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Background Melatonin is a ubiquitous molecule and exists across kingdoms. Studies on melatonin in plants have mainly focused on its physiological influence on growth and development, and on its biosynthesis. A number of studies have been conducted on the melatonin content and exogenous melatonin treatment of grapevine ( Vitis vinifera L.). However, key genes or enzymes of the melatonin biosynthetic pathway remain unclear. Results In this study, we cloned and identified the gene encoding serotonin N -acetyltransferase (SNAT) in grapevine ( VvSNAT2 ) . The VvSNAT2 protein was identified from a collection of 30 members of the grapevine GCN5-related N -acetyltransferase (GNAT) superfamily. Phylogenetic and protein sublocalization analyses showed that the candidate gene VvGNAT16 is VvSNAT2 . Characterization of VvSNAT2 showed that its enzymatic activity is highest at a pH of 8.8 and a temperature of 45 °C. Analysis of enzyme kinetics showed the values of K m and V max of VvSNAT2 using serotonin were 392.5 μM and 836 pmol/min/mg protein, respectively. The expression of VvSNAT2 was induced by melatonin treatment and pathogen inoculation. Overexpression of VvSNAT2 in Arabidopsis resulted in greater accumulation of melatonin and chlorophyll and enhanced resistance to powdery mildew in the transgenic plants compared with the wild type (WT). Additionally, our data showed that the marker genes in the salicylic acid (SA) signaling pathway were expressed to higher levels in the transgenic plants compared with the WT. Conclusions The VvSNAT2 gene was cloned and identified in grapevine for the first time. Our results indicate that VvSNAT2 overexpression activates the SA and JA signaling pathways; however, the SA pathway plays a central role in VvSNAT2 -mediated plant defense.

Background Resveratrol is a naturally occurring plant stilbene that exhibits a wide range of valuable biological and pharmacological properties. Although the beneficial effects of trans-resveratrol to human health and plant protection against fungal pathogens and abiotic stresses are well-established, yet little is known about the molecular mechanisms regulating stilbene biosynthesis in plant defense progress. Results Here, we cloned and identified the Chinese wild grape ( Vitis davidii ) R2R3-MYB transcription factor VdMYB1 , which activates defense responses against invading pathogen. VdMYB1 transcripts were significantly upregulated after inoculation with the grapevine powdery mildew fungus Erysiphe necator (Schw.) Burr. Transient expression analysis using onion epidermal cells and Arabidopsis thaliana protoplasts showed that VdMYB1 was localized in the nucleus. Yeast one-hybrid assays revealed that VdMYB1 acts as a transcriptional activator. Grapevine leaves transiently overexpressing VdMYB1 showed a lower number of fungal conidiophores compared with wild-type leaves. Overexpression of VdMYB1 in grapevine leaves did not alter the expression of genes in salicylic acid- and jasmonate-dependent pathways, but affected the expression of stilbene synthase ( STS ) genes, key regulators of flavonoid metabolism. Results of electrophoretic mobility shift assays and in vivo transcriptional activation assays showed that VdMYB1 binds to the MYB binding site (MYBBS) in the STS2 gene promoter, thus activating STS2 transcription. In heterologous expression assays using tobacco leaves, VdMYB1 activated STS2 gene expression and increased the accumulation of resveratrol. Conclusions Our study showed that VdMYB1 activates STS2 gene expression to positively regulate defense responses, and increases the content of resveratrol in leaves.

Water-conserving mining is an effective way to alleviate the contradiction between fragile ecological environment and high-intensity coal mining in the arid and semi-arid region of northwest China. It needs to consider the engineering and geological conditions, hydrogeological conditions and mining methods of coal seams. From the three aspects, this paper systematically analyzes the influencing factors and establishes an identification model with multi-level structures. The model includes three primary factors (including the engineering and geological conditions, hydrogeological conditions and mining methods), nine secondary factors (including overlying strata thickness, aquiclude, mining parameters and etc.), sixteen third-tier factors (including the faults, aquiclude thickness and effective mining height and etc.) and twelve fourth-tier factors (including the fault throw exponent, aquiclude permeability and coal pillar sizes and etc.). On the basis, the analytic hierarchy process is used to build the judgment matrix and obtain the weight of each influencing factor. The results indicate that the overlying strata thickness, aquiclude and effective mining height are the most important factors among the primary factors of engineering and geological conditions, hydrogeological conditions and mining methods, respectively. The research results could provide theoretical references for the water-conserving mining of coal resources in northwest China.

Activities of traditional longwall mining will result in ground subsidence and therefore cause issues such as damages to buildings and farmlands, water pollution and loss, and potential ecological and environmental problems in the mining region. With advantages of the longwall backfill mining method, as well as the room and pillar mining method, a continuous excavation and continuous backfill (CECB) method in longwall mining is recommended to effectively control the ground subsidence. In this method, mining roadways (MRs) are initially planned in a panel, and then they are excavated and backfilled in several stages until the whole panel is mined out and backfilled. According to the geologic conditions of an underground coal mine, and the elastic foundation beam theory, a mechanical model was built to study the subsidence of the roof while using this new mining method. In addition, methods to calculate roof subsidence in various stages in CECB were also provided. The mechanical parameters of backfilling materials, which were used in the theoretical calculation and the numerical analysis for mutual check, were defined through analyzing the stability conditions of the coal pillars and the filling bodies. The control effect for the ground subsidence of using the newly proposed mining method was analyzed based on both simulation results and site monitoring results, including the ground subsidence, horizontal displacement, tilt, curvature and horizontal strain. This research could provide suggestions to effectively control ground subsidence for a mine site with similar geologic conditions.

Ubiquitin-mediated regulation responds rapidly to specific stimuli; this rapidity is particularly important for defense responses to pathogen attack. Here, we investigated the role of the E3 ubiquitin ligase Erysiphe necator-induced RING finger protein 1 (EIRP1) in the defense response of Chinese wild grapevine Vitis pseudoreticulata. The regulatory function of E3 ubiquitin ligase EIRP1 was investigated using molecular, genetic and biochemical approaches. EIRP1 encodes a C3HC4-type Really Interesting New Gene (RING) finger protein that harbors E3 ligase activity. This activity requires the conserved RING domain, and VpWRKY11 also interacts with EIRP1 through the RING domain. VpWRKY11 localizes to the nucleus and activates W-box-dependent transcription in planta. EIRP1 targeted VpWRKY11 in vivo, resulting in VpWRKY11 degradation. The expression of EIRP1 and VpWRKY11 responds rapidly to powdery mildew in Vitis pseudoreticulata grapevine; also, overexpression of EIRP1 in Arabidopsis confers enhanced resistance to the pathogens Golovinomyces cichoracearum and Pseudomonas syringae pv tomato DC3000. Our data suggest that the EIRP1 E3 ligase positively regulates plant disease resistance by mediating proteolysis of the negative regulator VpWRKY11 via degradation by the 26S proteasome.

To better protect the ecological environment during large scale underground coal mining operations in the northwest of China, the authors have proposed a water-conservation coal mining (WCCM) method. This case study demonstrated the successful application of WCCM in the Yu-Shen mining area. Firstly, by using the analytic hierarchy process (AHP), the influencing factors of WCCM were identified and the identification model with a multilevel structure was developed, to determine the weight of each influencing factor. Based on this, the five maps: overburden thickness contour, stratigraphic structure map, water-rich zoning map of aquifers, aquiclude thickness contour and coal seam thickness contour, were analyzed and determined. This formed the basis for studying WCCM in the mining area. Using the geological conditions of the Yu-Shen mining area, the features of caved zone, water conductive fractured zone (WCFZ) and protective zone were studied. The equations for calculating the height of the “three zones” were proposed. Considering the hydrogeological condition of Yu-Shen mining area, the criteria were put forward to evaluate the impact of coal mining on groundwater, which were then used to determine the distribution of different impact levels. Using strata control theory, the mechanism and applicability of WCCM methods, including height-restricted mining, (partial) backfill mining and narrow strip mining, together with the applicable zone of these methods, were analyzed and identified. Under the guidance of “two zoning” (zoning based on coal mining’s impact level on groundwater and zoning based on applicability of WCCM methods), the WCCM practice was carried out in Yu-Shen mining area. The research findings will provide theoretical and practical instruction for the WCCM in the northwest mining area of China, which is important to reduce the impact of mining on surface and groundwater.

Objective The primary objective of this study was to elucidate the underlying pharmacological mechanisms by which Jingui Shenqi Wan (JGSQW) alleviates postmenopausal osteoporosis (PMOP). Through a systematic investigation, we sought to identify the specific molecular pathways through which JGSQW modulates the progression of PMOP, thereby providing a scientific basis for its clinical application. Methods We established an ovariectomized (OVX) mouse model to simulate estrogen deficiency-induced PMOP. Initially, micro-CT imaging and Alcian blue hematoxylin/orange G (ABH/OG) staining were employed to assess the effects of JGSQW on bone microarchitecture and bone mass preservation. Immunohistochemistry (IHC) was then utilized to evaluate the expression of osteogenic markers, including Osterix (OSX), Runx2, and Osteopontin (OPN). Additionally, Tartrate - Resistant Acid Phosphatase (TRAP) staining was performed to visualize and quantify osteoclasts. We further investigated the potential role of JGSQW in modulating the pyroptosis pathway. Results JGSQW effectively alleviates the destruction of bone microstructure and the loss of bone mass caused by estrogen deficiency, an effect that appears to be mediated by promoting osteogenesis. Additionally, JGSQW significantly downregulates the expression of GSDMD in osteoblasts and mitigates the abnormal release of inflammatory factors, thereby maintaining the normal functional activities of osteoblasts. Conclusion JGSQW may effectively mitigate the progression of estrogen deficiency-induced PMOP by inhibiting the dysregulated activation of osteoblast pyroptosis.

Background RING is one of the largest E3 ubiquitin ligase families and C3H2C3 type is the largest subfamily of RING, which plays an important role in plant growth and development, and growth and responses to biotic and abiotic stresses. Results A total of 143 RING C3H2C3-type genes ( RCHCs ) were discovered from the grapevine genome and separated into groups (I-XI) according to their phylogenetic analysis, and these genes named according to their positions on chromosomes. Gene replication analysis showed that tandem duplications play a predominant role in the expansion of VvRCHCs family together. Structural analysis showed that most VvRCHCs (67.13 %) had no more than 2 introns, while genes clustered together based on phylogenetic trees had similar motifs and evolutionarily conserved structures. Cis -acting element analysis showed the diversity of VvRCHCs regulation. The expression profiles of eight DEGs in RNA-Seq after drought stress were like the results of qRT-PCR analysis. In vitro ubiquitin experiment showed that VyRCHC114 had E3 ubiquitin ligase activity, overexpression of VyRCHC114 in Arabidopsis improved drought tolerance. Moreover, the transgenic plant survival rate increased by 30 %, accompanied by electrolyte leakage, chlorophyll content and the activities of SOD, POD, APX and CAT were changed. The quantitative expression of AtCOR15a , AtRD29A , AtERD15 and AtP5CS1 showed that they participated in the response to drought stress may be regulated by the expression of VyRCHC114 . Conclusions This study provides valuable new information for the evolution of grapevine RCHCs and its relevance for studying the functional characteristics of grapevine VyRCHC114 genes under drought stress.

Stilbene synthase is a plant-specific polyketide synthase, and plays important roles in diverse metabolic processes. The genomic stilbene synthase gene was cloned from accession “Baihe-35-1” of Chinese wild Vitis pseudoreticulata, and a stilbene synthase of V. pseudoreticulata (VpSTS) transcripts expressed in the grape-powdery mildew interaction were determined by semi-quantitative RT-PCR. To monitor VpSTS expression in plant, the promoter region flanking the 5′ VpSTS coding region was isolated from the genomic DNA of Chinese wild V. pseudoreticulata accession Baihe-35-1. Alignment of the VpSTS promoter sequence showed a 56.4% identity to Vitis vinifera. To identify the upstream region of the VpSTS gene required for promoter activity, a series of VpSTS promoter deletion derivatives was constructed. Each deletion construct was analyzed by Agrobacterium-mediated transient transformation in grapevine and tobacco leaves after infection by Uncinula necator and Alternaria alternata. In transiently transformed grapevine leaves, GUS activity was also determined after treatment with salicylic acid (SA) and 4°C cold. Analysis of a series of 5′ deletions of the VpSTS promoter in grapevine leaves indicated that the proximal 162 bp from the transcription initiation site was proved to be necessary for establishing both the constitutive and induced pattern of expression.

The gene encoding stilbene synthase (STS) plays a central role in many biochemical and physiological actions, and its metabolite resveratrol possesses broad-spectrum resistance to pathogens, as well as diverse pharmacological properties, notably an anticancer effect. Here, we report the expression analysis of the gene encoding STS and its promoter function from a powdery mildew (PM)-resistant Chinese wild Vitis pseudoreticulata, and compare it with two PM-susceptible cultivated grapevines, Vitis vinifera cvs. Carignane and Thompson Seedless. We show an unusual expression pattern of STS in V. pseudoreticulata, which differs markedly from that of the cultivated species. Sequence comparisons reveal that the genomic DNA sequences encoding STS in the three grapevines are highly conserved, but a novel residue mutation within the key motif of STS is solely present in V. pseudoreticulata. Moreover, the STS promoter in V. pseudoreticulata displays a significantly different structure from that found in the two V. vinifera. The three promoter-driven GUS differential expression patterns in transformed tobacco plants induced with Alternaria alternata, methyl jasmonate, and wounding indicated that the structurally different STS promoter of V. pseudoreticulata is responsible for its specific regulatory function. We also demonstrate that the expression of STS genes from their native promoters are functional in transformed tobacco and retain pathogen inducibility. Importantly, the genomic DNA-2 of V. pseudoreticulata under its native promoter shows good induction and the maximum level of resveratrol content. These findings further our understanding of the regulation of STS expression in a resistant grapevine and provide a new pathogen-inducible promoter system for the genetic improvement of plant disease resistance.