity was positive and oxidase activity was weakly positive. Aesculin, CM-cellulose, xylan and starch were hydrolysed but casein and gelatin were not. Acetoin production was weakly positive and nitrate reduction was negative. Urease production was negative. Growth was supported by many carbohydrates and organic acids as carbon sources. MK-7 was the predominant menaquinone and anteiso-C(15 : 0), iso-C(16 : 0) and iso-C(15 : 0) were the major fatty acids. Major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, a glycolipid, six phospholipids, an unidentified lipid and two unknown aminophosphoglycolipids. meso-Diaminopimelic acid was not detected in the peptidoglycan. The DNA G+C content of the isolate was 52.9 mol%. Phylogenetic, chemotaxonomic and phenotypic analyses showed that strain PW21(T) should be considered to represent a novel species of the genus Paenibacillus, for which the name Paenibacillus prosopidis sp. nov. is proposed. The type strain is PW21(T) (=LMG 25259(T) =CECT 7506(T) =DSM 22405(T)).

Reduced nodulation in alfalfa induced by arsenic correlates with altered expression of early nodulins.

Arsenic (As) reduces legume nodulation by affecting the first stages of the symbiotic interaction, which causes a 90% decrease in rhizobial infections. In this paper, we examine molecular mechanisms underlying this toxic effect, using the model system Medicago sativa-Sinorhizobium. In the presence and absence of As, the expression patterns of seven nodulin genes, markers for the different events leading to nodule formation, were analyzed by RT-PCR and by real-time RT-PCR. A significant decrease was observed, especially from days 1-5 after the inoculation, in the expression of four early nodulins: the genes coding the Nod factor receptor (nork), the transcription factor NIN and the markers for infection progression (N6) and nodule organogenesis (Enod2). On the contrary, the expression of markers for primordium initiation (Enod40) and differentiation (ccs52) was not significantly altered. Finally, the expression of a marker for nitrogen fixation (Legbrc) was also reduced, probably due to the reduction in nodule number induced by As. These results suggest that As affects the expression of nodulation genes that have been associated with processes that take place in the epidermis and the outer cortical cells, and that the expression of genes associated with events that take place in the inner cortical cells is less affected. This is the first report showing changes in the expression of nodulin genes induced by the presence of any toxic metal(loid).

The Symbiosis Interactome: a computational approach reveals novel components, functional interactions and modules in Sinorhizobium meliloti.

BACKGROUND: Rhizobium-Legume symbiosis is an attractive biological process that has been studied for decades because of its importance in agriculture. However, this system has undergone extensive study and although many of the major factors underpinning the process have been discovered using traditional methods, much remains to be discovered. RESULTS: Here we present an analysis of the 'Symbiosis Interactome' using novel computational methods in order to address the complex dynamic interactions between proteins involved in the symbiosis of the model bacteria Sinorhizobium meliloti with its plant hosts. Our study constitutes the first large-scale analysis attempting to reconstruct this complex biological process, and to identify novel proteins involved in establishing symbiosis. We identified 263 novel proteins potentially associated with the Symbiosis Interactome. The topology of the Symbiosis Interactome was used to guide experimental techniques attempting to validate novel proteins involved in different stages of symbiosis. The contribution of a set of novel proteins was tested analyzing the symbiotic properties of several S. meliloti mutants. We found mutants with altered symbiotic phenotypes suggesting novel proteins that provide key complementary roles for symbiosis. CONCLUSION: Our 'systems-based model' represents a novel framework for studying host-microbe interactions, provides a theoretical basis for further experimental validations, and can also be applied to the study of other complex processes such as diseases.

From pollen tubes to infection threads: recruitment of Medicago floral pectic genes for symbiosis.

While the biology of nitrogen-fixing root nodules has been extensively studied, little is known about the evolutionary events that predisposed legume plants to form symbiosis with rhizobia. We have studied the presence and the expression of two pectic gene families in Medicago, polygalacturonases (PGs) and pectin methyl esterases (PMEs) during the early steps of the Sinorhizobium meliloti-Medicago interaction and compared them with related pollen-specific genes. First, we have compared the expression of MsPG3, a PG gene specifically expressed during the symbiotic interaction, with the expression of MsPG11, a highly homologous pollen-specific gene, using promoter-gus fusions in transgenic M. truncatula and tobacco plants. These results demonstrated that the symbiotic promoter functions as a pollen-specific promoter in the non-legume host. Second, we have identified the presence of a gene family of at least eight differentially expressed PMEs in Medicago. One subfamily is represented by one symbiotic gene (MtPER) and two pollen-expressed genes (MtPEF1 and MtPEF2) that are clustered in the M. truncatula genome. The promoter-gus studies presented in this work and the homology between plant PGs, together with the analysis of the PME locus structure and MtPER expression studies, suggest that the symbiotic MsPG3 and MtPER could have as ancestors pollen-expressed genes involved in polar tip growth processes during pollen tube elongation. Moreover, they could have been recruited after gene duplication in the symbiotic interaction to facilitate polar tip growth during infection thread formation.

Expression of MsPG3-GFP fusions in Medicago truncatula'hairy roots' reveals preferential tip localization of the protein in root hairs.

Tip growth is a specialized type of polar growth where new cell wall is deposited in a localized region of the cell, the growing tip. These cells show a characteristic zonation, with a high accumulation of secretory vesicles containing cell wall components at the tip, followed by an organelle-enriched zone. MsPG3 is a Medicago sativa polygalacturonase gene isolated in our laboratory, specifically expressed during the interaction of this plant with its symbiotic partner Sinorhizobium meliloti and which might participate in tip growth processes during symbiosis. We have used MsPG3-GFP fusions to study in vivo protein transport processes and localization during root hair growth. Different MsPG3-GFP fusions were expressed in Medicago truncatula'hairy roots' following a protocol developed for this study and also tested by transient expression in onion epidermal cells. Preferential accumulation of an MsPG3-GFP fusion protein in the tip of the growing root hair at different developmental stages was found, confirming the delivery of MsPG3 to the newly synthesized cell wall. This indicates that this protein may participate in tip growth processes during symbiosis and, in addition, that this fusion could be a useful tool to study this process in plants.