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Metal-resistant bacteria can survive exposure to high metal concentrations without any negative impact on their growth. Biosorption is considered to be one of the more effective detoxification mechanisms acting in most bacteria. However, molecular-scale characterization of metal biosorption by wild metal-resistant bacteria has been limited. In this study, the Pb(II) biosorption behavior of Serratia Se1998 isolated from Pb-contaminated soil was investigated through macroscopic and microscopic techniques. A four discrete site non-electrostatic model fit the potentiometric titration data best, suggesting a distribution of phosphodiester, carboxyl, phosphoryl, and amino or hydroxyl groups on the cell surface. The presence of these functional groups was verified by the attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy, which also indicated that carboxyl and phosphoryl sites participated in Pb(II) binding simultaneously. The negative enthalpy (−9.11 kJ mol−1) and large positive entropy (81.52 J mol−1 K−1) of Pb(II) binding with the bacteria suggested the formation of inner-sphere complexes by an exothermic process. X-ray absorption fine structure (XAFS) analysis further indicated monodentate inner-sphere binding of Pb(II) through formation of C−O−Pb and P−O−Pb bonds. We inferred that C−O−Pb bonds formed on the flagellar surfaces, establishing a self-protective barrier against exterior metal stressors. This study has important implications for an improved understanding of metal-resistance mechanisms in wild bacteria and provides guidance for the construction of genetically engineered bacteria for remediation purposes.

Rhizobia and arbuscular mycorrhiza fungi (AMF) are important symbiotic microbes that are advantageous to plants growing in metal-contaminated soil. However, it remains unclear how inoculated microbes affect rhizosphere microbial communities or whether subsequent changes in rhizosphere microbiomes contribute to improving plant resistance under metal stress. This study investigated the effects of rhizobia and AMF inoculation on alfalfa resistance to Cd stress. The response of rhizosphere microbial communities to inoculation and its role in increasing alfalfa' ability to cope with stress were further analyzed using high-throughput sequencing of 16S and ITS rRNA genes. Results showed that single rhizobia or AMF inoculation significantly improved alfalfa resistance to Cd stress, while their co-inoculation resulted in the greatest overall improvement. Improved resistance was reflected by the significant mitigation of Cd-induced lipid peroxidation and reactive oxygen species (ROS) stress caused by increases in antioxidant enzyme activities along with co-inoculation. Furthermore, co-inoculation significantly altered the rhizosphere microbial community structure by decreasing fungal community diversity and increasing bacterial community diversity. Results of partial least squares path modeling (PLS-PM) and variation partitioning analysis (VPA) showed that the rhizosphere bacterial community predominated over the fungal community with respected to improvements in resistance to Cd stress under the co-inoculation treatments. This improvement was specifically seen in the enrichment of certain key bacterial taxa (including Proteobacteria, Actinobacteria, Acidobacteria, and Chloroflexi) induced by the rhizobia and AMF co-inoculation, enhancing alfalfa' ability to uptake rhizosphere nutrients and reduce its release of photosynthetically-derived carbon (C) into soil. Our findings revealed that the co-inoculation of multiple symbiotic microbes can assist plants to effectively cope with Cd stress, providing a greater understanding of rhizosphere bacterial taxa in the microbe-induced phytomanagement.

The diversity patterns and drivers of soil microbial communities in altitudinal gradients have recently received much attention. The rhizosphere is a focus of soil microbial communities, but the patterns and drivers of these communities have rarely been studied in alpine ecosystems. We used high-throughput Illumina sequencing to examine the community variations of bacteria, archaea and fungi between the rhizosphere and bulk soil along an altitudinal gradient in an Abies fabri (Mast.) community on Mount Gongga of the eastern Tibetan Plateau. Microbial alpha diversity and community structure varied significantly with altitude but not between the rhizosphere and bulk soil. Soil temperature and the carbon:nitrogen ratio were the primary drivers of the structures of the bacterial, archaeal and fungal communities, and altitude (geographic distance) contributed a small part (

Plant growth-promoting rhizobacteria (PGPR) and rhizobia are potentially advantageous in improving plant growth in heavy metal contaminated soils. However, only limited information is available in literature on the manner through which the co-inoculation of PGPR and rhizobia can potentially supply nutrients to benefit plant growth in heavy metal contaminated soil. Accordingly, this study investigated the effects of Paenibacillus mucilaginosus (PGPR) and Sinorhizobium meliloti (rhizobia) co-inoculation on soil nutrients, enzyme activities, and microbial biomass in copper (Cu) contaminated soil planted with alfalfa (Medicago sativa). Moreover, we assessed soil bacterial community structure using high-throughput Illumina sequencing of 16S rRNA genes. Results showed that PGPR and/or rhizobia inoculation improved alfalfa growth. In particular, we found that this co-inoculation approach decreased Cu accumulation (48.6%) in shoots compared to the control (uninoculated). Both partial least squares path modeling (PLS-PM) and the relative importance of regressors in the linear models identified that enzyme activities, microbial biomass, and microbial community structure in Cu contaminated soil were major controlling variables of soil nutrient availability. The co-inoculation treatment significantly increased soil carbon (C) and nitrogen (N) concentrations by increasing urease (55.6%), saccharase (29.5%), and β-glucosidase (31.4%) activities compared to the control. Furthermore, the rhizosphere microbial community structure in the co-inoculation treatment was mainly regulated by soil N concentrations (i.e., both total N and available N) while altering alpha diversity (α-diversity). The relative abundances of Firmicutes (including biomarkers of the Bacillus genus) and Acidobacteria were enriched in the co-inoculated treatment, which can potentially improve soil nutrient availability and subsequently benefit plant growth. These findings indicated that the co-inoculation of PGPR and rhizobia plays an important role in promoting plant growth in Cu contaminated soil. This is because this approach can increase soil nutrient availability by enhancing soil enzyme activities and regulating rhizosphere microbial community structure.

Metal contaminants present persistent and deleterious threats to environmental ecosystems and human health. Microorganisms can rapidly develop protective mechanisms against metal toxicity, such as metallothionein production. The identification of biological factors related to these protective mechanisms is essential for effective metal remediation. This study presents a robust pathway to rapidly locate and characterize a Pb-binding flagellin in Serratia Se1998, which can bind Pb at a 16:1 Pb: protein ratio. A column gel electrophoresis system hyphenated with inductively coupled plasma mass spectrometry (ICP MS) was constructed to efficiently separate and identify Pb-binding proteins from the whole bacterial proteome. PCR and transgenic assays were used to elucidate the exact sequences and biological function of Pb-binding proteins and heterogeneous expression of Pb-binding flagellin in E. coli could significantly enhance Pb removal from aqueous solution by approximately 45%. This method provides a benchmark procedure to rapidly identify biological factors responsible for metal biosorption. Identification of this unique Pb-binding flagellin highlights that microorganisms can survive high metal stresses due to various complex biological pathways for metal detoxification and remediation.

The effects and regulatory mechanisms of co-inoculation of plant-growth-promoting rhizobacteria (PGPRs) and rhizobium in plant-soil systems remain unclear, despite numerous reports that PGPRs or rhizobium can alleviate metal toxicity. We used the co-inoculation of the PGPR Paenibacillus mucilaginosus and the metal-resistant rhizobium Sinorhizobium meliloti for exploring the physiological and biochemical responses of the plant-soil system in metal-contaminated soil. The co-inoculation with the PGPR and rhizobium significantly increased the nutrient (N, P, and K) contents in plant tissues and promoted plant growth in soil contaminated with copper (Cu). Stress from Cu-induced reactive oxygen species and lipid peroxidation were largely attenuated by the co-inoculation by increasing the activities of antioxidant enzymes. The contents and uptake of Cu in plant tissues increased significantly in the co-inoculation treatment compared with the uninoculated control and individual inoculation treatment. Co-inoculation with PGPR and rhizobium significantly increased soil microbial biomass, enzymatic activities, total nitrogen, available phosphorus, and soil organic matter contents compared with the uninoculated control. Interestingly, co-inoculation also affected the composition of the rhizospheric microbial community, and slightly increased rhizospheric microbial diversity. These improvements of the soil fertility and biological activity also had a beneficial impact on plant growth under Cu stress. Our results suggested that alfalfa co-inoculated with PGPR and rhizobium could increase plant growth and Cu uptake in metal-contaminated soil by alleviating plant Cu stress and improving soil biochemical properties. These results indicate that the co-application of PGPR and rhizobium can have a positive effect on the biochemical responses of alfalfa-soil systems in soil contaminated by heavy metals and can provide an efficient strategy for the phytomanagement of metal-contaminated land.

Low temperature is a vital environmental factor that affects plant growth, development and crop yields. Ethylene response factor (ERF) is a plant specific transcription factor with important roles in stress adaptation. However, the regulatory mechanism on how grapevine responds to cold stress is still unclear. In this study, we isolated and characterized two ERF genes from Vitis amurensis designated as VaERF080 and VaERF087. The function of these two genes was investigated in transgenic Arabidopsis. We found that both VaERF080 and VaERF087 were induced by cold stress and ethylene. The overexpression of these genes in Arabidopsis resulted in enhanced cold tolerance. The VaERF080- and VaERF087-overexpressing (OE) Arabidopsis lines showed lower levels of malondialdehyde (MDA) content and higher activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) under cold stress compared to wild-type (WT) Arabidopsis. Nine representative cold-responsive genes, CBF1, CBF2, ICE1, ZAT12, KIN1, SIZ1, RD29A, COR15A, and COR47, were significantly upregulated in OE Arabidopsis. These results revealed that both VaERF080 and VaERF087 may improve the cold tolerance of transgenic Arabidopsis through increasing antioxidant enzyme activities and regulating the expression of cold-related genes.

Enzymes in the soil are useful for assessing heavy metal soil pollution. We analyzed the activity of a number of enzymes, including urease, protease, catalase, and alkaline phosphatase, in three types of land (farmland, woodland, and grassland) to evaluate soil pollution by heavy metals (Pb, Zn, and Cd). Our results showed that the tested soil was polluted by a combination of Pb, Zn, and Cd, but the primary pollutant was Cd. An ecological dose analysis demonstrated that urease was the most sensitive enzyme to Pb and Cd in the farmland, and catalase and phosphatase were the most sensitive enzymes to Pb, Zn, and Cd in the woodland and grassland. The ecological risk of Cd (E Cd ) was the smallest in all three types of land, suggesting that Cd was the major metal inhibiting enzyme activity. Electrical conductivity (EC) was shown to be a negative regulator, while nitrogen, phosphorus, and clay contents were positive regulators of soil enzyme activity. The total enzyme index (TEI) inhibition rates in the woodland were 22.2 and 38.6% under moderate and heavy pollution, respectively, which were lower than those of the other two types of land. Therefore, woodlands might be the optimum land use choice in relieving heavy metal pollution. Taken together, this study identified the key metal pollutant inhibiting soil enzyme activity and suitable land use patterns around typical metal mine. These results provide possible improvement strategies to the phytomanagement of metal-contaminated land around world.

Being signaling molecules, nitric oxide (NO) and hydrogen sulfide (H2S) can mediate a wide range of physiological processes caused by plant metal toxicity. Moreover, legume-rhizobium symbiosis has gained increasing attention in mitigating heavy metal stress. However, systematic regulatory mechanisms used for the exogenous application of signaling molecules to alter the resistance of legume-rhizobium symbiosis under metal stress are currently unknown. In this study, we examined the exogenous effects of sodium nitroprusside (SNP) as an NO donor additive and sodium hydrosulfide (NaHS) as a H2S donor additive on the phytotoxicity and soil quality of alfalfa (Medicago sativa)-rhizobium symbiosis in lead/cadmium (Pb/Cd)-contaminated soils. Results showed that rhizobia inoculation markedly promoted alfalfa growth by increasing chlorophyll content, fresh weight, and plant height and biomass. Compared to the inoculated rhizobia treatment alone, the addition of NO and H2S significantly reduced the bioaccumulation of Pb and Cd in alfalfa-rhizobium symbiosis, respectively, thus avoiding the phytotoxicity caused by the excessive presence of metals. The addition of signaling molecules also alleviated metal-induced phytotoxicity by increasing antioxidant enzyme activity and inhibiting the level of lipid peroxidation and reactive oxygen species (ROS) in legume-rhizobium symbiosis. Also, signaling molecules improved soil nutrient cycling, increased soil enzyme activities, and promoted rhizosphere bacterial community diversity. Both partial least squares path modeling (PLS-PM) and variation partitioning analysis (VPA) identified that using signaling molecules can improve plant growth by regulating major controlling variables (i.e., soil enzymes, soil nutrients, and microbial diversity/plant oxidative damage) in legume-rhizobium symbiosis. This study offers integrated insight that confirms that the exogenous application of signaling molecules can enhance the resistance of legume-rhizobium symbiosis under metal toxicity by regulating the biochemical response of the plant-soil system, thereby minimizing potential health risks.

Expression of VaNAC17 improved drought tolerance in transgenic Arabidopsis by upregulating stress-responsive genes, modulating JA biosynthesis, and enhancing ROS scavenging. Water deficit severely affects the growth and development of plants such as grapevine (Vitis spp.). Members of the NAC (NAM, ATAF1/2, and CUC2) transcription factor (TF) family participate in drought-stress-induced signal transduction in plants, but little is known about the roles of NAC genes in drought tolerance in grapevine. Here, we explored the role of VaNAC17 in Vitis amurensis, a cold-hardy, drought-tolerant species of grapevine. VaNAC17 was strongly induced in grapevine by drought, exogenous abscisic acid (ABA), and methyl jasmonate (MeJA). A transient expression assay in yeast indicated that VaNAC17 functions as a transcriptional activator. Notably, heterologous expression of VaNAC17 in Arabidopsis thaliana enhanced drought tolerance. VaNAC17-expressing Arabidopsis plants showed decreased reactive oxygen species (ROS) accumulation compared to wild-type plants under drought conditions. RNA-seq analysis indicated that VaNAC17 expression increased the transcription of downstream stress-responsive genes after 5 days of drought treatment, especially genes involved in jasmonic acid (JA) biosynthesis (such as LOX3, AOC1 and OPR3) and signaling (such as MYC2, JAZ1, VSP1 and CORI3) pathways. Endogenous JA levels increased in VaNAC17-OE plants under drought stress. Taken together, these results indicate that VaNAC17 plays a positive role in drought tolerance by modulating endogenous JA biosynthesis and ROS scavenging.

The utilization of forages grown on metal-contaminated soil can increase the risk of heavy metals entering the food chain and affecting human health because of elevated toxic metal concentrations. Meanwhile, hydrogen sulfide (H2S) and nitric oxide (NO) as signaling molecules are known to promote plant growth in metal-contaminated soils. However, the regulatory mechanisms of such molecules in plant physiology and soil biochemistry have not been well-documented. Hence, we investigate the role of the exogenous application of H2S and NO on alfalfa growth in lead/cadmium (Pb/Cd)-contaminated soil. Our results indicate that the signaling molecules increase the alfalfa chlorophyll and biomass content and improve alfalfa growth. Further, H2S and NO reduce the translocation and bioconcentration factors of Pb and Cd, potentially reducing the risk of heavy metals entering the food chain. These signaling molecules reduce metal-induced oxidative damage to alfalfa by mitigating reactive oxygen species accumulation and increasing antioxidant enzyme activities. Their exogenous application increases soil enzymatic activities, particularly of catalase and polyphenol oxidase, without significantly changing the composition and structure of rhizosphere bacterial communities. Interestingly, H2S addition enriches the abundance of plant-growth-promoting rhizobacteria in soil, including Nocardioides, Rhizobium, and Glycomyces. H2S is more effective than NO in improving alfalfa growth and reducing heavy-metal contamination of the food chain. These results provide new insights into the exogenous application of signaling molecules in alleviating metal-induced phytotoxicity, including an efficient strategy for the safe use of forages.

Grazing prohibition is an effective measure in improving soil stability and ecological quality. However, only a limited number of studies have been published on the dominant factors that impact soil aggregate stability and their associated effects on nutrient distribution for different size soil aggregates under long-term grazing prohibition management. In this study, we investigated variation in soil aggregate stability and nutrient distribution characteristics in semiarid grassland sites under different grazing prohibition timeframes (0 years [GP0], 11 years [GP11], 26 years [GP26], and 36 years [GP36]). Results showed that organic carbon (C) and total nitrogen (TN) concentrations in soil aggregates decreased at GP11 before progressively increasing and reaching its highest value at GP36, and the total phosphorus (TP) concentration did not change significantly. Most nutrients accumulated in macroaggregates (> 0.25 mm) under grazing prohibition, and the nutrient stoichiometry in soil aggregates increased after 26 years. Compared to the control (GP0), the mean weight diameter (MWD) value of the soil stability index increased at GP11 (21.7%) and decreased at GP26 (18.9%). Attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) also showed that the proportion of stable organic C-related functional groups (i.e., alkene-C and aromatic-C) in macroaggregates were higher at GP11 and GP36 than at GP26. Furthermore, principal component analysis (PCA), partial least squares path modeling (PLS-PM), and the relative importance of regressors all showed that glomalin-related soil proteins (GRSP) and nutrients indirectly improved aggregate stability in semiarid grassland through their influence on the GRSP accumulation potential and nutrient stoichiometry. Generally, after 26 years grazing prohibition had a positive effect on soil aggregate stability and nutrient accumulation in the semiarid grassland sites investigated for this study. Results from this study provide a theoretical basis to select appropriate grazing prohibition timeframes under grassland management initiatives to optimize ecological quality measures in semiarid regions.

Highlight VaNAC26, a member of the NAC gene family from the wild species Vitis amurensis, plays an important role in drought tolerance by positively modulating jasmonic acid synthesis and enhancing the antioxidant system., The growth and fruit quality of grapevines are widely affected by abnormal climatic conditions such as water deficits, but many of the precise mechanisms by which grapevines respond to drought stress are still largely unknown. Here, we report that VaNAC26, a member of the NAC transcription factor family, was upregulated dramatically during cold, drought and salinity treatments in Vitis amurensis, a cold and drought-hardy wild Vitis species. Heterologous overexpression of VaNAC26 enhanced drought and salt tolerance in transgenic Arabidopsis. Higher activities of antioxidant enzymes and lower concentrations of H2O2 and O2 − were found in VaNAC26-OE lines than in wild type plants under drought stress. These results indicated that scavenging by reactive oxygen species (ROS) was enhanced by VaNAC26 in transgenic lines. Microarray-based transcriptome analysis revealed that genes related to jasmonic acid (JA) synthesis and signaling were upregulated in VaNAC26-OE lines under both normal and drought conditions. VaNAC26 showed a specific binding ability on the NAC recognition sequence (NACRS) motif, which broadly exists in the promoter regions of upregulated genes in transgenic lines. Endogenous JA content significantly increased in the VaNAC26-OE lines 2 and 3. Our data suggest that VaNAC26 responds to abiotic stresses and may enhance drought tolerance by transcriptional regulation of JA synthesis in Arabidopsis.

Chelants application can increase the bioavailability of metals, subsequently limiting plant growth and reducing the efficiency of phytoremediation. Plant growth-promoting rhizobacteria (PGPRs) and rhizobium have substantial potential to improve plant growth and plant tolerance to metal stress. We evaluated the effects of co-inoculation with a PGPR strain (Paenibacillus mucilaginosus) and a Cu-resistant rhizobium strain (Sinorhizobium meliloti) on the efficiency of biodegradable chelant (S,S-ethylenediaminedisuccinic acid; EDDS) assisted phytoremediation of a Cu contaminated soil using alfalfa. The highest total Cu extraction by alfalfa was observed in the EDDS-treated soil upon co-inoculation with the PGPR and rhizobium strains, which was 1.2 times higher than that without co-inoculation. Partial least squares path modeling identified plant oxidative damage and soil microbial biomass as the key variables influencing Cu uptake by alfalfa roots. Co-inoculation significantly reduced the oxidative damage to alfalfa by mitigating the accumulation of malondialdehyde and reactive oxygen species, and improving the antioxidation capacity of the plant in the presence of EDDS. EDDS application decreased microbial diversity in the rhizosphere, whereas co-inoculation increased microbial biomass carbon and nitrogen, and microbial community diversity. Increased relative abundances of Actinobacteria and Bacillus and the presence of Firmicutes taxa as potential biomarkers demonstrated that co-inoculation increased soil nutrient content, and improved plant growth. Co-inoculation with PGPR and rhizobium can be useful for altering plant–soil biochemical responses during EDDS-enhanced phytoremediation to alleviate phytotoxicity of heavy metals and improve soil biochemical activities. This study provides an effective strategy for improving phytoremediation efficiency and soil quality during chelant assisted phytoremediation of metal-contaminated soils.

Revegetation of semiarid lands depends upon soil microbial communities to supply nutrients for successive plant species, but microbial activity can be constrained by insufficient water. The objective of this study was to quantify the metabolic limitation of microbes by extracellular enzymatic stoichiometry, and to determine how this affected microbial carbon use efficiency (CUE) with biogeochemical equilibrium model. The study occurred in long-term revegetation experiment with seven successional stages (0, 11, 35, 60, 100, 130 and 150 years) in the Loess Plateau, China. Microbes maintained stoichiometric homeostasis in all successional stages, but plants did not. Microbial metabolism was limited by low soil phosphorus (P) concentration throughout the succession, whereas plants were limited by low soil P during the late successional stages (from 60 to 150 years) only. An increase in soil moisture during succession was associated with greater P limitation in microbes and plants. There was less microbial P limitation at the 35-year successional stage, and the greatest microbial P limitation occurred at the 130-year successional stage. The microbial C limitation followed a unimodal pattern through the vegetation succession and reached a maximum at 100 years of succession (the early forest stage). This coincided with the lowest microbial CUE at 100 years of succession (CUE was from 0.24 to 0.41), suggesting a change in the physiological responses from microbes (such as enzyme synthesis and the priming effect), that tended to reduce soil C sequestration. Our results indicate that soil moisture regulated microbial C and P metabolism during the vegetation succession in this semiarid region, which has implications for understanding how microbial metabolism affects soil C dynamics under vegetation restoration.

Legume-rhizobium symbiosis has been heavily investigated for their potential to enhance plant metal resistance in contaminated soil. However, the extent to which plant resistance is associated with the nitrogen (N) supply in symbiont is still uncertain. This study investigates the effect of urea or/and rhizobium (Sinorhizobium meliloti) application on the growth of Medicago sativa and resistance in metals contaminated soil (mainly with Cu). The results show that Cu uptake in plant shoots increased by 41.7%, 69%, and 89.3% with urea treatment, rhizobium inoculation, and their combined treatment, respectively, compared to the control group level. In plant roots, the corresponding values were 1.9-, 1.7-, and 1.5-fold higher than the control group values, respectively. Statistical analysis identified that N content was the dominant variable contributing to Cu uptake in plants. Additionally, a negative correlation was observed between plant oxidative stress and N content, indicating that N plays a key role in plant resistance. Oxidative damage decreased after rhizobium inoculation as the activities of antioxidant enzymes (catalase and superoxide dismutase in roots and peroxidase in plant shoots) were stimulated, enhancing plant resistance and promoting plant growth. Our results suggest that individual rhizobium inoculation, without urea treatment, is the most recommended approach for effective phytoremediation of contaminated land.

VaPAT1 functions as a stress-inducible GRAS gene and enhanced cold, drought and salt tolerance in transgenic Arabidopsis via modulation of the expression of a series of stress-related genes. The plant-specific GRAS transcription factor family regulates diverse processes involved in plant growth, development and stress responses. In this study, VaPAT1, a GRAS gene from Vitis amurensis was isolated and functionally characterized. Sequence alignment and phylogenetic analysis showed that VaPAT1 has a high sequence identity to CmsGRAS and OsCIGR1, which belong to PAT1 branch of GRAS family and function in stress resistance. The transcription of VaPAT1 was markedly induced by stress-related phytohormone abscisic acid (ABA) and various abiotic stress treatments such as cold, drought and high salinity, however, it was repressed by exogenous gibberellic acid (GA) application. Overexpression of VaPAT1 increased the cold, drought and high salinity tolerance in transgenic Arabidopsis. When compared with wild type (WT) seedlings, the VaPAT1-overexpression lines accumulated higher levels of proline and soluble sugar under these stress treatments. Moreover, stress-related genes such as AtSIZ1, AtCBF1, AtATR1/MYB34, AtMYC2, AtCOR15A, AtRD29A and AtRD29B showed higher expression levels in VaPAT1 transgenic lines than in WT Arabidopsis under normal growth conditions. Together, our results indicated that VaPAT1 functions as a positive transcriptional regulator involved in grapevine abiotic stress responses.

Despite numerous reports that legume-rhizobium symbiosis alleviates Cu stress in plants, the possible roles of legume-rhizobium symbiosis and the regulatory mechanisms in counteracting Cu toxicity remain unclear. Here, Sinorhizobium meliloti CCNWSX0020 was used for analyzing the effects of rhizobium inoculation on plant growth in Medicago sativa seedlings under Cu stress. Our results showed that rhizobium inoculation alleviated Cu-induced growth inhibition, and increased nitrogen concentration in M. sativa seedlings. Moreover, the total amount of Cu uptake in inoculated plants was significantly increased compared with non-inoculated plants, and the increase in the roots was much higher than that in the shoots, thus decreasing the transfer coefficient and promoting Cu phytostabilization. Cu stress induced lipid peroxidation and reactive oxygen species production, but rhizobium inoculation reduced these components' accumulation through altering antioxidant enzyme activities and regulating ascorbate-glutathione cycles. Furthermore, legume-rhizobium symbiosis regulated the gene expression involved in antioxidant responses, phytochelatin (PC) biosynthesis, and metallothionein biosynthesis in M. sativa seedlings under Cu stress. Our results demonstrate that rhizobium inoculation enhanced Cu tolerance by affecting Cu uptake, regulating antioxidant enzyme activities and the ascorbate-glutathione cycle, and influencing PC biosynthesis-related gene expression in M. sativa. The results provide an efficient strategy for phytoremediation of Cu-contaminated soils.

Soil microorganisms are crucial to indicate ecosystem functions of terrestrial ecosystems. However, the responses of microbial communities to soil nutrient limitation in desert-grassland are still poorly understood. Hence, we investigated soil microbial community structures and metabolic characteristics in a desert-grassland ecological transition zone from the northern Loess Plateau, China, and explored the association of microbial communities with nutrient limitation via high-throughput sequencing. Threshold elemental ratios (TER) indicated that the microbial communities were strongly limited by nitrogen (N) under A. ordosica and P. tabuliformis communities. The phosphorus (P) limitation of microbial communities was observed in the aeolian sandy soil. The results imply that soil microbial communities had strong nutrient competition for N and P with aboveground vegetation in arid and oligotrophic ecosystems. The LEfSe and linear regression analysis revealed that the microbial taxa of Micrococcales, Micrococcaceae and Herpotrichiellaceae were significantly correlated with microbial N limitation. The Thermoleophilia taxa were significantly correlated with microbial P limitation. These biomarkers related to microbial nutrient limitation could be considered as the key microbial taxa to shape microbial communities and functions. Furthermore, N form had different effects on microbial communities, which NH4+-N strongly affected bacterial communities, whereas NO3--N had a significant influence on fungal communities. The different responses indicate that soil microorganisms had corresponding nutrient preferences for bacterial and fungal communities, which might alleviate the nutrient limitations and environmental stress. This study provided important insights on microbial community structures linking to community functions and on the mechanisms governing microbial N and P limitation in arid land ecosystems.

Intercropping can increase the biomass of plants and reduce the accumulation of heavy metals in plants. However, the mechanisms of intercropping increasing plant biomass and resistance to heavy metals are still unclear. Therefore, the pot experiment had been conducted to investigate the effect of intercropping treatment on the growth of gramineous pasture ryegrass (Lolium perenne L.) and leguminous forage alfalfa (Medicago sativa L.) in metal-contaminated soil. Our results showed that intercropping alleviated inhibition of heavy metals to plant growth and increased nitrogen and chlorophyll contents in the shoots and roots. Moreover, the Pb concentrations in the shoots and roots of ryegrass and alfalfa in the intercropping were significantly lower than those in the monoculture. And, the contents of saccharase and alkaline phosphatase were significantly increased in the intercropping treatment. Additionally, the intercropping treatment could reduce the oxidative damage and increase enzymatic antioxidant activities to improve the resistance of plants in contaminated soil. The intercropping treatment can increase the resistance of plants to heavy metals through reduction of plant oxidative damage and increase of antioxidant activity. It could provide us with a strategy that intercropping of ryegrass and alfalfa can increase biomass and reduce the absorption of Pb on forage plants.

Enzymes in the soil are vital for assessing heavy metal soil pollution. Although the presence of heavy metals is thought to change the soil enzyme system, the distribution of enzyme activities in heavy metal polluted-soil is still unknown. For the first time, using soil zymography, we analyzed the distribution of enzyme activities of alfalfa rhizosphere and soil surface in the metal-contaminated soil. The results showed that the growth of alfalfa was significantly inhibited, and an impact that was most pronounced in seedling biomass and chlorophyll content. Catalase activity (CAT) in alfalfa decreased with increasing heavy metal concentrations, while malondialdehyde (MDA) content continually increased. The distribution of enzyme activities showed that both phosphatase and β-glucosidase activities were associated with the roots and were rarely distributed throughout the soil. In addition, the total hotspot areas of enzyme activities were the highest in extremely heavy pollution soil. The hotspot areas of phosphatase were 3.4%, 1.5% and 7.1% under none, moderate and extremely heavy pollution treatment, respectively, but increased from 0.1% to 0.9% for β-glucosidase with the increasing pollution levels. Compared with the traditional method of enzyme activities, zymography can directly and accurately reflect the distribution and extent of enzyme activity in heavy metals polluted soil. The results provide an efficient research method for exploring the interaction between enzyme activities and plant rhizosphere.

Article history:Received 29 November 2013Received in revised form 11 August 2014Accepted 12 August 2014Available online 22 August 2014Editor: J. FeinKeywords:EPSAdsorptionCu(II)ATR-FTIRITCXAFS Biosorption mechanisms of Cu(II) by extracellular polymeric substances (EPS) from Bacillus subtilis were inves-tigated using a combination of batch experiments, attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy, isothermal titration calorimetry (ITC), and X-ray absorption fine structure (XAFS) spectros-copy.Athreediscretesitenon-electrostaticmodelfitthepotentiometrictitrationdatabest,withthepK a valuesof4.12 ± 0.12, 6.60 ± 0.06, and 9.09 ± 0.03, and site concentrations of 4.81 ± 0.62 × 10 −3 , 2.16 ± 0.14 × 10 ,and2.87±0.21×10 −3 molpergramdrymassofEPS,respectively.TheATR–FTIRresultsconfirmedthepresenceofthe functional groups with above pK a valueswithin the EPSmolecules, and indicatedthat Cu(II) binding ontothe EPS involves either phosphoryl or carboxyl sites, or both. The calculated enthalpies and entropies of Cu(II)adsorption onto EPS suggest that Cu(II) binds with anionic oxygen-bearing ligands and forms inner-spherecomplexes with the EPS functional groups. The XAFS results were consistent with inner-sphere binding ofCu(II) by carboxyl sites with 2.03 C atoms at distance of 2.96 A in the second shell, which further suggests thatthe carboxyl groups are the dominant sites for Cu(II) adsorption by EPS at pH 5.0, and that a five-memberedchelate ring structure is the most likely binding environment for Cu(II) bound to EPS. The molecular bindingmechanisms obtained in this study will add fundamental knowledge of understanding the fate of heavy metalsin natural environments.© 2014 Published by Elsevier B.V.

Legume–rhizobium symbiosis plays an important role in agriculture and ecological restoration. However, the regulatory mechanisms of rhizobium in alleviating heavy metal stress through the biochemical response of plant-soil system is limited. In this study, alfalfa was inoculated with a copper (Cu)-resistant rhizobium, and its effect on plant growth and the spatial distribution of phosphatase in the rhizosphere under Cu stress was assessed. Our results showed that rhizobium inoculation alleviated Cu-induced growth inhibition, and increased the nitrogen and phosphorus content in alfalfa tissues. Moreover, inoculated plants had a higher Cu uptake than non-inoculated plants, with a much higher increase in the roots than in the shoots; thus, inoculation with rhizobium was shown to decrease the transfer coefficient and promote Cu phytostabilization. The zymograms illustrated that the distribution of phosphatase activities was associated with the presence of roots. Compared with the non-inoculated treatment, the rhizobium inoculation increased the hotspot areas of phosphatase by 26.1% and 39.3% at the Cu 0 and Cu 800 treatments, respectively. In addition, the available phosphorus in the soil showed negative correlations with soil phosphatase activity (p

Long non-coding RNAs (lncRNAs) were found to widely exist in eukaryotes and play important roles in most biological processes. Trees belonging to Populus spp. are economically important because of their fast-growing characteristics and wide use for wood, pulp, paper, and fuel. However, the prevalence of leaf rust disease caused by Melampsora spp. on some species of Populus severely affects their growth and decreases wood production. Therefore, the identification and characterization of lncRNAs involved in the defense of Melampsora spp. would not only help us to understand plant-pathogen interactions but also provide genetic elements for producing disease-resistant poplars. In the present study, an RNA-Seq transcriptome analysis was performed for poplar leaves in response to early infection by M. larici-populina. The hybrid variety “NL895” (P. × euramericana) was used as plant material. A total of 3994 lncRNAs were identified by mining the RNA-Seq data. The identified lncRNAs had lower abundance, fewer exons, and shorter lengths when compared to protein-coding genes. In addition, 53 lncRNAs were differentially expressed between treatments and controls. Two of these 53 lncRNAs were predicted to compete to be the target of miRNAs. Moreover, the differentially expressed lncRNAs were found to be preferentially located in close proximity to the protein-coding genes they co-expressed. In this study, we were able to show that lncRNAs may play important roles in plant-pathogen interactions in the poplar. This study also improves our understanding of how plants deploy their defense system when encountering biotic stresses.

Grapevine is an important fruit crop that has undergone a long history of evolution. Analysis of the whole genome sequence of grapevine has revealed presence of an early palaeo-hexaploid along with three complements. Thus, gene duplication and genome expansion are common in this genome. In this study, we identified 17,922 duplicated genes in the whole grapevine genome. Among these, 2,039; 628; 1,428; 722; and 2,942 were identified respectively as produced by genome-wide, tandem, proximal, retrotransposed, and DNA-based transposed duplications. Analyses of the evolutionary patterns for different types of duplication using non-synonymous and synonymous substitution rates uncovered a series of underlying rules. Thereafter, all the grapevine genes were classified into families, and the contributions of different types of duplication to the expansion of large families were revealed. No duplication type was solely responsible for the formation of any large gene family, but some families showed enrichment of a special type of duplication. On the basis of this study, we believe that uncovering the underlying rules for gene duplications, expansions of gene families, and their evolutionary styles will contribute significantly to a comprehensive understanding of the features of the grapevine genome.

Ethylene (ET) is a gaseous plant hormone that plays essential roles in biotic and abiotic stress responses in plants. However, the role of ET in cold tolerance varies in different species. This study revealed that low temperature promotes the release of ET in grapevine. The treatment of exogenous 1-aminocyclopropane-1-carboxylate increased the cold tolerance of grapevine. By contrast, the application of the ET biosynthesis inhibitor aminoethoxyvinylglycine reduced the cold tolerance of grapevine. This finding suggested that ET positively affected cold stress responses in grapevine. The expression of VaERF057, an ET signaling downstream gene, was strongly induced by low temperature. The overexpression of VaERF057 also enhanced the cold tolerance of Arabidopsis. Under cold treatment, malondialdehyde content was lower and superoxide dismutase, peroxidase, and catalase activities were higher in transgenic lines than in wild-type plants. RNA-Seq results showed that 32 stress-related genes, such as CBF1-3, were upregulated in VaERF057-overexpressing transgenic line. Yeast one-hybrid results further demonstrated that VaERF057 specifically binds to GCC-box and DRE motifs. Thus, VaERF057 may directly regulate the expression of its target stress-responsive genes by interacting with a GCC-box or a DRE element. Our work confirmed that ET positively regulates cold tolerance in grapevine by modulating the expression of VaERF057.

The extraction protocol of flavonoids from lotus (Nelumbo nucifera) leaves was optimized through an orthogonal design. The solvent was the most important factor comparing solvent, solvent:tissue ratio, extraction time, and temperature. The highest yield of flavonoids was achieved with 70% methanol-water and a solvent:tissue ratio of 30:1 at 4 °C for 36 h. The optimized analytical method for HPLC was a multi-step gradient elution using 0.5% formic acid (A) and CH₃CN containing 0.1% formic acid (B), at a flow rate of 0.6 mL/min. Using this optimized method, thirteen flavonoids were simultaneously separated and identified by high performance liquid chromatography coupled with photodiode array detection/electrospray ionization mass spectrometry (HPLC/DAD/ESI-MS(n)). Five of the bioactive compounds are reported in lotus leaves for the first time. The flavonoid content of the leaves of three representative cultivars was assessed under the optimized extraction and HPLC analytical conditions, and the seed-producing cultivar 'Baijianlian' had the highest flavonoid content compared with rhizome-producing 'Zhimahuoulian' and wild floral cultivar 'Honglian'.

The effect of extracellular polymeric substances (EPS) of Gram-positive Bacillus subtilis and Gram-negative Pseudomonas putida on Cu(II) adsorption was investigated using a combination of batch adsorption, potentiometric titrations, Fourier transform infrared spectroscopy. Both the potentiometric titrations and the Cu(II) adsorption experiments indicated that the presence of EPS in a biomass sample significantly enhance Cu(II) adsorption capacity. Surface complexation modeling showed that the pKa values for the three functional groups (carboxyl, phosphate and hydroxyl) were very similar for untreated and EPS-free cells, indicating no qualitative difference in composition. However, site concentrations on the untreated cell surface were found to be significantly higher than those on the EPS-free cell surface. Infrared analysis provided supporting evidence and demonstrated that carboxyl and phosphate groups are responsible for Cu(II) adsorption on the native and EPS-free cells.

In this study, Fourier transform infrared spectroscopy, potentiometric titration, along with sorption experiments using chemically modified bacteria were conducted to compare the behavior of Gram-positive Bacillus thuringiensis and Gram-negative Escherichia coli as sorbents for Cu 2+ and Cd 2+ ions. The IR spectra showed that there were obvious changes connected with the–COOH groups for Cu(II)- and Cd(II)-loaded B. thuringiensis . A three site non-electrostatic model provides an excellent fit to the titration curves of both E. coli and B. thuringiensis with the first corresponding p K a values of 4.16 ± 0.18 and 3.30 ± 0.24, respectively, implying that B. thuringiensis contains more carboxyl groups than E. coli . Chemical modification and metal sorption experiments further confirmed that carboxyl groups may play a more important role in the binding Cd(II) and Cu(II) for B. thuringiensis than for E. coli , which could be attributed to the higher concentration of carboxyl sites of B. thuringiensis than E. coli . These results show that the main chemical functional group responsible for the binding of metal ions on B. thuringiensis and E. coli is obviously different because of the different composition and structure of bacterial cell walls. The concentrations of the chemical functional groups on bacterial cell walls are regarded to govern their role in the binding of metal ions and affect the affinity between the bacterial cells and metal ions.

Background QTLs controlling individual sugars and acids (fructose, glucose, malic acid and tartaric acid) in grape berries have not yet been identified. The present study aimed to construct a high-density, high-quality genetic map of a winemaking grape cross with a complex parentage (V. vinifera × V. amurensis) × ((V. labrusca × V. riparia) × V. vinifera), using next-generation restriction site-associated DNA sequencing, and then to identify loci related to phenotypic variability over three years. Results In total, 1 826 SNP-based markers were developed. Of these, 621 markers were assembled into 19 linkage groups (LGs) for the maternal map, 696 for the paternal map, and 1 254 for the integrated map. Markers showed good linear agreement on most chromosomes between our genetic maps and the previously published V. vinifera reference sequence. However marker order was different in some chromosome regions, indicating both conservation and variation within the genome. Despite the identification of a range of QTLs controlling the traits of interest, these QTLs explained a relatively small percentage of the observed phenotypic variance. Although they exhibited a large degree of instability from year to year, QTLs were identified for all traits but tartaric acid and titratable acidity in the three years of the study; however only the QTLs for malic acid and β ratio (tartaric acid-to-malic acid ratio) were stable in two years. QTLs related to sugars were located within ten LGs (01, 02, 03, 04, 07, 09, 11, 14, 17, 18), and those related to acids within three LGs (06, 13, 18). Overlapping QTLs in LG14 were observed for fructose, glucose and total sugar. Malic acid, total acid and β ratio each had several QTLs in LG18, and malic acid also had a QTL in LG06. A set of 10 genes underlying these QTLs may be involved in determining the malic acid content of berries. Conclusion The genetic map constructed in this study is potentially a high-density, high-quality map, which could be used for QTL detection, genome comparison, and sequence assembly. It may also serve to broaden our understanding of the grape genome. Electronic supplementary material The online version of this article (doi:10.1186/s12870-015-0428-2) contains supplementary material, which is available to authorized users.

High and low resveratrol (Res) contents in two cultivars are correlated with the expression abundance of Myb14 , which could directly activate transcriptional expression of stilbene synthase gene ( STS ). Resveratrol (3,5,4′-trihydroxystilbene) is one of the natural polyphenols produced by secondary metabolism in some plants. Stilbene synthase (STS) is the key enzyme for the final step of precursor formation of resveratrol (Res) in grapevines. In this study, we found that Res contents in ripe berry skin were completely different in two grape cultivars, namely, ‘Z168’ (Vitis monticola × Vitis riparia) with high-Res and ‘Jingzaojing’ (Vitis vinifera) with low-Res. Moreover, the level of expression of STS gene was higher in the ripe berry skin of ‘Z168’ than in that of ‘Jingzaojing’. To further investigate the underlying mechanisms, we conducted a co-expression analysis through transcriptomic data. We confirmed that Myb14, an R2R3 Myb transcription factor, is the direct regulator of STS by binding to Box-L5 motif. Moreover, the expression pattern of Myb14 is associated with the variation of Res content. To test this prediction, we conducted a number of experiments in vivo and in vitro. The expression patterns of Myb14 and STS in grapevine leaves were identical under a series of stimulus. Myb14 showed higher expression in the ripe berry skin of ‘Z168’ than in that of ‘Jingzaojing’. Yeast one-hybrid assay indicated that grapevine Myb14 could interact with the promoter of STS in vitro, and the transient overexpression of Myb14 promoted the expression of STS. Furthermore, co-expressing 35S::Myb14 in transgenic Arabidopsis could activate GUS expression promoted by STS promoter. Thus, Myb14 is the direct activator of STS, and its expression pattern is associated with Res content variation in grapes.

Background WRKY transcription factors are one of the largest families of transcriptional regulators in plants. WRKY genes are not only found to play significant roles in biotic and abiotic stress response, but also regulate growth and development. Grapevine (Vitis vinifera) production is largely limited by stressful climate conditions such as cold stress and the role of WRKY genes in the survival of grapevine under these conditions remains unknown. Results We identified a total of 59 VvWRKYs from the V. vinifera genome, belonging to four subgroups according to conserved WRKY domains and zinc-finger structure. The majority of VvWRKYs were expressed in more than one tissue among the 7 tissues examined which included young leaves, mature leaves, tendril, stem apex, root, young fruits and ripe fruits. Publicly available microarray data suggested that a subset of VvWRKYs was activated in response to diverse stresses. Quantitative real-time PCR (qRT-PCR) results demonstrated that the expression levels of 36 VvWRKYs are changed following cold exposure. Comparative analysis was performed on data from publicly available microarray experiments, previous global transcriptome analysis studies, and qRT-PCR. We identified 15 VvWRKYs in at least two of these databases which may relate to cold stress. Among them, the transcription of three genes can be induced by exogenous ABA application, suggesting that they can be involved in an ABA-dependent signaling pathway in response to cold stress. Conclusions We identified 59 VvWRKYs from the V. vinifera genome and 15 of them showed cold stress-induced expression patterns. These genes represented candidate genes for future functional analysis of VvWRKYs involved in the low temperature-related signal pathways in grape.

Grape is one of the most important fruit crops worldwide. The suitable geographical locations and productivity of grapes are largely limited by temperature. Vitis amurensis is a wild grapevine species with remarkable cold-tolerance, exceeding that of Vitis vinifera, the dominant cultivated species of grapevine. However, the molecular mechanisms that contribute to the enhanced freezing tolerance of V. amurensis remain unknown. Here we used deep sequencing data from restriction endonuclease-generated cDNA fragments to evaluate the whole genome wide modification of transcriptome of V. amurensis under cold treatment. Vitis vinifera cv. Muscat of Hamburg was used as control to help investigate the distinctive features of V. amruensis in responding to cold stress. Approximately 9 million tags were sequenced from non-cold treatment (NCT) and cold treatment (CT) cDNA libraries in each species of grapevine sampled from shoot apices. Alignment of tags into V. vinifera cv. Pinot noir (PN40024) annotated genome identified over 15,000 transcripts in each library in V. amruensis and more than 16,000 in Muscat of Hamburg. Comparative analysis between NCT and CT libraries indicate that V. amurensis has fewer differential expressed genes (DEGs, 1314 transcripts) than Muscat of Hamburg (2307 transcripts) when exposed to cold stress. Common DEGs (408 transcripts) suggest that some genes provide fundamental roles during cold stress in grapes. The most robust DEGs (more than 20-fold change) also demonstrated significant differences between two kinds of grapevine, indicating that cold stress may trigger species specific pathways in V. amurensis. Functional categories of DEGs indicated that the proportion of up-regulated transcripts related to metabolism, transport, signal transduction and transcription were more abundant in V. amurensis. Several highly expressed transcripts that were found uniquely accumulated in V. amurensis are discussed in detail. This subset of unique candidate transcripts may contribute to the excellent cold-hardiness of V. amurensis.

KEY MESSAGE : Genome-wide identification of grapevine NAC domain genes and investigation of their chromosome locations, gene structures, duplication, evolution, phylogeny and expression profiles. Grapevine is a widely used fruit crop. NAC (NAM, ATAF1/2 and CUC2) domain genes are plant-specific transcription factors (TFs) that comprise a conserved NAM domain in the N-terminus. Members of this gene family have been reported to contribute to plant development. During this study, 74 NAC genes were identified from 12× assembled grapevine genomic sequences. The duplication patterns, genomic structures and phylogeny of these 74 grapevine NAC genes were investigated. To understand the roles of VvNAC during grapevine development, their expression profiles in different tissues including leaf, tendril, inflorescence, stem, root and veraison berry skin were tested using quantitative real-time PCR. Analysis revealed expression diversity of various VvNAC genes among different grapevine tissues. To identify candidate grapevine NAC genes with a role in response to stress, publicly available microarray data were obtained to calculate their expression change under abiotic and biotic treatments, with a number of VvNAC genes displaying up-regulation after stress induction. Therefore, this study has uncovered more knowledge relating to the gene structures, chromosome organizations, evolution, expression profiles and functions of VvNAC genes.

The role of extracellular polymeric substances (EPS) in Cd adsorption by Bacillus subtilis and Pseudomonas putida was investigated using a combination of batch adsorption experiments, potentiometric titrations, Fourier transform infrared spectroscopy (FTIR). An increased adsorption capacity of Cd was observed for untreated bacteria relative to that for EPS-free bacteria. Surface complexation modeling of titration data showed the similar pKa values of functional groups (carboxyl, phosphate and hydroxyl) between untreated and EPS-free bacteria. However, site concentrations on the untreated bacteria were found to be higher than those on the EPS-free bacteria. FTIR spectra also showed that no significant difference in peak positions was observed between untreated and EPS-free bacteria and carboxyl and phosphate groups were responsible for Cd adsorption on bacterial cells. The information obtained in this study is of fundamental significance for understanding the interaction mechanisms between heavy metals and biofilms in natural environments.

Background Genetic mapping and QTL detection are powerful methodologies in plant improvement and breeding. Construction of a high-density and high-quality genetic map would be of great benefit in the production of superior grapes to meet human demand. High throughput and low cost of the recently developed next generation sequencing (NGS) technology have resulted in its wide application in genome research. Sequencing restriction-site associated DNA (RAD) might be an efficient strategy to simplify genotyping. Combining NGS with RAD has proven to be powerful for single nucleotide polymorphism (SNP) marker development. Results An F1 population of 100 individual plants was developed. In-silico digestion-site prediction was used to select an appropriate restriction enzyme for construction of a RAD sequencing library. Next generation RAD sequencing was applied to genotype the F1 population and its parents. Applying a cluster strategy for SNP modulation, a total of 1,814 high-quality SNP markers were developed: 1,121 of these were mapped to the female genetic map, 759 to the male map, and 1,646 to the integrated map. A comparison of the genetic maps to the published Vitis vinifera genome revealed both conservation and variations. Conclusions The applicability of next generation RAD sequencing for genotyping a grape F1 population was demonstrated, leading to the successful development of a genetic map with high density and quality using our designed SNP markers. Detailed analysis revealed that this newly developed genetic map can be used for a variety of genome investigations, such as QTL detection, sequence assembly and genome comparison.