Constitutive Overexpression of an NB-ARC Gene from Wild Chinese Vitis quinquangularis in Arabidopsis thaliana Enhances Resistance to Phytopathogenic Oomycete and Bacteria.

Resistance (R) genes were used to recognize pathogen effectors directly or indirectly in plants and activate defense signal pathways. Most of these R proteins consist of a nucleotide-binding adaptor (NB-ARC) domain, a leucine-rich repeat (LRR) domain and some also have a coiled-coil (CC) structure. In this study, we cloned a gene which encodes the CC-NB-ARC-LRR R protein (VqCNL) from Chinese wild grapevine Vitis. quinquangularis accession 'Dan-2'. The transcript of VqCNL was obviously induced by inoculation with Plasmopara viticola and the salicylic acid (SA) treatment. The results of sequence analysis showed that the VqCNL gene contained a CC domain at the N-terminus, along with an NB-ARC and an LRR domain at the C-terminus. We transferred this gene into wildtype Arabidopsis and treated transgenic lines with Hyaloperonospora arabidopsidis (Hpa) and Pseudomonas syringae pv. tomato DC3000 (Pst DC3000); the results demonstrated that VqCNL promotes broad spectrum resistance to pathogens. Furthermore, qPCR analysis displayed that VqCNL may display a significant function in disease resistance via activating SA signaling pathways. In general, these conclusions primarily demonstrated that VqCNL enhances the disease resistance level in plants and contributes to future research of the R gene identification for grape breeding biotechnology.

Optimization of In Vitro Embryo Rescue and Development of a Kompetitive Allele-Specific PCR (KASP) Marker Related to Stenospermocarpic Seedlessness in Grape (Vitis vinifera L.).

Seedlessness is one of the highest valued agronomic traits in grapes. Embryo rescue in combination with marker-assisted selection have been widely applied in seedless grape breeding due to the advantages of increasing the ratio of seedless progenies and shortening the breeding cycle. However, the large number of deformed seedlings produced during embryo rescue and the lack of fast, efficient, and low-cost markers severely inhibit the process of seedless grape breeding. In this study, a total of eighty-three grape cultivars (51 seedless and 32 seeded) with diverse genetic backgrounds and two populations derived from embryo rescue, including 113 F1 hybrid individuals (60 seedless and 53 seeded), were utilized. We screened suitable media for converting malformed seedlings into normal seedlings, analyzed the association between the SNP in VviAGL11 and seeded/seedless phenotype, and developed a KASP marker related to stenospermocarpic seedlessness. Our results indicated that the transformation rate of 37.8% was obtained with MS medium supplemented with 2.0 mg·L-1 of 6-BA and 0.5 mg·L-1 of IBA. The presence of an A nucleotide allele at position chr18:26889437 was further confirmed to be fully associated with the stenospermocarpic seedlessness phenotype. The developed KASP marker, based on the verified SNP locus in VviAGL11, successfully distinguished the seedless and seeded genotypes with high precision and throughput. The results will contribute to enhancing the efficiency of embryo rescue and facilitate parent selection and early selection of seedless offspring with molecular markers, thereby accelerating the breeding process in seedless table grapes.

Heterologous VvDREB2c Expression Improves Heat Tolerance in Arabidopsis by Inducing Photoprotective Responses.

Extreme temperatures limit grape production and sustainability. Dehydration-responsive element-binding (DREB) transcription factors affect plant responses to temperature related stresses. Therefore, we investigated the role of VvDREB2c, a DREB-coding gene, found in grapes (Vitis vinifera L.). Protein characterization revealed that VvDREB2c is localized to the nucleus and that its AP2/ERF domain contains three ß-sheets and one α-helix sheet. Analysis of the VvDREB2c promoter region revealed the presence of light-, hormone-, and stress-related cis-acting elements. Furthermore, we observed that the heterologous expression of VvDREB2c in Arabidopsis improved growth, drought tolerance, and heat tolerance. Furthermore, it improved the leaf quantum yield of regulated energy dissipation [Y(NPQ)], elevated the activities of RuBisCO, and phosphoenolpyruvate carboxylase and reduced the quantum yield of non-regulated energy dissipation [Y(NO)] in plants exposed to high temperatures. VvDREB2c-overexpressing lines also specifically upregulated several photosynthesis-related genes (CSD2, HSP21, and MYB102). In addition, VvDREB2c-overexpressing lines reduced light damage and enhanced photoprotective ability by dissipating excess light energy and converting it into heat, which eventually improves tolerance to high temperature. The contents of abscisic acid, jasmonic acid, and salicylic acid and differentially expressed genes (DEGs) in the mitogen-activated protein kinase (MAPK) signaling pathway were affected by heat stress in VvDREB2c-overexpressing lines, which indicated that VvDREB2c positively regulates heat tolerance via a hormonal pathway in Arabidopsis. VvDREB2c promotes heat tolerance in Arabidopsis by exerting effects on photosynthesis, hormones, and growth conditions. This study may provide useful insights into the enrichment of the heat-tolerance pathways in plants.

Preparation and Properties of PED-TDI Polyurethane-Modified Silicone Coatings.

To explore the influence mechanisms of polyurethane soft segments on modified silicone coatings, a series of modified coatings was prepared by introducing different contents of hydroxypropyl-terminated polydimethylsiloxane (PDMS2200) into the soft segment of polyurethane. ATR-FTIR, NMR, CLSM, AFM, contact angle measurement, the tensile test, bacterial adhesion, and the benthic diatom adhesion test were used to investigate the structure, morphology, roughness, degree of microphase separation, surface energy, tensile properties, and antifouling properties of the modified coatings. The results show that PDMS2200 could aggravate the microphase separation of the modified coatings, increase the surface-free energy, and reduce its elastic modulus; when the microphase separation exceeded a certain degree, increasing PDMS2200 would decrease the tensile properties. The PED-TDI polyurethane-modified silicone coating prepared with the formula of PU-Si17 had the best tensile properties and antifouling properties among all modified coatings.

Rapid identification of the short-lived intermediates in alternating-current electrolysis by mass spectrometry.

Herein, we report the rapid mass spectrometric identification of the short-lived intermediates generated under AC electrolysis via combining bipolar electrochemistry with nanoelectrospray ionization in a hybrid ultramicroelectrode/ion emitter. The key reactive intermediates involved in the C-O/O-H cross-metathesis between 4-alkoxy anilines and alcohols were successfully captured and identified for the first time, providing direct evidence for the previously proposed mechanism.

Colored Shade Nets Can Relieve Abnormal Fruit Softening and Premature Leaf Senescence of "Jumeigui" Grapes during Ripening under Greenhouse Conditions.

High temperature causes premature grape leaf senescence, abnormal berry softening, and shortening of the fruiting period. Furthermore, the fruit quality and yield are severely affected. Here, the "Jumeigui" grape quality and leaf senescence were evaluated under shading; green, blue, black, and gray nets were used for shading, and their spectra were measured. At the same density, the shade-net color significantly affected cooling and shading efficiencies, with gray nets showing the best light transmission and cooling effect. Shading significantly alleviated abnormal heat-induced grape softness. The total soluble solids (TSS) content and grape coloration were affected under gray, blue, and green shade nets. Nonetheless, TSS exceeded 18 °Brix under gray, blue, and green nets, as required of first-class high-quality fruit. The peel color was not significantly affected under gray or blue shade nets, whereas unshaded grapes showed clear heat-stress damage, especially on the edges of unshaded bottom leaves, in which the net photosynthesis rate was significantly lower than that under shading, indicating that high light intensity and heat caused premature leaf senescence. Colored shade nets reduced greenhouse temperature and light intensity, thereby alleviating the premature senescence of grape plants. Grape quality under black shade nets was poor, whereas superior quality was achieved using gray or blue shade nets.

Interaction of VvbZIP60s and VvHSP83 in response to high-temperature stress in grapes.

The occurrence of frequent, extreme high temperatures affects agriculture and causes irreversible damage during the ripening period of grapes. Breeding high-temperature-tolerant varieties of grapes is the main way to deal with this challenge, thus necessitating research on the regulatory mechanism of high-temperature tolerance. Extreme high temperature causes the mismatch of proteins in the endoplasmic reticulum in plant cells and initiates the unfolded protein response (UPR). The transcription factor bZIP60 participates in the UPR process. In the present study, VvbZIP60 and VvbZIP60s (unconventional splicing of VvbZIP60) were cloned and expressed in a transgenic system to verify heat tolerance. VvbZIP60s was found to be a key gene in adapting to heat stress. VvbZIP60s/60u interacted with VvHSP83 as observed in two yeast hybrids, with bimolecular fluorescence complementation and pull-down assays. VvHSP83 is also a key gene for plants to adapt to heat stress by participating in the renaturation and degradation of denatured proteins under adversity, causing plants to resist high temperatures. This study provides a basis for analyzing the mechanism of high-temperature tolerance in grapes.

Effect of Short-Time High-Temperature Treatment on the Photosynthetic Performance of Different Heat-Tolerant Grapevine Cultivars.

To explore the photosynthetic response of short-term high-temperature treatments to the leaves of different grape varieties (Shenyue and Shenfeng), we performed 45°C treatments on annual potted grapes in an artificial climate chamber to simulate the summer high-temperature period. We measured the rapid A-Ci response curve, 1,5-ribulose bisphosphate carboxylase/oxygenase (Rubisco) activity and chlorophyll a fluorescence. Photosynthetic performance-related parameters of grapevine leaves were found to be adversely affected by high temperature; the maximum rates of carboxylation of Rubisco and ribulose diphosphate regeneration and Rubisco activity were all significantly reduced. The JI of the JIP-test curve disappeared under high-temperature treatment, and the K peak appeared, indicating that the oxygen-evolving complex (OEC) had been destroyed. Principal component analysis showed that PSII activity (Fv /Fm , DFABS , φEo , ABS/RC) was greatly affected by high temperature. It was also found that the Rubisco activity and OEC destruction strength were greater in one of the cultivars, and we were able to determine heat resistance based on the phenotype and photosynthetic performance. It can be seen from the above that Shenyue leaves have higher heat tolerance and stronger photosynthetic performance than Shenfeng leaves. This shows that photosynthetic performance is strongly correlated with a grapevine's tolerance to abiotic stress.

Ectopic expression of a grape nitrate transporter VvNPF6.5 improves nitrate content and nitrogen use efficiency in Arabidopsis.

BACKGROUND: Nitrate plays an important role in grapevines vegetative and reproductive development. However, how grapevines uptake, translocate and utilize nitrate and the molecular mechanism still remains to be investigated. RESULTS: In this study, we report the functional characterization of VvNPF6.5, a member of nitrate transporter 1/peptide transporter family (NRT1/PTR/NPF) in Vitis vinifera. Subcellular localization in Arabidopsis protoplasts indicated that VvNPF6.5 is plasma membrane localized. Quantitative RT-PCR analysis indicated that VvNPF6.5 is expressed predominantly in roots and stems and its expression is rapidly induced by nitrate. Functional characterization using cRNA-injected Xenopus laevis oocytes showed that VvNPF6.5 uptake nitrate in a pH dependent way and function as a dual-affinity nitrate transporter involved in both high- and low-affinity nitrate uptake. Further ectopic expression of VvNPF6.5 in Arabidopsis resulted in more 15NO3- accumulation in shoots and roots and significantly improved nitrogen use efficiency (NUE). Moreover, VvNPF6.5 might participate in the nitrate signaling by positively regulating the expression of primary nitrate response genes. CONCLUSION: Our results suggested that VvNPF6.5 encodes a pH-dependent, dual-affinity nitrate transporter. VvNPF6.5 regulates nitrate uptake and allocation in grapevines and is involved in primary nitrate response.

[Diagnosis of a case of 2q37 deletion syndrome by whole exome sequencing combined with whole genome low-coverage sequencing method].

OBJECTIVE: To analyze the clinical and genetic characteristics in a girl with 2q37 deletion syndrome. METHODS: Genomic DNA was extracted from peripheral blood samples taken from the patient and her parents, and was subjected to whole exome sequencing (WES) and low-coverage massively parallel copy number variation sequencing (CNV-seq). Candidate CNVs were verified by chromosomal karyotyping analysis and fluorescence quantitative PCR. RESULTS: The child was found to harbor a 6 Mb heterozygous deletion in 2q37 by WES and CNV-seq. The deletion has encompassed 98 genes with a range from GBX2 to LINC01881, and was de novo in origin. The result of fluorescence quantitative PCR was consistent with that of WES and CNV-seq. However, karyotyping analysis has failed to detect the deletion. CONCLUSION: The patient was diagnosed with 2q37 deletion syndrome. Combined WES and CNV-seq method features high resolution, high throughput, and high sensitivity, which can significant raise the diagnostic rate for patients with mental disorder, multiple malformations and unknown syndromes.

Dlg4 and Vamp2 are involved in comorbid epilepsy and attention-deficit hyperactivity disorder: A microarray data study.

OBJECTIVE: Children with epilepsy exhibit a significantly higher risk for attention-deficit hyperactivity disorder (ADHD), which is often associated with lower quality of life. In this study, we aimed to identify molecular mechanisms associated with both epilepsy and ADHD. MATERIALS AND METHODS: Gene expression profiles of GSE12457 and GSE47752 were downloaded from the gene expression omnibus (GEO) database. Differentially expressed genes (DEGs) were separately screened in epilepsy and ADHD samples and compared with controls. Weighted gene coexpression network analysis (WGCNA) was used to identify candidate modules associated with the two disorders. Functional annotation and analysis of hub genes and molecular complex detection (MCODE) was also performed. RESULTS: Three modules closely related to epilepsy and ADHD were screened using WGCNA; DEGs in this module were involved in the synaptic vesicle cycle, axon and neuron regeneration, and neurotransmission. The Dlg4 and Vamp2 genes were selected as common candidate factors in epilepsy and ADHD pathogenesis. CONCLUSION: Dlg4 and Vamp2 could play essential roles in comorbidity between epilepsy and ADHD.

Influence of cluster thinning and girdling on aroma composition in 'Jumeigui' table grape.

Cluster thinning and girdling are common and simple practices applied to improve berry quality in table grape cultivation. However, there is limited information about the accumulation and biosynthesis of the entire aromatic profile under cluster thinning and girdling, notably in table grapes. This research investigated the influences of cluster thinning and girdling (alone or in combination) on aroma profiles, particularly the changes in biosynthesis and accumulation of Muscat-flavored related compounds from véraison to harvest in 'Jumeigui' grape. Cluster thinning and girdling (alone or in combination) significantly increased the concentrations of total soluble solids (TSS) and key aromatic compounds at harvest, with higher concentrations of both under cluster thinning than girdling. Berry weight and titratable acidity (TA) were unaffected by cluster thinning, girdling, or in combination at harvest. Linalool, the most abundant and active odorant related to Muscat flavor, accumulated in 28.6% and 20.2% higher concentrations from cluster thinning than control and girdling at maturity, respectively. Furthermore, higher DXS3 transcript abundance in cluster thinning groups might contribute to the increased accumulation of terpenes and linalool in 'Jumeigui' grape. The results will contribute to further understand the mechanism of source/sink ratio modulation on aroma accumulation and better apply cluster thinning and girdling for grape production.

A key 'foxy' aroma gene is regulated by homology-induced promoter indels in the iconic juice grape 'Concord'.

'Concord', the most well-known juice grape with a parentage of the North American grape species Vitis labrusca L., possesses a special 'foxy' aroma predominantly resulted from the accumulation of methyl anthranilate (MA) in berries. This aroma, however, is often perceived as an undesirable attribute by wine consumers and rarely noticeable in the common table and wine grape species V. vinifera. Here we discovered homology-induced promoter indels as a major genetic mechanism for species-specific regulation of a key 'foxy' aroma gene, anthraniloyl-CoA:methanol acyltransferase (AMAT), that is responsible for MA biosynthesis. We found the absence of a 426-bp and/or a 42-bp sequence in AMAT promoters highly associated with high levels of AMAT expression and MA accumulation in 'Concord' and other V. labrusca-derived grapes. These promoter variants, all with direct and inverted repeats, were further confirmed in more than 1,300 Vitis germplasm. Moreover, functional impact of these indels was validated in transgenic Arabidopsis. Superimposed on the promoter regulation, large structural changes including exonic insertion of a retrotransposon were present at the AMAT locus in some V. vinifera grapes. Elucidation of the AMAT genetic regulation advances our understanding of the 'foxy' aroma trait and makes it genetically trackable and amenable in grapevine breeding.

Recurrent seizures cause immature brain injury and changes in GABA a receptor α1 and γ2 subunits.

OBJECTIVE: Recurrent seizures can cause brain damage and affect the cognitive outcome, particularly in developing children. We aimed to determine the effects of recurrent seizures on the expression of gamma-aminobutyric acid A receptor (GABAAR) α1 and γ2 subunit and neurodevelopment in immature rats. The role of the GABAAR agonist clonazepam and antagonist/partial agonist flumazenil in seizure-induced brain injury was also studied. METHODS: Recurrent seizures (RS) were induced by flurothyl inhalation in immature rats. Clonazepam (CZP) and flumazenil (FMZ) were administered to modulate GABAAR subunit expression in different experimental groups. Neurobehavioral changes and GABAAR α1 and γ2 subunit expression were studied. RESULTS: Inhalation of flurothyl for five days triggered RS and caused reflex delay, inability to adapt to new environments in adulthood, and deficits in long-term learning and memory ability in rats. Down-regulation of GABAAR α1 and γ2 subunits occurred after seizure onset and persisted for a long time. CZP treatment decreased the expression of GABAAR α1 and γ2 subunits and delayed neurodevelopment of the immature rats, whereas FMZ did not show any significant effects. CONCLUSIONS: Changes in GABAAR α1 and γ2 subunit expression and neurodevelopment were related to recurrent seizures and administration of CZP. Thus, GABAAR α1 and γ2 subunits likely play a significant role in the development of immature rats with RS and provide a novel target for therapeutic intervention.

Carbide Precipitation during Tempering and Its Effect on the Wear Loss of a High-Carbon 8 Mass% Cr Tool Steel.

In this paper, the precipitation of carbide and wear loss of high-carbon 8 mass% Cr tool steel at two tempering conditions (i.e., 773⁻803 K and 823⁻853 K) were studied by INCA Steel, EPMA-1720H, XRD, and ML-10 tester. The results show that the particles of test steels include the carbides (Cr7C3 and Cr23C6) and carbides nucleated on Al2O3. When carbides are of the same size, the number of carbides in test steel at a tempering temperature of 773⁻803 K is greater than that at a tempering temperature of 823⁻853 K, especially when the size of carbides is less than 5 µm. Compared with the test steel tempered at 823⁻853 K, the distance between adjacent actual particles reduced by 80.6 µm and the maximum amount of reduction was 9.4% for single wear loss at the tempering temperature of 773⁻803 K. It can be concluded from thermodynamics results that Al2O3 inclusions began to precipitate in liquid, and the precipitation of carbides was at the solid⁻liquid region. Al2O3 can be used as the nucleation interface of carbide, thus promoting the formation of carbides. During the cooling of molten steel, a lower temperature can increase the difference of actual solubility product bigger than equilibrium solubility product, thus promoting the carbide formation.

Systematic Identification and Analysis of Lysine Succinylation in Strawberry Stigmata.

The various post-translational modifications (PTMs) of plant proteins have important regulatory roles in development. We therefore examined various modified proteins from strawberry stigmata and found that succinylation of lysine residues was the most abundant type of modification. We then subjected proteins from strawberry stigmata to an efficient enrichment method for succinylated peptides and identified 200 uniquely succinylated lysines (Suks) in 116 proteins. A bioinformatics analysis revealed that these proteins are involved in important biological processes, including stress responses, vesicular transport, and energy metabolism. Proteomics, combined with immunoprecipitation and immunoblotting, revealed an obvious increase in succinylation of the assembly polypeptide 2 (AP2) and clathrin from 0.5 to 2 h after pollination, suggesting that succinylation is involved in the recognition of pollen-stigma signaling substances and vesicular transport. These results suggest that AP2/clathrin-mediated vesicular transport processes are regulated by lysine succinylation during pollen recognition.

Impact of cluster thinning on transcriptional regulation of anthocyanin biosynthesis-related genes in 'Summer Black' grapes.

Cluster thinning is an agronomic practice that strongly affects anthocyanin biosynthesis in the skin of grape berries. However, the impact of cluster thinning on anthocyanin biosynthesis has not been fully elucidated at the molecular level. Here, we investigated its effects on the berry quality, the biosynthesis of anthocyanins, and the expression levels of related genes from the onset of véraison to harvest in 'Summer Black' grapes. It was observed that the total soluble solid and anthocyanin content in berry skin significantly increased under cluster thinning, whereas the berry weight and titratable acidity showed no differences from the beginning of véraison to harvest. The expression level of most anthocyanin biosynthesis-related genes was significantly up-regulated by cluster thinning from the beginning of véraison and was higher at its end compared to the control. Up-regulation of flavonoid 3',5'-hydroxylase (F3'5'H) and O-methyltransferase (OMT) expression, and down-regulation of flavonoid 3'-hydroxylase (F3'H) expression were observed, which might be the cause of shift in the anthocyanin profile. These findings provide insights into the molecular basis of the relationship between cluster thinning and anthocyanin biosynthesis in the grape berry skin.

High Temperature Affects Photosynthetic and Molecular Processes in Field-Cultivated Vitis vinifera L. × Vitis labrusca L.

High-temperature stress markedly influences grape growth and development. However, how high-temperature stress response differs between controlled and field-cultivated grape is poorly understood. In this study, the effects of heat treatment on grapevines were studied for changes in photosystem II (PSII) activity and expression levels of heat-responsive genes and heat shock protein HSP21. July 31st, 2015 was considered as the post high-temperature treatment ("42°C"; temperatures above 40°C for a period of time each day ranging from 1-7 h) under field cultivation in our experiment. The recovery of chlorophyll fluorescence indicators and the increasing expression of heat-responsive genes and the heat shock protein HSP21 suggested the development of heat tolerance in the form of acclimation in grape. Changes in various parameters of photosynthetic pigment fluorescence and of the electron transport chain (Fv/Fm, PIABS , Wk , RCQA , ΦPo , and ΦEo ) between "42°C" and the 45°C treatment demonstrated that the donor side, reaction center, and acceptor side of PSII were influenced by a critical high temperature. Furthermore, the difference between the two cultivation conditions studied was attributed to other environmental factors and inherent tree vigor.

Changes in the protective mechanism of photosystem II and molecular regulation in response to high temperature stress in grapevines.

The response to high temperature stress, which influences the growth and development of grapes, varies between laboratory conditions and ambient growth conditions, and is poorly understood. In the present study, we investigated the effects of high temperature on grapevines (Vitis vinifera L. × Vitis labrusca L.) grown under artificial and ambient conditions. A temperature of 35 °C did not alter Photosystem II (PS II) activity and the expression of some heat-shock protein (HSPs) genes. These changes were, however, observed at 45 °C under artificial conditions, as well as when the ambient natural temperature was greater than 40 °C. Interestingly, these changes corresponded to shifts in PS II activity and HSPs expression. The protective mechanism of PS II was induced by temperatures greater than 40 °C. These data indicating that the expression of HSFA2, GLOS1 and some heat-shock protein (sHSPs) genes were more sensitive to the heat stress. Unlike the Kyoho grapevines, the Jumeigui grapevines showed rapid and dramatically deterioration in PS II activity and the expression of some heat response genes and HSP21, indicating that the Jumeigui grapevines could not counter the heat stress. These were some differences in PSII activity and the expression of heat response genes between the two cultivated conditions could be attributed to other environmental factors, inherent plant vigor, and the adaptation mechanism.