New perspectives on microbiome and nutrient sequestration in soil aggregates during long-term grazing exclusion.

Grazing exclusion alters grassland soil aggregation, microbiome composition, and biogeochemical processes. However, the long-term effects of grazing exclusion on the microbial communities and nutrient dynamics within soil aggregates remain unclear. We conducted a 36-year exclusion experiment to investigate how grazing exclusion affects the soil microbial community and the associated soil functions within soil aggregates in a semiarid grassland. Long-term (36 years) grazing exclusion induced a shift in microbial communities, especially in the <2 mm aggregates, from high to low diversity compared to the grazing control. The reduced microbial diversity was accompanied by instability of fungal communities, extended distribution of fungal pathogens to >2 mm aggregates, and reduced carbon (C) sequestration potential thus revealing a negative impact of long-term GE. In contrast, 11-26 years of grazing exclusion greatly increased C sequestration and promoted nutrient cycling in soil aggregates and associated microbial functional genes. Moreover, the environmental characteristics of microhabitats (e.g., soil pH) altered the soil microbiome and strongly contributed to C sequestration. Our findings reveal new evidence from soil microbiology for optimizing grazing exclusion duration to maintain multiple belowground ecosystem functions, providing promising suggestions for climate-smart and resource-efficient grasslands.

Deciphering microbiomes dozens of meters under our feet and their edaphoclimatic and spatial drivers.

Microbes inhabiting deep soil layers are known to be different from their counterpart in topsoil yet remain under investigation in terms of their structure, function, and how their diversity is shaped. The microbiome of deep soils (>1 m) is expected to be relatively stable and highly independent from climatic conditions. Much less is known, however, on how these microbial communities vary along climate gradients. Here, we used amplicon sequencing to investigate bacteria, archaea, and fungi along fifteen 18-m depth profiles at 20-50-cm intervals across contrasting aridity conditions in semi-arid forest ecosystems of China's Loess Plateau. Our results showed that bacterial and fungal α diversity and bacterial and archaeal community similarity declined dramatically in topsoil and remained relatively stable in deep soil. Nevertheless, deep soil microbiome still showed the functional potential of N cycling, plant-derived organic matter degradation, resource exchange, and water coordination. The deep soil microbiome had closer taxa-taxa and bacteria-fungi associations and more influence of dispersal limitation than topsoil microbiome. Geographic distance was more influential in deep soil bacteria and archaea than in topsoil. We further showed that aridity was negatively correlated with deep-soil archaeal and fungal richness, archaeal community similarity, relative abundance of plant saprotroph, and bacteria-fungi associations, but increased the relative abundance of aerobic ammonia oxidation, manganese oxidation, and arbuscular mycorrhizal in the deep soils. Root depth, complexity, soil volumetric moisture, and clay play bridging roles in the indirect effects of aridity on microbes in deep soils. Our work indicates that, even microbial communities and nutrient cycling in deep soil are susceptible to changes in water availability, with consequences for understanding the sustainability of dryland ecosystems and the whole-soil in response to aridification. Moreover, we propose that neglecting soil depth may underestimate the role of soil moisture in dryland ecosystems under future climate scenarios.

Warming-induced shifts in alpine soil microbiome: An ecosystem-scale study with environmental context-dependent insights.

Climate warming is a pressing global issue with substantial impacts on soil health and function. However, the influence of environmental context on the responses of soil microorganisms to warming remains largely elusive, particularly in alpine ecosystems. This study examined the responses of the soil microbiome to in situ experimental warming across three elevations (3850 m, 4100 m, and 4250 m) in the meadow of Gongga Mountain, eastern Tibetan Plateau. Our findings demonstrate that soil microbial diversity is highly resilient to warming, with significant impacts observed only at specific elevations. Furthermore, the influence of warming on the composition of the soil microbial community is also elevation-dependent, underscoring the importance of local environmental context in shaping microbial evolution in alpine soils under climate warming. Notably, we identified soil moisture at 3850 m and carbon-to-nitrogen ratio at 4250 m as indirect predictors regulating the responses of microbial diversity to warming at specific elevations. These findings underscore the paramount importance of considering pre-existing environmental conditions in predicting the response of alpine soil microbiomes to climate warming. Our study provides novel insights into the intricate interactions between climate warming, soil microbiome, and environmental context in alpine ecosystems, illuminating the complex mechanisms governing soil microbial ecology in these fragile and sensitive environments.

Crop residue return sustains global soil ecological stoichiometry balance.

Although soil ecological stoichiometry is constrained in natural ecosystems, its responses to anthropogenic perturbations are largely unknown. Inputs of inorganic fertilizer and crop residue are key cropland anthropogenic managements, with potential to alter their soil ecological stoichiometry. We conducted a global synthesis of 682 data pairs to quantify the responses of soil carbon (C), nitrogen (N), and phosphorus (P) and grain yields to combined inputs of crop residue plus inorganic fertilizer compared with only inorganic fertilizer application. Crop residue inputs enhance soil C (10.5%-12%), N (7.63%-9.2%), and P (2.62%-5.13%) contents, with an increase in C:N (2.51%-3.42%) and C:P (7.27%-8.00%) ratios, and grain yields (6.12%-8.64%), indicating that crop residue alleviated soil C limitation caused by inorganic fertilizer inputs alone and was able to sustain balanced stoichiometry. Moreover, the increase in soil C and C:N(P) ratio reached saturation in ~13-16 years after crop residue return, while grain yield increase trend discontinued. Furthermore, we identified that the increased C, N, and P contents and C:N(P) ratios were regulated by the initial pH and C content, and the increase in grain yield was not only related to soil properties, but also negatively related to the amount of inorganic N fertilizer input to a greater extent. Given that crop residual improvement varies with soil properties and N input levels, we propose a predictive model to preliminary evaluate the potential for crop residual improvement. Particularly, we suggest that part of the global budget should be used to subsidize crop residue input management strategies, achieving to a win-win situation for agricultural production, ecological protection, and climate change mitigation.

Microbial communities in terrestrial surface soils are not widely limited by carbon.

Microbial communities in soils are generally considered to be limited by carbon (C), which could be a crucial control for basic soil functions and responses of microbial heterotrophic metabolism to climate change. However, global soil microbial C limitation (MCL) has rarely been estimated and is poorly understood. Here, we predicted MCL, defined as limited availability of substrate C relative to nitrogen and/or phosphorus to meet microbial metabolic requirements, based on the thresholds of extracellular enzyme activity across 847 sites (2476 observations) representing global natural ecosystems. Results showed that only about 22% of global sites in terrestrial surface soils show relative C limitation in microbial community. This finding challenges the conventional hypothesis of ubiquitous C limitation for soil microbial metabolism. The limited geographic extent of C limitation in our study was mainly attributed to plant litter, rather than soil organic matter that has been processed by microbes, serving as the dominant C source for microbial acquisition. We also identified a significant latitudinal pattern of predicted MCL with larger C limitation at mid- to high latitudes, whereas this limitation was generally absent in the tropics. Moreover, MCL significantly constrained the rates of soil heterotrophic respiration, suggesting a potentially larger relative increase in respiration at mid- to high latitudes than low latitudes, if climate change increases primary productivity that alleviates MCL at higher latitudes. Our study provides the first global estimates of MCL, advancing our understanding of terrestrial C cycling and microbial metabolic feedback under global climate change.

Nitrogen removal in improved subsurface wastewater infiltration system: Mechanism, microbial indicators and the limitation of phosphorus.

To enhance the nitrogen removal capacity, scrap iron filings and Si-Al porous clay mineral material (PCMW) was used to improve a subsurface wastewater infiltration system (SWIS). The results showed TN and NH4+-N removal efficiencies of improved SWIS were 20.72% and 5.49% higher than those of the control SWIS, respectively. Based on the response of the removal performance, microbial community and function analysis of 16s rRNA amplicon sequencing results, the amending soil matrix substantially enriched the nitrogen removal bacteria (Rhizobiales_Incertae_Sedis and Gemmatimonadaceae), and significantly improved the activities of key enzymes (Hao, NasAB, NarGHI, NirK, NorBC, NirA and NirBD), particularly at co-occurrence zone of nitrification and denitrification (70-130 cm depth). The amending soil matrix not only extended the growth space of microbes, but also provided additional electrons and carbon sources for denitrifying bacteria by regulating the structure and function of the microbial community. In addition, amending soil matrix could enhance phosphate metabolism genes and phosphate solubilizing microbes in the denitrification zone by increasing the phosphorus source, thus strengthening nitrogen metabolism. Nitrospiraceae, Rhizobiales_Incertae_Sedis and Gemmatimonadaceae related to nitrogen removal and Bacillaceae with phosphate-solubilizing ability could be used as microbial indicators of nitrogen removal in SWISs. The reciprocal action of environmental on microbial characteristics exhibited microbial functional were related to DO, Fe2+, TOC, TP, TN, NH4+-N and NO3--N. Those could be used as physicochemical and biological indicators for application and monitoring of SWIS. In conclusion, this study provided a low-cost and efficient enhancement approach for the application of SWIS in decentralized domestic sewage treatment, and furnished theoretical support for subsequent applications.

Dissolved Organic Matter Promotes the Aging Process of Polystyrene Microplastics under Dark and Ultraviolet Light Conditions: The Crucial Role of Reactive Oxygen Species.

Microplastics (MPs) interact frequently with dissolved organic matter (DOM) commonly found in the environment, but information on the aging behavior of MPs under the participation of DOM is still lacking. Thus, the polystyrene microplastic (PSMP) aging process with DOM participation was systematically studied by electron paramagnetic resonance spectroscopy, high-performance liquid chromatography, Fourier transform infrared (FTIR) spectroscopy, and two-dimensional correlation spectroscopy analyses under dark and ultraviolet (UV) light conditions. DOM was found to promote electron transfer to generate reactive oxygen species (ROS) under dark conditions and the aging of PSMPs, while the process of DOM generating ROS under UV light was more susceptible to photoelectrons and accelerated the aging process of PSMPs. However, among the four DOM types, fulvic acid (FA) has a more significant promoting effect on the aging process of PSMPs than humic acid, which can be attributed to the stronger conversion ability of FA to semiquinone radicals. Density functional theory calculations are used to describe the difference in the aging process of different structures of plastics with the participation of DOM. This study provides a necessary theoretical basis for the study of the migration of MPs in groundwater and deep surface water.

Pollution characteristics and health risk assessment of potentially toxic elements in soils around China's gold mines: a meta-analysis.

Since toxic element pollution is widespread in soils near gold mines due to increasing mining activities, the adverse effects of potentially toxic elements (PTEs) in the soils on ecological systems and human health cannot be ignored. However, assessments of PTE pollution in soils and their ecological-health risks on a national scale are still limited. Here, the concentrations of eight PTEs in soils near gold mines throughout China were obtained from published articles. Based on these data, the pollution levels and ecological-health risks of the eight PTEs in soils were comprehensively estimated. The results showed that the average contents of As, Cr, Cd, Pb, Hg, Cu, Ni, and Zn were 81.62, 79.82, 1.04, 206.03, 2.05, 40.82, 71.82, and 130.42 mg kg-1, respectively, which exceeded the corresponding background values for soils. Most of the examined soils were heavily polluted by Hg and Cd, and higher pollution levels were found in the Henan and Shaanxi Provinces than in other regions. The average potential ecological risk value of all PTEs was 2534.71, indicating the presence of very high risks. Contribution of Hg to the potential ecological risk was more than 80%. For adults, all hazard index (HI) values of noncarcinogenic risks were below the safe level of 1.00. For children, none of the HI values exceeded the safe level, with the exception of As (HI = 1.81); nevertheless, four PTEs (As, Cr, Cu, and Ni) presented unacceptable carcinogenic risks. This study provides scientific basis for controlling PTE contamination and reducing the health risks in soils near gold mines worldwide.

Molecular Mechanisms of Lead Binding to Ferrihydrite-Bacteria Composites: ITC, XAFS, and µ-XRF Investigations.

Binding of Pb(II) to ferrihydrite-Bacillus subtilis composites formed in the presence of bacterial cells were investigated through macroscopic and microscopic techniques. Diffuse layer model (DLM) fitting and isothermal titration calorimetry (ITC) analysis indicated that the hydroxyl group played a key role in Pb(II) sorption onto composites by masking reactive sites, such as carboxyl and phosphoryl groups of bacterial cells. Negative enthalpy (from -39.29 to -57.87 kJ mol-1) and positive entropy (from 135.61 to 193.47 kJ mol-1) of Pb(II) sorption onto composites revealed that inner-sphere complexes formed through exothermic reactions and was driven by both entropy and enthalpy. Spatial distribution of these inner-sphere species at varied Pb(II) loading demonstrated that interactions between Pb(II) and bacterial cells preceded that of mineral components in composites, using microfocus X-ray fluorescence spectroscopy (µ-XRF) maps and microfocus X-ray absorption near edge structure (µ-XANES) spectra. Combined with bulk Pb LIII-edge X-ray absorption fine structure (XAFS) spectrum, we inferred that mononuclear bidentate edge-sharing hydroxyl-Pb complexes, monodentate mononuclear carboxyl-Pb and phosphoryl-Pb complexes predominantly contributed to Pb(II) inner-sphere binding with mineral and bacterial fractions in composites, respectively. The molecular binding mechanisms obtained in this study provide further insight into the sequestration and migration of toxic metals in natural environments.

The transcription factor VaNAC17 from grapevine (Vitis amurensis) enhances drought tolerance by modulating jasmonic acid biosynthesis in transgenic Arabidopsis.

KEY MESSAGE: 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.

Impact of co-inoculation with plant-growth-promoting rhizobacteria and rhizobium on the biochemical responses of alfalfa-soil system in copper contaminated soil.

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.

Application of signaling molecules in reducing metal accumulation in alfalfa and alleviating metal-induced phytotoxicity in Pb/Cd-contaminated soil.

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.

Proton and Copper Binding to Humic Acids Analyzed by XAFS Spectroscopy and Isothermal Titration Calorimetry.

Proton and copper (Cu) binding to soil and lignite-based humic acid (HA) was investigated by combining X-ray absorption fine structure (XAFS) spectroscopy, isothermal titration calorimetry (ITC), and nonideal-competitive-adsorption (NICA) modeling. NICA model calculations and XAFS results showed that bidentate and monodentate complexation occurred for Cu binding to HA. The site-type-specific thermodynamic parameters obtained by combining ITC measurements and NICA calculations revealed that copper binding to deprotonated carboxylic-type sites was entropically driven and that to deprotonated phenolic-type sites was driven by entropy and enthalpy. Copper binding to HA largely depended on the site-type and coordination environment, but the thermodynamic binding mechanisms for Cu binding to the specific site-types were similar for the different HAs studied. By comparing the site-type-specific thermodynamic parameters of HA-Cu complexation with those of low molar mass organic acids, the Cu coordination could be further specified. Bidentate carboxylic-Cu complexes made the dominating contributions to Cu binding to HA. The present study not only yields molecular-scale mechanisms of ion binding to carboxylic- and phenolic-type sites of HA but also provides the new insight that the universal nature of site-type-specific thermodynamic data enables quantitative estimation of the binding structures of heavy metal ions to humic substances.

Reveal the response of enzyme activities to heavy metals through in situ zymography.

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 ß-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 ß-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.

Rhizobium inoculation enhances copper tolerance by affecting copper uptake and regulating the ascorbate-glutathione cycle and phytochelatin biosynthesis-related gene expression in Medicago sativa seedlings.

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.

Natural succession on abandoned cropland effectively decreases the soil erodibility and improves the fungal diversity.

Changes in plants and soils during natural succession have been evaluated, but little is known about the effects of succession on the activities of soil microbes and their interactions with soil erodibility. We conducted a field study on the Chinese Loess Plateau, typical of this semiarid area, to determine the effect of secondary succession on the stability of soil structure against erosion and on the composition of soil fungal communities. Characteristics of plant, soil, and fungal communities were assessed across a 30-yr chronosequence of grassland developed from abandoned cropland. The diversity and composition of the fungal communities were determined using high-throughput sequencing of the internal transcribed spacer. Six grasslands were selected to represent different successional age classes: 0 (cropland), 5, 10, 15, 20, and 30 yr. Short-term decreases (initial 5 yr) in the amounts of soil organic carbon, total nitrogen, available phosphorus, and fungal biomass and in fungal diversity had returned to original levels (i.e., cropland) within 15 yr and were much higher after continued succession. Abandoning cropland for succession caused the soil erodibility (K) decrease and the aboveground coverage, soil nutrient levels, content of larger (>5 mm) water-stable aggregate, mean aggregate weight diameter, and diversity of the fungal communities improvement including arbuscular mycorrhizas (AMF), ectomycorrhizas (EMF), and saprotrophs. The fungal communities were dominated by Ascomycota, Zygomycota, Basidiomycota, and Glomeromycota during the succession. The successional patterns of the plant and fungal communities were similar, although distinct fungal communities were not observed in the two initial stages, suggesting that fungal succession may develop more slowly than plant succession. Plant root biomass, EMF, and soil organic carbon content accounted for most of the variation of soil erodibility (28.6%, 19.5%, and 11.8%, respectively), indicating their importance in shaping soil structure to prevent erosion. Our results demonstrated that abandoning cropland for natural succession could decrease soil erodibility and increase fungal diversity. EMF plays an important role in soil stability against erosion in the Loess Plateau. Abandoning cropland for natural succession should be recommended for alleviating soil erosion and improving the degraded soils in this area.

Expression of Vitis amurensis NAC26 in Arabidopsis enhances drought tolerance by modulating jasmonic acid synthesis.

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.

Overexpression of VaPAT1, a GRAS transcription factor from Vitis amurensis, confers abiotic stress tolerance in Arabidopsis.

KEY MESSAGE: 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.

Construction of a high-density genetic map and QTLs mapping for sugars and acids in grape berries.

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 ß 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 ß 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.

Genome-wide identification of WRKY family genes and their response to cold stress in Vitis vinifera.

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.