Microbial Consortium of PGPR, Rhizobia and Arbuscular Mycorrhizal Fungus Makes Pea Mutant SGECdt Comparable with Indian Mustard in Cadmium Tolerance and Accumulation

Cadmium (Cd) is one of the most widespread and toxic soil pollutants that inhibits plant growth and microbial activity. Polluted soils can be remediated using plants that either accumulate metals (phytoextraction) or convert them to biologically inaccessible forms (phytostabilization). The phytoremediation potential of a symbiotic system comprising the Cd-tolerant pea (Pisum sativum L.) mutant SGECdt and selected Cd-tolerant microorganisms, such as plant growth-promoting rhizobacterium Variovorax paradoxus 5C-2, nodule bacterium Rhizobium leguminosarum bv. viciae RCAM1066, and arbuscular mycorrhizal fungus Glomus sp. 1Fo, was evaluated in comparison with wild-type pea SGE and the Cd-accumulating plant Indian mustard (Brassica juncea L. Czern.) VIR263. Plants were grown in pots in sterilized uncontaminated or Cd-supplemented (15 mg Cd kg−1) soil and inoculated or not with the microbial consortium. Cadmium significantly inhibited growth of uninoculated and particularly inoculated SGE plants, but had no effect on SGECdt and decreased shoot biomass of B. juncea. Inoculation with the microbial consortium more than doubled pea biomass (both genotypes) irrespective of Cd contamination, but had little effect on B. juncea biomass. Cadmium decreased nodule number and acetylene reduction activity of SGE by 5.6 and 10.8 times, whereas this decrease in SGECdt was 2.1 and 2.8 times only, and the frequency of mycorrhizal structures decreased only in SGE roots. Inoculation decreased shoot Cd concentration and increased seed Cd concentration of both pea genotypes, but had little effect on Cd concentration of B. juncea. Inoculation also significantly increased concentration and/or accumulation of nutrients (Ca, Fe, K, Mg, Mn, N, P, S, and Zn) by Cd-treated pea plants, particularly by the SGECdt mutant. Shoot Cd concentration of SGECdt was twice that of SGE, and the inoculated SGECdt had approximately similar Cd accumulation capacity as compared with B. juncea. Thus, plant–microbe systems based on Cd-tolerant micro-symbionts and plant genotypes offer considerable opportunities to increase plant HM tolerance and accumulation

Water relations responses of the pea

Mercury (Hg) is one of the most toxic heavy metals and has multiple impacts on plant growth and physiology, including disturbances of plant water status. The impact of Hg on water relations was assessed by exposing the unique Hg-sensitive pea (Pisum sativum L.) mutant SGECdt and its wild-type (WT) line SGE in hydroponic culture. When the plants were grown in the presence of 1 or 2 µM HgCl2 for 11 days, the SGECdt mutant had lower whole plant transpiration rate and increased leaf temperature, indicating stomatal closure. Shoot removal of Hg-untreated plants resulted in greater root-pressure induced xylem sap flow in the SGECdt mutant than WT plants. Treating these plants with 50 µM HgCl2 (an inhibitor of aquaporins) for 1 h decreased xylem sap flow of both genotypes by about 5 times and eliminated differences between WT and mutant. Adding 1 mM dithiothreitol (the reducing thiol reagent used for opening aquaporins) to the nutrient solution of Hg-treated plants partially restored xylem sap flow in SGECdt roots only, suggesting genotypic differences in aquaporin function. Thus root water uptake is important in mediating sensitivity of SGECdt to toxic Hg

Rhizosphere bacteria containing 1-aminocyclopropane-1-carboxylate deaminase increase growth and photosynthesis of pea plants under salt stress by limiting Na+ accumulation

Although plant salt tolerance has been improved by soil inoculation with rhizobacteria containing the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase (which metabolises ACC, the immediate precursor of the phytohormone ethylene), it is not always clear whether ion homeostasis and plant water relations are affected. When pea (Pisum sativum L. cv. Alderman) was grown with 70 and 130 mM NaCl, the ACC-deaminase containing rhizobacterium Variovorax paradoxus 5C-2 increased total biomass by 25 and 54% respectively. Nutrient flow modelling showed that V. paradoxus 5C-2 increased K uptake and root to shoot K flow, but decreased Na flow and increased Na deposition in roots. Thus, shoot K+ : Na+ ratio increased following V. paradoxus 5C-2 inoculation. At 70 and 130 mM NaCl, rhizobacterial inoculation decreased stomatal resistance by 14 and 31% and decreased xylem balancing pressure by 7 and 21% respectively. Furthermore, rhizobacterial inoculation improved photosynthetic efficiency (F-v/F-m) by 12 and 19% and increased maximal electron transport rate (ETR) by 18 and 22% at 70 and 130 mM NaCl respectively. Thus V. paradoxus 5C-2 mitigates salt stress by improving water relations, ion homeostasis and photosynthesis of pea plants, and may provide an economic means of promoting growth of plants exposed to salt stress

The cadmium-tolerant pea (Pisum sativum L.) mutant SGECd(t) is more sensitive to mercury:assessing plant water relations

Heavy metals have multiple effects on plant growth and physiology, including perturbation of plant water status. These effects were assessed by exposing the unique Cd-tolerant and Cd-accumulating pea (Pisum sativum L.) mutant SGECd(t) and its wild-type (WT) line SGE to either cadmium (1, 4 mu M CdCl2) or mercury (0.5, 1, 2 mu M HgCl2) in hydroponic culture for 12 days. When exposed to Cd, SGECd(t) accumulated more Cd in roots, xylem sap, and shoot, and had considerably more biomass than WT plants. WT plants lost circa 0.2 MPa turgor when grown in 4 mu M CdCl2, despite massive decreases in whole-plant transpiration rate and stomatal conductance. In contrast, root Hg accumulation was similar in both genotypes, but WT plants accumulated more Hg in leaves and had a higher stomatal conductance, and root and shoot biomass compared with SGECd(t). Shoot excision resulted in greater root-pressure induced xylem exudation of SGECd(t) in the absence of Cd or Hg and following Cd exposure, whereas the opposite response or no genotypic differences occurred following Hg exposure. Exposing plants that had not been treated with metal to 50 mu M CdCl2 for 1 h increased root xylem exudation of WT, whereas 50 mu M HgCl2 inhibited and eliminated genotypic differences in root xylem exudation, suggesting differences between WT and SGECd(t) plants in aquaporin function. Thus, root water transport might be involved in mechanisms of increased tolerance and accumulation of Cd in the SGECd(t) mutant. However, the lack of cross-tolerance to Cd and Hg stress in the mutant indicates metal-specific mechanisms related to plant adaptation

The rhizosphere bacterium Variovorax paradoxus 5C-2 containing ACC deaminase does not increase systemic ABA signaling in maize (Zea mays L.).

Soil inoculation with the ACC deaminase-containing rhizobacterium Variovorax paradoxus 5C-2 increased pea (Pisum sativum) growth and yield in both well watered and drying soil, with an attenuation of systemic ACC signaling likely key in the latter case.1 However, inoculated plants also had increased xylem ABA concentrations (which may also promote growth) in drying soil. Possible mediation of ABA levels by V. paradoxus 5C-2 was investigated in two experiments in which maize (Zea mays) growth was promoted. Xylem ABA concentration of both inoculated and uninoculated plants increased similarly as leaf water potential decreased. Furthermore, hormone flow modeling showed a decreased phloem flow of ABA back to the root. Thus Variovorax paradoxus 5C-2 does not intensify ABA signaling in planta

Aluminum-Immobilizing Rhizobacteria Modulate Root Exudation and Nutrient Uptake and Increase Aluminum Tolerance of Pea Mutant E107 (<i>brz</i>)

It is well known that plant-growth-promoting rhizobacteria (PGPRs) increase the tolerance of plants to abiotic stresses; however, the counteraction of Al toxicity has received little attention. The effects of specially selected Al-tolerant and Al-immobilizing microorganisms were investigated using pea cultivar Sparkle and its Al-sensitive mutant E107 (brz). The strain Cupriavidus sp. D39 was the most-efficient in the growth promotion of hydroponically grown peas treated with 80 µM AlCl3, increasing the plant biomass of Sparkle by 20% and of E107 (brz) by two-times. This strain immobilized Al in the nutrient solution and decreased its concentration in E107 (brz) roots. The mutant showed upregulated exudation of organic acids, amino acids, and sugars in the absence or presence of Al as compared with Sparkle, and in most cases, the Al treatment stimulated exudation. Bacteria utilized root exudates and more actively colonized the root surface of E107 (brz). The exudation of tryptophan and the production of IAA by Cupriavidus sp. D39 in the root zone of the Al-treated mutant were observed. Aluminum disturbed the concentrations of nutrients in plants, but inoculation with Cupriavidus sp. D39 partially restored such negative effects. Thus, the E107 (brz) mutant is a useful tool for studying the mechanisms of plant–microbe interactions, and PGPR plays an important role in protecting plants against Al toxicity

Rhizosphere bacteria containing ACC deaminase decrease root ethylene emission and improve maize root growth with localized nutrient supply

Localized nutrient supply can enhance maize root proliferation, but also increase root ethylene production. Whether engineering ethylene signalling in the rhizosphere can further enhance root growth and nutrient uptake remains unknown. Here, field and column experiments for maize (Zea mays. L) were designed as different nutrient treatments (broadcast or localized nutrient supply containing ammonium and phosphorus) with or without inoculation with rhizobacterium Variovorax paradoxus 5C-2 containing the 1-aminocyclopropane-1-carboxylate (ACC) deaminase. Rhizobacterial inoculation increased shoot biomass by 12% and root length density by 50% with localized nutrient supply. Meanwhile, localized nutrient supply increased root ethylene production by 54% compared with broadcast, and rhizobacterial inoculation prevented the increase in root ethylene. Reduced root ethylene production following V. paradoxus 5C-2 inoculation was highly associated with a greater proportion of fine root proliferation under localized nutrient supply, which may account for the increased nitrogen and phosphorus uptake. Our work sheds light on the understanding of the interactions between root and microbe through taking hormone into consideration to dissect the relationship between below ground and above ground. It is useful to explore the strategy of soil–crop management by introducing rhizosphere microorganisms to regulate plant ethylene signal and then benefit sustainable agriculture

Abscisic acid metabolizing rhizobacteria decrease ABA concentrations in planta and alter plant growth

Although endogenous phytohormones such as abscisic acid (ABA) regulate root growth, and many rhizobacteria can modulate root phytohormone status, hitherto there have been no reports of rhizobacteria mediating root ABA concentrations and growth by metabolising ABA. Using a selective ABA-supplemented medium, two bacterial strains were isolated from the rhizosphere of rice (Oryza sativa) seedlings grown in sod-podzolic soil and assigned to Rhodococcus sp. P1Y and Novosphingobium sp. P6W using partial 16S rRNA gene sequencing and phenotypic patterns by the GEN III MicroPlate test. Although strain P6W had more rapid growth in ABA-supplemented media than strain P1Y, both could utilize ABA as a sole carbon source in batch culture. When rice seeds were germinated on filter paper in association with bacteria, root ABA concentration was not affected, but shoot ABA concentration of inoculated plants decreased by 14% (strain P6W) and 22% (strain P1Y). When tomato (Solanum lycopersicum) genotypes differing in ABA biosynthesis (ABA deficient mutants flacca - flc, and notabilis - not and the wild-type cv. Ailsa Craig, WT) were grown in gnotobiotic cultures on nutrient solution agar, rhizobacterial inoculation decreased root and/or leaf ABA concentrations, depending on plant and bacteria genotypes. Strain P6W inhibited primary root elongation of all genotypes, but increased leaf biomass of WT plants. In WT plants treated with silver ions that inhibit ethylene perception, both ABA-metabolising strains significantly decreased root ABA concentration, and strain P6W decreased leaf ABA concentration. Since these changes in ABA status also occurred in plants that were not treated with silver, it suggests that ethylene was probably not involved in regulating bacteria-mediated changes in ABA concentration. Correlations between plant growth and ABA concentrations in planta suggest that ABA-metabolising rhizobacteria may stimulate growth via an ABA-dependent mechanism

Rhizosphere bacteria containing ACC deaminase decrease root ethylene emission and improve maize root growth with localized nutrient supply

Localized nutrient supply can enhance maize root proliferation, but also increase root ethylene production. Whether engineering ethylene signalling in the rhizosphere can further enhance root growth and nutrient uptake remains unknown. Here, field and column experiments for maize (Zea mays. L) were designed as different nutrient treatments (broadcast or localized nutrient supply containing ammonium and phosphorus) with or without inoculation with rhizobacterium Variovorax paradoxus 5C-2 containing the 1-aminocyclopropane-1-carboxylate (ACC) deaminase. Rhizobacterial inoculation increased shoot biomass by 12% and root length density by 50% with localized nutrient supply. Meanwhile, localized nutrient supply increased root ethylene production by 54% compared with broadcast, and rhizobacterial inoculation prevented the increase in root ethylene. Reduced root ethylene production following V. paradoxus 5C-2 inoculation was highly associated with a greater proportion of fine root proliferation under localized nutrient supply, which may account for the increased nitrogen and phosphorus uptake. Our work sheds light on the understanding of the interactions between root and microbe through taking hormone into consideration to dissect the relationship between below ground and above ground. It is useful to explore the strategy of soil–crop management by introducing rhizosphere microorganisms to regulate plant ethylene signal and then benefit sustainable agriculture

The rhizobacterium Variovorax paradoxus 5C-2, containing ACC deaminase, promotes growth and development of Arabidopsis thaliana via an ethylene-dependent pathway

Many plant-growth-promoting rhizobacteria (PGPR) associated with plant roots contain the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase and can metabolize ACC, the immediate precursor of the plant hormone ethylene, thereby decreasing plant ethylene production and increasing plant growth. However, relatively few studies have explicitly linked ethylene emission and/or action to growth promotion in these plantmicrobe interactions. This study examined effects of the PGPR Variovorax paradoxus 5C-2 containing ACC deaminase on the growth and development of Arabidopsis thaliana using wild-type (WT) plants and several ethylene-related mutants (etr1-1, ein2-1, and eto1-1). Soil inoculation with V. paradoxus 5C-2 promoted growth (leaf area and shoot biomass) of WT plants and the ethylene-overproducing mutant eto1-1, and also enhanced floral initiation of WT plants by 2.5 days. However, these effects were not seen in ethylene-insensitive mutants (etr1-1 and ein2-1) even though bacterial colonization of the root system was similar. Furthermore, V. paradoxus 5C-2 decreased ACC concentrations of rosette leaves of WT plants by 59% and foliar ethylene emission of both WT plants and eto1-1 mutants by 42 and 37%, respectively. Taken together, these results demonstrate that a fully functional ethylene signal transduction pathway is required for V. paradoxus 5C-2 to stimulate leaf growth and flowering of A. thaliana