The endophytic bacterium Sphingomonas SaMR12 alleviates Cd stress in oilseed rape through regulation of the GSH-AsA cycle and antioxidative enzymes.

BACKGROUND: Microbes isolated from hyperaccumulating plants have been reported to be effective in achieving higher phytoextraction efficiency. The plant growth-promoting bacteria (PGPB) SaMR12 from the cadmium (Cd)/zinc hyperaccumulator Sedum alfredii Hance could promote the growth of a non-host plant, oilseed rape, under Cd stress. However, the effect of SaMR12 on Brasscia juncea antioxidative response under Cd exposure was still unclear. RESULTS: A hydroponic experiment was conducted to study the effects of Sphingomonas SaMR12 on its non-host plant Brassica juncea (L.) Czern. under four different Cd treatments. The results showed that SaMR12 could colonize and aggregate in the roots and then move to the shoots. SaMR12 inoculation promoted plant growth by up to 71% in aboveground biomass and 81% in root biomass over that of the non-inoculated plants. SaMR12-inoculated plants significantly enhanced root Cd accumulation in the 10 and 20 µM Cd treatments, with 1.72- and 0.86-fold increases, respectively, over that of the non-inoculated plants. SaMR12 inoculation not only decreased shoot hydrogen peroxide (H2O2) content by up to 38% and malondialdehyde (MDA) content by up to 60% but also reduced proline content by 7-30% in shoots and 17-32% in roots compared to the levels in non-inoculated plants. Additionally, SaMR12 inoculation promoted the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX) and facilitated the relative gene expression levels of dehydroascorbate reductase (DHAR) and glutathione reductase (GR) involved in the glutathione (GSH)-ascorbic acid (AsA) cycle. CONCLUSIONS: The results demonstrated that, under Cd stress, SaMR12 inoculation could activate the antioxidative response of B. juncea by decreasing the concentrations of H2O2, MDA and proline, increasing the activities of antioxidative enzymes, and regulating the GSH-AsA cycle. These results provide a theoretical foundation for the potential application of hyperaccumulator endophytic bacteria as remediating agents to improve heavy metal tolerance within non-host plant species, which could further improve phytoextraction efficiency.

Promotion of the root development and Zn uptake of Sedum alfredii was achieved by an endophytic bacterium Sasm05.

Endophyte-assisted phytoremediation has gained increasing attention. However, the interacting mechanisms of endophytes and metal hyperaccumulators are still not clear. An endophytic bacterium Pseudomonas fluorescens Sasm05 inoculation promoted Sedum alfredii Hance rooting and root development, in which the specific root length (SRL) and average number of root tips (ART) increased to 2.09- and 3.35-fold, respectively. Sasm05 inoculation promoted plant growth, increased the chlorophyll content, and elevated Zn uptake of plant at excess Zn supply. At 200 µM Zn treatment level, Sasm05 inoculation increased plant biomass and the chlorophyll content by more than 40%, and root Zn content by 40%. Furthermore, Sasm05 inoculation upregulated the expression of the Zn transporter SaIRT1 to 3.43-fold in the roots, while another transporter SaNramp1 expression was increased to 38.66-fold in the roots and 7.53-fold in the shoots. Time course study showed the best effects of Sasm05 on plant biomass and the chlorophyll content were detected at 30 d, while for Zn content at 3 d. These results firstly provided molecular evidences of endophytic bacteria in facilitating host plant Zn uptake, which will absolutely benefit the understanding of interacting mechanisms between hyperaccumulators and their endophytes.

Chromosome doubling of Sedum alfredii Hance: A novel approach for improving phytoremediation efficiency.

Sedum alfredii Hance is a cadmium (Cd)/zinc (Zn) hyperaccumulator native to China. However, its relatively low biomass restricted the large-scale application for heavy metal contamination remediation. The chromosome set doubling of S. alfredii in vitro was achieved by 0.1%-0.2% (W/V) colchicine treatment. The plant DNA ploidy was analyzed by flow cytometry and chromosome set doubling plants (CSD) were identified based on the obvious different sharp peak. A tissue culture experiment with different Cd treated levels and a field trial with natural polluted mined soil were conducted to study the effects of chromosome doubling on plant biomass and Cd accumulation in shoots. The results suggested that S. alfredii is a mixoploid. Compared with the wild type plants (WT), CSD exhibited typical "gigas" characteristics in morphology including stem thickness, root hair production, number of leaves and size of stoma guard cell. Fresh weight and dry weight of CSD were increased to 1.62-2.03-fold and 2.26-3.25-fold of WT. And Cd content of CSD showed a 17.49%-42.82% increase and 59% increase under tissue culture and field condition, accordingly. In addition, the TF and in BCF of CSD were 2.37- and 1.59-fold of WT, respectively. These results proved that it is feasible to promote phytoextraction efficiency of S. alfredii in Cd contaminated soils through chromosomal engineering, which provides a novel approach for hyperaccumulator application in phytoremediation.

Enhanced Cd extraction of oilseed rape (Brassica napus) by plant growth-promoting bacteria isolated from Cd hyperaccumulator Sedum alfredii Hance.

Four plant growth-promoting bacteria (PGPB) were used as study materials, among them two heavy metal-tolerant rhizosphere strains SrN1 (Arthrobacter sp.) and SrN9 (Bacillus altitudinis) were isolated from rhizosphere soil, while two endophytic strains SaN1 (Bacillus megaterium) and SaMR12 (Sphingomonas) were identified from roots of the cadmium (Cd)/zinc (Zn) hyperaccumulator Sedum alfredii Hance. A pot experiment was carried out to investigate the effects of these PGPB on plant growth and Cd accumulation of oilseed rape (Brassica napus) plants grown on aged Cd-spiked soil. The results showed that the four PGPB significantly boosted oilseed rape shoot biomass production, improved soil and plant analyzer development (SPAD) value, enhanced Cd uptake of plant and Cd translocation to the leaves. By fluorescent in situ hybridization (FISH) and green fluorescent protein (GFP), we demonstrated the studied S. alfredii endophytic bacterium SaMR12 were able to colonize successfully in the B. napus roots. However, all four PGPB could increase seed Cd accumulation. Due to its potential to enhance Cd uptake by the plant and to restrict Cd accumulation in the seeds, SaMR12 was selected as the most promising microbial partner of B. napus when setting up a plant-microbe fortified remediation system.

Inoculation of plant growth promoting bacteria from hyperaccumulator facilitated non-host root development and provided promising agents for elevated phytoremediation efficiency.

Plant growth promoting bacteria (PGPB) have been reported to have the ability to promote plant growth, development and increase heavy metals (HMs) uptake. Therefore, PGPB inoculation as soil remediation agents into plants with larger biomass and potential of phytoextraction is of great importance to increase bioremediation efficiency. In this study, 12 PGPB strains isolated from a cadmium (Cd)/zinc hyperaccumulator Sedum alfredii Hance were inoculated into non-host plant Brassica juncea and their effects on plant growth and Cd uptake were determined. The results showed that inoculation of most PGPB strains promoted plant growth, boosted root development and improved chlorophyll content in the absence of Cd. Inoculation of PGPB strains promoted plant growth up to 111% in shoot and 358% in root when treated with 2 µM Cd. In addition, PGPB inoculation not only ameliorated plant root morphology including the total root length (RL), total surface area (SA), total root volume (RV) and number of root tips (RT), but also facilitated Cd uptake up to 126%. Furthermore, inoculation of PGPB strains promoted plant Cd accumulation up to 261% in shoot and up to 8.93-fold increase in root. Among all the 12 PGPB strains, Burkholdria SaMR10 and Sphingomonas SaMR12 were identified as the promising microbes for improving phytoremediation efficiency of Cd contaminated soils. These results not only provided useful findings for further investigation of interacting mechanisms between different bacterial strains and plants, but also facilitated the development of microbe-assisted phytoremediation application for HM contaminated soil.

The Effects of the Endophytic Bacterium Pseudomonas fluorescens Sasm05 and IAA on the Plant Growth and Cadmium Uptake of Sedum alfredii Hance.

Endophytic bacteria have received attention for their ability to promote plant growth and enhance phytoremediation, which may be attributed to their ability to produce indole-3-acetic acid (IAA). As a signal molecular, IAA plays a key role on the interaction of plant and its endomicrobes. However, the different effects that endophytic bacteria and IAA may have on plant growth and heavy metal uptake is not clear. In this study, the endophytic bacterium Pseudomonas fluorescens Sasm05 was isolated from the stem of the zinc (Zn)/cadmium (Cd) hyperaccumulator Sedum alfredii Hance. The effects of Sasm05 and exogenous IAA on plant growth, leaf chlorophyll concentration, leaf Mg2+-ATPase and Ca2+-ATPase activity, cadmium (Cd) uptake and accumulation as well as the expression of metal transporter genes were compared in a hydroponic experiment with 10 µM Cd. The results showed that after treatment with 1 µM IAA, the shoot biomass and chlorophyll concentration increased significantly, but the Cd uptake and accumulation by the plant was not obviously affected. Sasm05 inoculation dramatically increased plant biomass, Cd concentration, shoot chlorophyll concentration and enzyme activities, largely improved the relative expression of the three metal transporter families ZRT/IRT-like protein (ZIP), natural resistance associated macrophage protein (NRAMP) and heavy metal ATPase (HMA). Sasm05 stimulated the expression of the SaHMAs (SaHMA2, SaHMA3, and SaHMA4), which enhanced Cd root to shoot translocation, and upregulated SaZIP, especially SaIRT1, expression to increase Cd uptake. These results showed that although both exogenous IAA and Sasm05 inoculation can improve plant growth and photosynthesis, Sasm05 inoculation has a greater effect on Cd uptake and translocation, indicating that this endophytic bacterium might not only produce IAA to promote plant growth under Cd stress but also directly regulate the expression of putative key Cd uptake and transport genes to enhance Cd accumulation of plant.

The effects of endophytic bacterium SaMR12 on Sedum alfredii Hance metal ion uptake and the expression of three transporter family genes after cadmium exposure.

A hydroponic experiment was conducted to investigate the effects of an endophytic bacterium SaMR12 on Sedum alfredii Hance metal ion accumulation, chlorophyll concentration, and the expression of three metal transporter families, zinc-regulated transporters, iron-regulated transporter-like protein (ZIP); natural resistance-associated macrophage protein; and heavy metal ATPase (HMA) at different Cd treatment levels. The results showed that at relatively low Cd conditions (≤25 µM), SaMR12 demonstrated a 19.5-27.5% increase in Fe, a 46.7-90.7% increase in Zn, and a 7.9-43.7% increase in Cu content in the shoot and elevated expression of SaIRT1, SaZIP3, SaHMA2, and SaNramp3 in the shoot and SaZIP1, SaHMA2, SaNramp1, and SaNramp3 in the root. At high Cd conditions (100 and 400 µM), SaMR12 demonstrated a 16.4-18.5% increase in leaf chlorophyll concentration, a 18.9-23.2% increase in Fe, and a 15.4-17.5% increase in Mg content in the shoot and elevated expression of SaZIP3, SaNramp6, SaHMA2, and SaHMA3 in the shoot and SaZIP3, SaNarmp1, SaNarmp3, and SaNarmp6 in the root. These results indicated that SaMR12 can elevate essential metal ion uptake and regulate the expression of transport genes to promote plant growth and enhance Cd tolerance and uptake to improve Cd accumulation up to 118-130%.

Endophytic bacterium Sphingomonas SaMR12 promotes cadmium accumulation by increasing glutathione biosynthesis in Sedum alfredii Hance.

A hydroponic experiment was conducted to verify the effects of inoculation with endophytic bacteria Sphingomonas SaMR12 on root growth, cadmium (Cd) uptake, reactive oxygen species (ROS), antioxidases, glutathione (GSH) and the related gene expression of Sedum alfredii Hance under different levels of Cd such as 0, 10, 25, 100 and 400 µM. The results showed that inoculation of SaMR12 improved Cd accumulation and upregulated glutathione synthase (GS) expression, but slightly reduced malondialdehyde (MDA) concentration and alleviated Cd-induced damage in roots. However it didn't alter the activities of antioxidant enzymes. When Cd concentration exceeded 25 µM, SaMR12 increased the concentration of GSH and the expression level of GSH1. At high Cd treatment levels (100 and 400 µM), SaMR12 significantly reduced H2O2 concentration and enhanced expression level of 1-Cys peroxiredoxin PER1 and ATPS genes. These results indicate that although SaMR12 has no significant effects on antioxidases activities, it reduces H2O2 concentration by enhancing GSH concentration and relevant genes expression, and subsequently improves Cd tolerance and accumulation.

An endophytic bacterium Acinetobacter calcoaceticus Sasm3-enhanced phytoremediation of nitrate-cadmium compound polluted soil by intercropping Sedum alfredii with oilseed rape.

Intensive agricultural system with high input of fertilizer results in high agricultural output. However, excessive fertilization in intensive agricultural system has great potential to cause nitrate and heavy metal accumulation in soil, which is adverse to human health. The main objective of the present study was to observe the effects of intercropping and inoculation of endophytic bacterium Acinetobacter calcoaceticus Sasm3 on phytoremediation of combined contaminated soil in oilseed rape (Brassica napus L.). The results showed that with Sasm3 inoculation, the biomass of rape was increased by 10-20% for shoot, 64% for root, and 23-29% for seeds while the nitrate accumulation in rape was decreased by 14% in root and by 12% in shoot. The cadmium concentration in rape increased significantly with mono-inoculating treatment, whereas it decreased significantly after intercropping treatment. By denaturing gradient gel electrophoresis (DGGE) and real-time quantitative PCR analysis, the diversity of bacterial community and the number of nirS and nirK gene copies increased significantly with inoculation or/and intercropping treatment. In conclusion, the endophytic bacterium Sasm3-inoculated intercropping system not only improved the efficiency of clearing cadmium from soil without obstructing crop production, but also improved the quality of crop.

Improvement of cadmium uptake and accumulation in Sedum alfredii by endophytic bacteria Sphingomonas SaMR12: effects on plant growth and root exudates.

Inoculating endophytic bacteria was proven as a promising way to enhance phytoremediation. By a hydroponic experiment, the role of this study was to clarify the effects of inoculating endophytic bacterium Sphingomonas SaMR12 on phytoremediation, with special emphasis on changes of cadmium uptake, plant growth, root morphology, and organic acids secretion at different cadmium treated levels (0, 5, 50, and 100 µM). The results showed that SaMR12 inoculation improved the accumulation of cadmium as well as plant biomass, length of roots, number of root tips, and root surface area. Root secretion of oxalic, citric, and succinic acids was also increased after inoculated, which may alleviate the cadmium toxicity to plant or inhibit the rising trend of oxidative stress of plant. The major finding of this work suggested that in the root, SaMR12 improves cadmium bioavailability and absorption facility by increasing root-soil contact area and root organic acid secretion; and in the shoot, SaMR12 increases cadmium tolerance by alleviating oxidative stress of plant, so as to enhance the capability of cadmium extraction by plant.

The endophytic bacterium, Sphingomonas SaMR12, improves the potential for zinc phytoremediation by its host, Sedum alfredii.

The endophytic bacterium, Sphingomonas SaMR12, isolated from Sedum alfredii Hance, appears to increase plant biomass and zinc-extraction from contaminated soil; however, the mechanism by which this occurs is not clear. Here, the ability of SaMR12 to promote zinc extraction and its effects on root morphology and exudation were examined in hydroponics. Zinc treatment increased shoot biomass by 30 to 45%, and by a further 10 to 19% when combined with SaMR12 inoculation. Zinc treatment also increased zinc accumulation modestly and this too was enhanced with SaMR12. Both biomass and zinc levels increased in a dose-dependent manner with significant effects seen at 50 µM zinc and apparent saturation at 500 µM. Zinc and the endophyte also increased levels of auxin but not at 50 µM and zinc increased levels of superoxide and hydrogen peroxide but mainly at 500 µM. As for root morphology, SaMR12 increased root branching, the number of root tips, and surface area. Zinc and SaMR12 also increased the exudation of oxalic acid. For most assays the effects of the endophyte and zinc were additive, with the notable exception of SaMR12 strongly reducing the production of reactive oxygen species at 500 µM zinc. Taken together, these results suggest that the promotion of growth and zinc uptake by exposure to zinc and to SaMR12 are independent of reactive oxygen and do not involve increases in auxin.

Improved yield and Zn accumulation for rice grain by Zn fertilization and optimized water management.

Zinc (Zn) deficiency and water scarcity are major challenges in rice (Oryza sativa L.) under an intensive rice production system. This study aims to investigate the impact of water-saving management and different Zn fertilization source (ZnSO4 and Zn-EDTA) regimes on grain yield and Zn accumulation in rice grain. Different water managements, continuous flooding (CF), and alternate wetting and drying (AWD) were applied during the rice growing season. Compared with CF, the AWD regime significantly increased grain yield and Zn concentrations in both brown rice and polished rice. Grain yield of genotypes (Nipponbare and Jiaxing27), on the average, was increased by 11.4%, and grain Zn concentration by 3.9% when compared with those under a CF regime. Zn fertilization significantly increased Zn density in polished rice, with a more pronounced effect of ZnSO4 being observed as compared with Zn-EDTA, especially under an AWD regime. Decreased phytic acid content and molar ratio of phytic acid to Zn were also noted in rice grains with Zn fertilization. The above results demonstrated that water management of AWD combined with ZnSO4 fertilization was an effective agricultural practice to elevate grain yield and increase Zn accumulation and bioavailability in rice grains.

Effect of silicate supplementation on the alleviation of arsenite toxicity in 93-11 (Oryza sativa L. indica).

Chronic exposure to arsenic (As) in rice has raised many health and environmental problems. As reported, great variation exists among different rice genotypes in As uptake, translocation, and accumulation. Under hydroponic culture, we find that the Chinese wild rice (Oryza rufipogon; acc. 104624) takes up the most arsenic among tested genotypes. Of the cultivated rice, the indica cv. 93-11 has the lowest arsenic translocation factor value but accumulates the maximum concentration of arsenic followed by Nipponbare, Minghui 86, and Zhonghua 11. Higher level of arsenite concentration (50 µM) can induce extensive photosynthesis and root growth inhibition, and cause severe oxidative stress. Interestingly, external silicate (Si) supplementation has significantly increased the net photosynthetic rate, and promoted root elongation, as well as strongly ameliorated the oxidative stress by increasing the activities of antioxidant enzymes superoxide dismutase, ascorbate peroxidase, and peroxidase in roots and/or leaves of 93-11 seedlings. Notably, 1.873 mM concentration of Si considerably decreases the total As uptake and As content in roots, but significantly increases the As translocation from roots to shoots. In contrast, Si supplementation with 1.0 mM concentration significantly increases the total As uptake and As concentrations in roots and shoots of 93-11 seedlings after 50 µM arsenite treatment for 6 days.