Grass xylan structural variation suggests functional specialization and distinctive interaction with cellulose and lignin.

Xylan is the most abundant non-cellulosic polysaccharide in grass cell walls, and it has important structural roles. The name glucuronoarabinoxylan (GAX) is used to describe this variable hemicellulose. It has a linear backbone of ß-1,4-xylose (Xyl) residues that may be substituted with α-1,2-linked (4-O-methyl)-glucuronic acid (GlcA), α-1,3-linked arabinofuranose (Araf), and sometimes acetylation at the O-2 and/or O-3 positions. The role of these substitutions remains unclear, although there is increasing evidence that they affect the way xylan interacts with other cell wall components, particularly cellulose and lignin. Here, we used substitution-dependent endo-xylanase enzymes to investigate the variability of xylan substitution in grass culm cell walls. We show that there are at least three different types of xylan: (i) an arabinoxylan with evenly distributed Araf substitutions without GlcA (AXe); (ii) a glucuronoarabinoxylan with clustered GlcA modifications (GAXc); and (iii) a highly substituted glucuronoarabinoxylan (hsGAX). Immunolocalization of AXe and GAXc in Brachypodium distachyon culms revealed that these xylan types are not restricted to a few cell types but are instead widely detected in Brachypodium cell walls. We hypothesize that there are functionally specialized xylan types within the grass cell wall. The even substitutions of AXe may permit folding and binding on the surface of cellulose fibrils, whereas the more complex substitutions of the other xylans may support a role in the matrix and interaction with other cell wall components.

Roots selectively decompose litter to mine nitrogen and build new soil carbon.

Plant-microbe interactions in the rhizosphere shape carbon and nitrogen cycling in soil organic matter (SOM). However, there is conflicting evidence on whether these interactions lead to a net loss or increase of SOM. In part, this conflict is driven by uncertainty in how living roots and microbes alter SOM formation or loss in the field. To address these uncertainties, we traced the fate of isotopically labelled litter into SOM using root and fungal ingrowth cores incubated in a Miscanthus x giganteus field. Roots stimulated litter decomposition, but balanced this loss by transferring carbon into aggregate associated SOM. Further, roots selectively mobilized nitrogen from litter without additional carbon release. Overall, our findings suggest that roots mine litter nitrogen and protect soil carbon.

Co-culture between Miscanthus x giganteus and Trifolium repens L. to enhance microbial activity, biomass and density in a PAH contaminated technosol.

Phytoremediation is a biological soil remediation technique using plants and their associated microorganisms to clean-up contaminated soils and improve soils' quality. We tested whether a co-culture between Miscanthus x giganteus (MxG) and Trifolium repens L. would enhance the soil biological quality. The objective was to determine the influence of MxG in mono- and in co-culture with white clover on the soil microbial activity, biomass and density. MxG was tested in mono- and in co-culture with white clover in a mesocosm over 148 days. The microbial respiration (CO2 production), the microbial biomass and the microbial density of the technosol were measured. Results showed that MxG induced an increase in microbial activity in the technosol compared to the non-planted condition with the co-culture having a greater impact. Regarding the bacterial density, MxG in mono- and in co-culture significantly increased the 16S rDNA gene copy number. The co-culture increased the microbial biomass, the fungal density and stimulated the degrading bacterial population, contrary to the monoculture and the non-planted condition. We can conclude the co-culture between MxG and white clover was more interesting than MxG monoculture in regards to the technosol biological quality and its potential for PAH remediation improvement.

Our precedent results have shown the benefits of using Miscanthus x giganteus in association with Trifolium repens L. to improve polycyclic aromatic hydrocarbons dissipation and decrease soil ecotoxicity compared to monocultures. In this study we focused on the plant species' influence on the soil's biological quality to improve MxG biomass productivity in the long term and phytoremediation. Many bioindicators were used such as microbial activity, microbial biomass as well as bacteria, fungi and PAH-degrading bacteria density.We showed it was more beneficial to use co-culture instead of MxG monoculture to improve biological technosol quality and in particular microbial activity and biomass as well as fungi and PAH-degrading bacteria density.

Stress reduction with co-culture of Miscanthus x giganteus and Pelargonium x hortorum in a pb contaminated soil to improve biomass production.

The industrial past of most regions in Lorraine and the intensification of activities on soils has increased the number of polluted sites. To rehabilitate these areas, several methods can be employed. In this study, co-culture of Miscanthus x giganteus and Pelargonium x hortorum was used to clean up a soil mainly contaminated by metallic elements including lead. The use of ornamental plants has been little studied, even if these species can be used to rehabilitate a site while improving its esthetics. At the end of the experiment, Pb concentrations were measured in the soil and plants. Furthermore, phytohormones were also measured to evaluate the defense mechanisms of the plants in front of pollutants. The results showed a reduction in Pb concentrations following the phytoremediation process implemented and that PxH was able to extract Pb from the soil. Results showed that co-culture was not beneficial to the development of MxG. Concerning the molecules synthesized by the plants under stress conditions, only salicin was found in MxG roots and aerial parts in particular for plants grown in individual culture. According to the results obtained, it seems that MxG is able to make compromises between the synthesis of protective molecules and its development.

This study will provide a better understanding of plant defence mechanisms in a phytoremediation context. It will allow us to better understand how Miscanthus x giganteus reacts and develops when confronted with stress induced by soil pollution or co-cultivation with Pelargonium x hortorum by involving phytohormones such as salicin. Furthermore, this study will enable us to better manage industrial wasteland by choosing the most appropriate plants.

MsHDZ23, a Novel Miscanthus HD-ZIP Transcription Factor, Participates in Tolerance to Multiple Abiotic Stresses.

The homeodomain-leucine zipper (HD-ZIP) transcription factors, representing one of the largest plant-specific superfamilies, play important roles in the response to various abiotic stresses. However, the functional roles of HD-ZIPs in abiotic stress tolerance and the underlying mechanisms remain relatively limited in Miscanthus sinensis. In this study, we isolated an HD-ZIP TF gene, MsHDZ23, from Miscanthus and ectopically expressed it in Arabidopsis. Transcriptome and promoter analyses revealed that MsHDZ23 responded to salt, alkali, and drought treatments. The overexpression (OE) of MsHDZ23 in Arabidopsis conferred higher tolerance to salt and alkali stresses compared to wild-type (WT) plants. Moreover, MsHDZ23 was able to restore the hb7 mutant, the ortholog of MsHDZ23 in Arabidopsis, to the WT phenotype. Furthermore, MsHDZ23-OE lines exhibited significantly enhanced drought stress tolerance, as evidenced by higher survival rates and lower water loss rates compared to WT. The improved drought tolerance may be attributed to the significantly smaller stomatal aperture in MsHDZ23-OE lines compared to WT. Furthermore, the accumulation of the malondialdehyde (MDA) under abiotic stresses was significantly decreased, accompanied by dramatically enhanced activities in several antioxidant enzymes, including superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) in the transgenic plants. Collectively, these results demonstrate that MsHDZ23 functions as a multifunctional transcription factor in enhancing plant resistance to abiotic stresses.

Photosynthetic variation and detoxification strategies based on cadmium uptake, non-protein thiols, and secondary metabolites in Miscanthus sacchariflorus under cadmium exposure.

Miscanthus sacchariflorus is previously demonstrated to be a potential candidate for remediation of cadmium (Cd) pollution. To explore its resistance strategy to Cd, a hydroponic experiment was conducted to determine the variations of photosynthetic activity in leaves and physiological response in roots of this plant. Results showed that the root of M. sacchariflorus was the primary location for Cd accumulation. The bioconcentration factor in the roots and rhizomes was >1, and the translocation factor from underground to aboveground was <1. Throughout the experimental period, treatment with 0.06 mM Cd2+ did not significantly alter the contents of chlorophyll a, chlorophyll b, or carotenoid. By contrast, treatment with 0.15 and 0.30 mM Cd2+ decreased the contents of chlorophyll a, chlorophyll b, and carotenoid; caused the deformation of the chlorophyll fluorescence transient curve; reduced the photochemical efficiency of photosystem II; and increased the contents of non-protein thiols, total flavone, and total phenol. These results indicate that M. sacchariflorus has good adaptability to 0.06 mM Cd2+. Moreover, the accumulation of the non-protein thiols, total flavone, and total phenol in roots may promote the chelation of Cd2+, thus alleviating Cd toxicity. This study provides theoretical support for using M. sacchariflorus to remediate Cd-polluted wetlands.

Application of Miscanthus substrates in the cultivation of Ganoderma lingzhi.

Ganoderma lingzhi is a traditional Chinese medicine that has been used to improve health and longevity for thousands of years. It is usually cultivated on hardwood log- or sawdust-based formulations. Conversely, in this study, we used Miscanthus sacchariflorus (MSF), M. floridulus, and M. sinensis (MSS), fast-growing perennial grasses widely distributed in China, for G. lingzhi cultivation. Mycelial growth rate, activities of lignin-degrading enzymes on colonized mushroom substrates, and expression levels of CAZymes and laccase genes based on different substrates were analyzed. Total triterpenoids, sterols, and polysaccharides content of fruiting bodies obtained from different substrates were investigated. The activities of laccase and manganese peroxidase in mycelia increased in the MSF- and MSS-based formulations compared with that in the sawdust-based formulation. The results of mycelial growth- and cultivation-related experiments showed that the Miscanthus substrates could be used as the substrates for cultivating G. lingzhi. The content of active ingredients, namely triterpenoids, sterols, and polysaccharides, in fruiting bodies cultivated on the Miscanthus substrates did not decrease compared with those in substrate obtained from the sawdust-based formulation. Therefore, the present study provides alternative substrates for the cultivation of G. lingzhi, and a reference for better utilization of inexpensive substrate in future.

Effects of hydrogen sulfide on the growth and physiological characteristics of Miscanthus sacchariflorus seedlings under cadmium stress.

As a gas signal molecule, hydrogen sulfide (H2S) can participate in many physiological and biochemical processes such as seed germination and photosynthesis regulation. In order to explore the regulatory effect of H2S on the growth of Miscanthus sacchariflorus under Cd stress and to provide sufficient theoretical basis for the complex action of H2S and energy plants to remediate soil pollution. In this experiment, the effects of different concentrations of H2S (10, 25, 50, 100, 300, 400, 500 µmol·L-1 (µM)) pretreatment on the growth index, lipid peroxidation degree, chlorophyll (Chl) content, osmoregulation substance content, antioxidant enzyme activity and non-enzymatic antioxidant content of M. sacchariflorus under Cd stress (50 µM) were studied. The results showed that under Cd stress, the reactive oxygen species (ROS) content in the body of M. sacchariflorus was unbalanced, and the growth were severely inhibited, the activities of antioxidant enzymes, such as catalase (CAT) and superoxide dismutase (SOD) significantly decreased, and the content of osmoregulation substance, ascorbic acid (AsA) and glutathione (GSH) significantly increased. With the increase of H2S concentration, its effect on resisting Cd stress can be shown as "low concentration promotes, high concentration inhibits". When the concentration of H2S ≤ 300 µM, although there was no significant difference in Cd content compared with Cd treatment alone, it can regulate the activities of peroxidase (POD), SOD, glutathione reductase (GR) and monodehydroascorbate reductase (MDHAR), increase the content of osmoregulation substances, oxidized glutathione (GSSG), and the transformation rate of AsA and dehydroascorbic acid (DHA) to reduce the oxidative damage and improve the growth and photosynthetic indicators of plants; when the concentration of H2S ≥ 400 µM, Cd content in the ground and root decreased significantly, but the transport factor increased significantly, the growth status of M. sacchariflorus were more severely inhibited by the combined stress of H2S and Cd. In this experiment, it was found that the concentration of H2S pretreatment ≤ 300 µM could regulate the growth of M. sacchariflorus under Cd stress to normal level, and when the treatment concentration was 50 µM, the effect was the best. It will provide a new idea for the treatment of contaminated soil by energy plants.

Recovery of microelements from municipal sewage sludge by reed canary grass and giant miscanthus.

The aim of the study was to evaluate the content, uptake, and utilization of Al, Mn, Fe, Co, and Mo from municipal sewage sludge by the reed canary grass (RCG) and the giant miscanthus (MG). The impact of sewage sludge on the mass ratios between microelements contained in the plant biomass was analyzed as well. The long-term field experiment consisted in application of four levels of fertilization with sewage sludge: 10, 20, 40, and 60 Mg DM·ha-1. A non-fertilized control object (0 Mg DM·ha-1) was used as well. The miscanthus biomass was characterized by higher content of Al and Mn, whereas higher levels of Fe, Co, and Mo were determined in the reed canary grass biomass. As in the case of the element content, the giant miscanthus was characterized by highest level of Al and Mn uptake, while the highest uptake of Fe, Co, and Mo was exhibited by the reed canary grass. In the group of the analyzed microelements, the giant miscanthus showed the highest the recovery of Mo (47%) followed by Mn (39%), Fe (35%), and Co (15%), and the lowest uptake was determined in the case of Al (5%).

In addition to the use of their biomass for the production of renewable energy, energy crops (reed canary grass, giant miscanthus) can be used to recover microelements from municipal sewage sludge. Energy crop biomass can therefore be a potential source of microelements in animal feed. Attention was paid to the mass ratios of Fe:Mn, Fe:Al, Fe:Co, Fe:Mo, Mn:Al, Mn:Co, Mn:Mo, and Co:Mo in the biomass yield of the species used.

Recent Advances in Miscanthus Macromolecule Conversion: A Brief Overview.

Miscanthus is a valuable renewable feedstock and has a significant potential for the manufacture of diverse biotechnology products based on macromolecules such as cellulose, hemicelluloses and lignin. Herein, we overviewed the state-of-the art of research on the conversion of miscanthus polymers into biotechnology products comprising low-molecular compounds and macromolecules: bioethanol, biogas, bacterial cellulose, enzymes (cellulases, laccases), lactic acid, lipids, fumaric acid and polyhydroxyalkanoates. The present review aims to assess the potential of converting miscanthus polymers in order to develop sustainable technologies.

Biosynthesis of Bacterial Nanocellulose from Low-Cost Cellulosic Feedstocks: Effect of Microbial Producer.

Biodegradable bacterial nanocellulose (BNC) is a highly in-demand but expensive polymer, and the reduction of its production cost is an important task. The present study aimed to biosynthesize BNC on biologically high-quality hydrolyzate media prepared from miscanthus and oat hulls, and to explore the properties of the resultant BNC depending on the microbial producer used. In this study, three microbial producers were utilized for the biosynthesis of BNC: individual strains Komagataeibacter xylinus B-12429 and Komagataeibacter xylinus B-12431, and symbiotic Medusomyces gisevii Sa-12. The use of symbiotic Medusomyces gisevii Sa-12 was found to have technological benefits: nutrient media require no mineral salts or growth factors, and pasteurization is sufficient for the nutrient medium instead of sterilization. The yield of BNCs produced by the symbiotic culture turned out to be 44-65% higher than that for the individual strains. The physicochemical properties of BNC, such as nanofibril width, degree of polymerization, elastic modulus, Iα allomorph content and crystallinity index, are most notably dependent on the microbial producer type rather than the nutrient medium composition. This is the first study in which we investigated the biosynthesis of BNC on hydrolyzate media prepared from miscanthus and oat hulls under the same conditions but using different microbial producers, and showed that it is advisable to use the symbiotic culture. The choice of a microbial producer is grounded on the yield, production process simplification and properties. The BNC production from technical raw materials would cover considerable demands of BNC for technical purposes without competing with food resources.

Miscanthus x giganteus stress tolerance and phytoremediation capacities in highly diesel contaminated soils.

Second generation biofuel crop Miscanthus x giganteus (Mxg) was studied as a candidate for petroleum hydrocarbons (PHs) contaminated soil phytomanagement. The soil was polluted by diesel in wide concentration gradient up to 50 g⋅kg-1 in an ex-situ pot experiment. The contaminated soil/plant interactions were investigated using plant biometric and physiological parameters, soil physico-chemical and microbial community's characteristics. The plant parameters and chlorophyll fluorescence indicators showed an inhibitory effect of diesel contamination; however much lower than expected from previously published results. Moreover, lower PHs concentrations (5 and 10 g⋅kg-1) resulted in positive reinforcement of electron transport as a result of hormesis effect. The soil pH did not change significantly during the vegetation season. The decrease of total organic carbon was significantly lower in planted pots. Soil respiration and dehydrogenases activity increased with the increasing contamination indicating ongoing PHs biodegradation. In addition, microbial biomass estimated by phospholipid fatty acids increased only at higher PHs concentrations. Higher dehydrogenases values were obtained in planted pots compared to unplanted. PHs degradation followed the first-order kinetics and for the middle range of contamination (10-40 g⋅kg-1) significantly lower PHs half-lives were determined in planted than unplanted soil pointing on phytoremediation. Diesel degradation was in range 35-70 % according to pot variant. Results confirmed the potential of Mxg for diesel contaminated soils phytomanagement mainly in PHs concentrations up to 20 g⋅kg-1 where phytoremediation was proved, and biomass yield was reduced only by 29 %.

Investigation of lactic acid production by pressurized liquid hot water from cultivated Miscanthus × giganteus.

The production of lactic acid, a polylactic acid monomer from energy crop Miscanthus × giganteus lignocellulosic biomass cultivated in Izmir was investigated. Liquid hot water (LHW) pretreatment was carried out at a temperature range of 140-200 °C, pressure 100 to 200 bar and reaction time of 15-45 min at a fixed flow rate of 2 mL/min using D-optimal experimental plan. The optimum conditions were elicited as 140 °C, 100 bar and 45 minutes, yielding the highest reducing sugar content of 77.32 mg/g, whereas 1.25 mg/mL arabinose and 1.35 mg/mL xylose as monomeric sugars. Subsequently, the enzymatic hydrolysis was applied to the solid fraction. The optimum conditions for enzymatic hydrolysis were determined as 5% (w/v) solid/liquid ratio, 20 FPU/mL enzyme loading and 72 hours, revealing the highest amount of reducing sugar as 200 mg/mL. LHW hydrolysate was used as a production medium for lactic acid manufacturing in submerged fermentation by Rhizopus oryzae. The maximum lactic acid content was found to be 6.8 g/L at 24 hours, whereas the lactic acid yield was 0.28 g/L.h. The sequential design of LHW, followed by enzymatic hydrolysis and submerged lactic acid fermentation can be utilized in industry, contributing to the bioeconomy.

CROPSR: an automated platform for complex genome-wide CRISPR gRNA design and validation.

BACKGROUND: CRISPR/Cas9 technology has become an important tool to generate targeted, highly specific genome mutations. The technology has great potential for crop improvement, as crop genomes are tailored to optimize specific traits over generations of breeding. Many crops have highly complex and polyploid genomes, particularly those used for bioenergy or bioproducts. The majority of tools currently available for designing and evaluating gRNAs for CRISPR experiments were developed based on mammalian genomes that do not share the characteristics or design criteria for crop genomes. RESULTS: We have developed an open source tool for genome-wide design and evaluation of gRNA sequences for CRISPR experiments, CROPSR. The genome-wide approach provides a significant decrease in the time required to design a CRISPR experiment, including validation through PCR, at the expense of an overhead compute time required once per genome, at the first run. To better cater to the needs of crop geneticists, restrictions imposed by other packages on design and evaluation of gRNA sequences were lifted. A new machine learning model was developed to provide scores while avoiding situations in which the currently available tools sometimes failed to provide guides for repetitive, A/T-rich genomic regions. We show that our gRNA scoring model provides a significant increase in prediction accuracy over existing tools, even in non-crop genomes. CONCLUSIONS: CROPSR provides the scientific community with new methods and a new workflow for performing CRISPR/Cas9 knockout experiments. CROPSR reduces the challenges of working in crops, and helps speed gRNA sequence design, evaluation and validation. We hope that the new software will accelerate discovery and reduce the number of failed experiments.

Investigation of genetic relationships within three Miscanthus species using SNP markers identified with SLAF-seq.

BACKGROUND: Miscanthus, which is a leading dedicated-energy grass in Europe and in parts of Asia, is expected to play a key role in the development of the future bioeconomy. However, due to its complex genetic background, it is difficult to investigate phylogenetic relationships in this genus. Here, we investigated 50 Miscanthus germplasms: 1 female parent (M. lutarioriparius), 30 candidate male parents (M. lutarioriparius, M. sinensis, and M. sacchariflorus), and 19 offspring. We used high-throughput Specific-Locus Amplified Fragment sequencing (SLAF-seq) to identify informative single nucleotide polymorphisms (SNPs) in all germplasms. RESULTS: We identified 257,889 SLAF tags, of which 87,162 were polymorphic. Each tag was 264-364 bp long. The obtained 724,773 population SNPs were used to investigate genetic relationships within three species of Miscanthus. We constructed a phylogenetic tree of the 50 germplasms using the obtained SNPs and grouped them into two clades: one clade comprised of M. sinensis alone and the other one included the offspring, M. lutarioriparius, and M. sacchariflorus. Genetic cluster analysis had revealed that M. lutarioriparius germplasm C3 was the most likely male parent of the offspring. CONCLUSIONS: As a high-throughput sequencing method, SLAF-seq can be used to identify informative SNPs in Miscanthus germplasms and to rapidly characterize genetic relationships within this genus. Our results will support the development of breeding programs with the focus on utilizing Miscanthus cultivars with elite biomass- or fiber-production potential for the developing bioeconomy.

Reference gene selection for quantitative RT-PCR in Miscanthus sacchariflorus under abiotic stress conditions.

BACKGROUND: Reference genes are necessary for quantitative real-time PCR (qRT-PCR) analysis and their stability can directly influence the accuracy of gene expression result. Miscanthus sacchariflorus, a perennial C4 grass that serves as promising biofuel plant for temperate climates, has not been explored for the identification of stable reference genes yet. MATERIALS AND METHODS: Nine potential reference genes (ACT, EF1a, FBOX, GAPDH, PP2A, SAND, TIP41, TUB and UBC) of M. sacchariflorus under different abiotic (salinity, drought and cadmium) stresses, as well as in two tissues (roots and leaves) were evaluated. The expression stability of these genes were analyzed by four commonly used software programs (geNorm, NormFinder, BestKeeper, ΔCt method and RefFinder). RESULTS: Our results found that FBOX and SAND are the most stable genes among all tested samples. FBOX and EF1a are suitable for gene expression normalization of cadmium-treated samples and salinity-treated leaves. FBOX and PP2A are appropriate reference genes for salt-stressed roots and PEG-treated leaves. The traditional reference gene ACT and GAPDH exhibited the most variable pattern, which would not be recommended for qRT-PCR analysis under different abiotic stresses. Furthermore, the expression levels of PIP2, NHX1 and MT2a under drought, salt and cadmium treatment were detected with above reference genes. CONCLUSIONS: This work identified the appropriate reference genes for qRT-PCR in M. sacchariflorus and FBOX was recommended to be effective internal control for gene expression normalization in M. sacchariflorus in response to different abiotic stresses.

Comparison of the effects of different pretreatments on the structure and enzymatic hydrolysis of Miscanthus.

Miscanthus is regarded as a desired bioenergy crop with enormous lignocellulose residues for biofuels and other chemical products. In this study, the effect of different pretreatments (including microwave, NaOH, CaO, and microwave + NaOH/CaO) on sugar yields was investigated, leading to largely varied hexose yields at 4.0-73.4% (% cellulose) released from enzymatic hydrolysis of pretreated Miscanthus residues. Among them, the highest yield of 73.4% for hexoses was obtained from 12% NaOH (w/v) solution pretreatment, whereas 1% CaO (w/w) and microwave pretreatment resulted in a lower hexose yield than the control (without pretreatment). The sugar yield from microwave followed with 1% NaOH pretreatment was 4.3 times higher than that of microwave followed with 1% CaO. However, the enzymatic hydrolysis efficiencies of the sample were 15.2% and 58.5% under microwave pretreatment followed by 12% NaOH or 12.5% CaO, respectively, which were lower than those of the same concentration of alkali (NaOH and CaO) pretreatments. To investigate the mechanism of varied enzymatic saccharification under different pretreatments, the changes in the surface structure and porosity of the Miscanthus-pretreated lignocelluses were studied by means of Fourier transform infrared, Congo red staining, and scanning electron microscopy analysis. The results show that the different pretreatments destroy the cell wall cladding structure and reduce the bonding force between cellulose, hemicellulose, and lignin to different degrees, therefore increasing the accessibility of cellulose and enhancing cellulose digestion.

Heteroauxin-producing bacteria enhance the plant growth and lead uptake of Miscanthus floridulus (Lab.).

Soil lead (Pb) contamination has caused severe environmental threats and is in urgent need of remediation. This study was aimed to explore the feasibility of using the Miscanthus-microbe combination to reduce Pb pollution in the farmland surrounding a lead-zinc mining area. We have screened three heteroauxin (IAA)-producing microbes (Lelliottia jeotgali MR2, Klebsiella michiganensis TS8, and Klebsiella michiganensis ZR1) with high Pb tolerance. The IAA-producing ability of the mixed-species was stronger than that of the single bacterium. In pot experiments, the mixed-species of MR2-ZR1 and MR2-TS8 had better performance in enhancing the weight of Miscanthus grass (increased by 22.2-53.6% compared to the control group without inoculating microbes). The remediation efficiency of Pb was significantly higher in the MR2 (30.79%), MR2-TS8 (24.96%), and TS8-ZR1 (21.10%) groups than that in the control group (6.75%). We speculated that MR2 and mixed species of MR2-TS8 and TS8-ZR1 could promote the percentages of activated Pb fractions in soils and increase the Pb uptake of M. floridulus (Lab.). These results implied that the MR2-TS8 mixed-species might be selected as the effective microbial agent to simultaneously enhance the remediation efficiency of Pb-contaminated soils and the biomass of M. floridulus (Lab.).

This study investigated the effects of single and mixed IAA-producing bacteria on the phytoremediation of Pb-contaminated soils and the growth of energy crop-Miscanthus floridulus (Lab.). The results showed that the bacterial combination of MR2-TS8 may be most suitable as a microbial agent to simultaneously enhance the remediation efficiency of Pb in soils and the biomass of M. floridulus (Lab.) compared to the single strain. This study significantly contributes to offering a potential and effective strategy for Pb remediation of farmland around mine areas.

Genome-Wide Characterization and Expression Profiling of the GRAS Gene Family in Salt and Alkali Stresses in Miscanthus sinensis.

The GRAS family genes encode plant-specific transcription factors that play important roles in a diverse range of developmental processes and abiotic stress responses. However, the information of GRAS gene family in the bioenergy crop Miscanthus has not been available. Here, we report the genome-wide identification of GRAS gene family in Micanthus sinensis. A total of 123 MsGRAS genes were identified, which were divided into ten subfamilies based on the phylogenetic analysis. The co-linearity analysis revealed that 59 MsGRAS genes experienced segmental duplication, forming 35 paralogous pairs. The expression of six MsGRAS genes in responding to salt, alkali, and mixed salt-alkali stresses was analyzed by transcriptome and real-time quantitative PCR (RT-qPCR) assays. Furthermore, the role of MsGRAS60 in salt and alkali stress response was characterized in transgenic Arabidopsis. The MsGRAS60 overexpression lines exhibited hyposensitivity to abscisic acid (ABA) treatment and resulted in compromised tolerance to salt and alkali stresses, suggesting that MsGRAS60 is a negative regulator of salt and alkali tolerance via an ABA-dependent signaling pathway. The salt and alkali stress-inducible MsGRAS genes identified serve as candidates for the improvement of abiotic stress tolerance in Miscanthus.

Physiological and biochemical characteristic of Miscanthus × giganteus grown in heavy metal - oil sludge co-contaminated soil.

The effect of oil sludge and zinc, present in soil both separately and as a mixture on the physiological and biochemical parameters of Miscanthus × giganteus plant was examined in a pot experiment. The opposite effect of pollutants on the accumulation of plant biomass was established: in comparison with uncontaminated control the oil sludge increased, and Zn reduced the root and shoot biomass. Oil sludge had an inhibitory effect on the plant photosynthetic apparatus, which intensified in the presence of Zn. The specific antioxidant response of M. × giganteus to the presence of both pollutants was a marked increase in the activity of superoxide dismutase (mostly owing to oil sludge) and glutathione-S-transferase (mostly owing to zinc) in the shoots. The participation of glutathione-S-transferase in the detoxification of both the organic and the inorganic pollutants was assumed. Zn inhibited the activity of laccase-like oxidase, whereas oil sludge promoted laccase and ascorbate oxidase activities. This finding suggests that these enzymes play a part in the oxidative detoxification of the organic pollutаnt. With both pollutants used jointly, Zn accumulation in the roots increased 6-fold, leading to increase in the efficiency of soil clean-up from the metal. In turn, Zn did not significantly affect the soil clean-up from oil sludge. This study shows for the first time the effect of co-contamination of soil with oil sludge and Zn on the physiological and biochemical characteristics of the bioenergetic plant M. × giganteus. The data obtained are important for understanding the mechanisms of phytoremediation with this plant.