miR156b-targeted VvSBP8/13 functions downstream of the abscisic acid signal to regulate anthocyanins biosynthesis in grapevine fruit under drought.

Anthocyanins are the primary color components of grapevine berries and wines. In cultivation practices, a moderate water deficit can promote anthocyanin accumulation in red grape skins. Our previous study showed that abscisic acid (ABA) plays a key role in this process. Herein, we identified a microRNA, vv-miR156b, that is generated in grapevine berries in response to drought stress, along with increasing anthocyanin content and biosynthetic structural gene transcripts. In contrast, vv-miR156b short tandem target mimic (STTM) function-loss callus exhibits the opposite phenotype. Results from in vivo and in vitro experiments revealed that the ABA-signaling-regulated transcription factor VvAREB2 binds directly to the ABA-responsive element (ABRE) of the MIR156b promoter and activates miR156b expression. Furthermore, two miR156b downstream targets, VvSBP8 and VvSBP13, exhibited reduced grape anthocyanin content in their overexpressors but there was a contrary result in their CRISPR-edited lines, the decrease in anthocyanin content was rescued in miR156b and SBP8/13 double overexpressors. We further demonstrated that both VvSBP8 and VvSBP13, encoding transcriptional repressors, displayed sufficient ability to interact with VvMYC1 and VvMYBA1, thereby interfering with MYB-bHLH-WD (MBW) repeat transcriptional complex formation, resulting in the repression of anthocyanin biosynthesis. Our findings demonstrate a direct functional relationship between ABA signaling and the miR156-SBP-MBW complex regulatory module in driving drought-induced anthocyanin accumulation in grape berries.

Manganese sulfate application promotes berry flavonoid accumulation in Vitis vinifera cv. 'Cabernet Sauvignon' by regulating flavonoid metabolome and transcriptome profiles.

BACKGROUND: Flavonoids are vital for the development of high-quality grapes and wine, and manganese deficiency decreases grape berry coloration. However, the effects and underlying mechanisms of action of manganese sulfate on grape metabolic profiles have not been adequately researched. In this study, three concentrations of manganese sulfate solutions, 0.5 µmol·L-1 (low, L), 5 µmol·L-1 (middle, M - the standard manganese concentration of Hoagland nutrient solution, control), and 1000 µmol·L-1 (high, H), were applied to the 'Cabernet Sauvignon' grapevine (Vitis vinifera L.) to explore the effect on berry composition. RESULTS: Manganese application improved manganese concentration effectively in grape organs. Furthermore, the concentrations of malvidin 3-O-(6-O-acetyl)-glucoside, malvidin 3-O-glucoside, malvidin-trans-3-O-(6-O-p-coumaryl)-glucoside, and peonidin 3-O-(6-O-acetyl)-glucoside increased significantly under H treatment. Weighted gene co-expression network analysis (WGCNA) revealed that the structural genes (VvDFR, VvUFGT, and VvOMT) of flavonoid biosynthesis were upregulated under H treatment, and their transcription levels correlated positively with malvidin- and peonidin-derived anthocyanin concentrations. CONCLUSIONS: This study suggested that manganese application regulates berry transcriptional and flavonoid metabolic profiles, providing a theoretical basis for improving the color of red grapes and wines. © 2023 Society of Chemical Industry.

Inter- and intra-varietal genetic variations co-shape the polyphenol profiles of Vitis vinifera L. grapes and wines.

Inheritance and mutations are important factors affecting grape phenolic composition. To investigate the inter- and intra-varietal differences in polyphenolic compounds among grapes and wines, 27 clones belonging to eight varieties of Vitis vinifera L. were studied over two consecutive years. A total of 24 polyphenols (nine anthocyanins, three flavanols, five flavonols, and seven phenolic acids) were analyzed, and the physicochemical parameters of the grapes and wines were determined. Polyphenol profiles showed significant varietal and clonal polymorphisms, and malvidin-3-O-glucoside, peonidin-3-O- glucoside, and epicatechin were identified as key biomarkers distinguishing different grapes and wines when using an orthogonal partial least squares discriminant analysis. Further multivariate analysis classified these genotypes into three subclasses, and a somatic variant of 'Malbec', MBVCR6, had the most abundant polyphenolic compounds that were related to the titratable acid content. The current results reveal that varietal and clonal variations are important for obtaining wines with high polyphenol content.

Maturity degree assessment of hospital ward system using integrated fuzzy AHP-TOPSIS model.

BACKGROUND: The hospital ward system is the core service unit of a hospital and an important aspect of hospital management. The maturity of the hospital ward system represents the level of development and improvement in ward management and services. In order to improve the quality of hospital services, it is significant to assess the maturity of the ward system. Although various assessment methods have been proposed in existing studies, there are some issues such as a single-dimensional factor system, subjectivity in qualitative factor values, and insufficient objectivity in the evaluation methods. METHODS: Therefore, based on the data collection of the factors used in the existing literature, this study made correlation analysis, determined the similarity of factors, and established a maturity assessment factor system satisfying distinctiveness and comprehensiveness. Furthermore, an evaluation method for the weights of each factor was proposed based on the fuzzy analytic hierarchy process, while taking account of the ambiguity of expert information. In addition, through the technique for order of preference by similarity to ideal solution method, an objective evaluation method for the maturity of the hospital ward system was established. Finally, a case study involving 6 hospitals in a specific location was conducted. RESULTS: There are a total of 16 factors used to assess the maturity of the hospital ward system, among which the most important is Doctor service. The maturity degree of the 6 researched hospitals are 0.4517, 0.0035, 0.4254, 0.8681, 0.6636, 0.1586, and the maturity degree of the 6 researched hospitals are II, I, II, IV, III, I. CONCLUSION: The factor system constructed in this study effectively reflected the importance of human-related factors in the development process of ward system. The evaluation results were consistent with the actual situation, demonstrating the effectiveness of the proposed method.

[A comparative study of the effects of citrate and heparin anticoagulation on coagulation function and efficacy in children with septic shock undergoing continuous blood purification].

OBJECTIVE: To compare the effects of citrate and heparin anticoagulation on coagulation function and efficacy in children with septic shock undergoing continuous blood purification (CBP), and to provide guidance for CBP anticoagulation in children with septic shock. METHODS: A case control study was conducted. Thirty-seven children with septic shock admitted to the pediatric intensive care unit (PICU) of the First Affiliated Hospital of Gannan Medical University from July 2019 to September 2022 were enrolled as the research subjects. The patients were divided into citrate local anticoagulation group and heparin systemic anticoagulation group according to different anticoagulation methods. The baseline data, the level of coagulation indicators [prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), fibrinogen (Fib), D-dimer] before treatment and 1 day after weaning from CBP, serum inflammatory mediators [interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), hypersensitivity C-reactive protein (hs-CRP), procalcitonin (PCT)], bleeding complications during CBP and 7-day mortality were collected. RESULTS: A total of 37 cases were enrolled finally, including 17 cases with citric acid local anticoagulation and 20 cases with heparin systemic anticoagulation. There was no statistically significant difference in general data such as gender, age, and body weight of children between the two groups. There were no statistically significant differences in baseline levels of coagulation indicators and inflammatory mediators before treatment of children between the two groups. One day after weaning from CBP, both groups showed varying degrees of improvement in coagulation indicators compared with those before treatment. Compared with before treatment, the PT of the heparin systemic anticoagulation group was significantly shortened after 1 day of weaning (s: 11.82±2.05 vs. 13.64±2.54), APTT and TT were significantly prolonged [APTT (s): 51.54±12.69 vs. 35.53±10.79, TT (s): 21.95±4.74 vs. 19.30±3.33], D-dimer level was significantly reduced (mg/L: 1.92±1.58 vs. 4.94±3.94), with statistically significant differences (all P < 0.05). While in the citrate local anticoagulation group, only APTT was significantly prolonged after treatment compared with that before treatment (s: 49.28±10.32 vs. 34.34±10.32, P < 0.05). There were no statistically significant differences in other coagulation indicators compared with before treatment. Compared with the citric acid local anticoagulation group, the PT of the heparin systemic anticoagulation group was significantly shortened after treatment (s: 11.82±2.05 vs. 13.61±3.05, P < 0.05), and the D-dimer level was significantly reduced (mg/L: 1.92±1.58 vs. 3.77±2.38, P < 0.01). The levels of inflammatory mediators in both groups were significantly reduced 1 day after CBP weaning compared with those before treatment [citric acid local anticoagulation group: hs-CRP (mg/L) was 12.53±5.44 vs. 22.65±7.27, PCT (µg/L) was 1.86±1.20 vs. 3.30±2.34, IL-6 (ng/L) was 148.48±34.83 vs. 202.32±48.62, TNF-α (ng/L) was 21.38±7.71 vs. 55.14±15.07; heparin systemic anticoagulation group: hs-CRP (mg/L) was 11.82±4.93 vs. 21.62±8.35, PCT (µg/L) was 1.90±1.08 vs. 3.18±1.97, IL-6 (ng/L) was 143.81±33.41 vs. 194.02±46.89, TNF-α (ng/L) was 22.44±8.17 vs. 56.17±16.92, all P < 0.05]. However, there was no statistically significant difference between the two groups (all P > 0.05). There was no statistically significant difference in bleeding complication during CBP and 7-day mortality in children between the citrate local anticoagulation group and the heparin systemic anticoagulation group (5.9% vs. 30.0%, 17.6% vs. 20.0%, both P > 0.05). CONCLUSIONS: Heparin for systemic anticoagulation and regional citrate anticoagulation can significantly reduce the levels of IL-6, TNF-α, hs-CRP and PCT in children with septic shock, and relieve inflammatory storm. Compared with citric acid local anticoagulation, heparin systemic anticoagulation can shorten the PT and reduce the level of D-dimer in children with septic shock, which may benefit in the prevention and treatment of disseminated intravascular coagulation (DIC).

The effect of inter-varietal variation in sugar hydrolysis and transport on sugar content and photosynthesis in Vitis vinifera L. leaves.

Sugar synthesis from photosynthesis and its utilization through sugar metabolism jointly determine leaf sugar content, and in contrast, excess sugar represses leaf photosynthesis. Although plant photosynthesis is affected by leaf sugar metabolism, the relationship between sugar metabolism and photosynthetic capacity of different grape genotypes remains unclear. In this study, two grape (Vitis vinifera L.) genotypes 'Riesling' (RI, high sugar content in leaf) and 'Petit Manseng' (PM, low sugar content in leaf) were used to evaluate the relationship between sugar metabolism and photosynthesis. Sugar content, chlorophyll content, photosynthetic parameters, enzyme activity, and gene expression related to sucrose metabolism in leaves were measured, and the correlations between photosynthesis and sugar metabolism were assessed. The contents of sucrose and glucose were significantly higher in RI leaves than in PM leaves, while the fructose content pattern was reversed. Cell wall invertase activity for sucrose hydrolysis and the transcript levels of VvCWINV, VvHTs, VvTMT1, VvFKs, and VvHXK2 were also higher in RI leaves than in PM leaves, whereas that of VvHXK1 mediating glucose phosphorylation, was lower in RI leaves than in PM leaves. Net photosynthetic rate, stomatal conductance, transpiration rate, and chlorophyll content were lower in RI leaves than in PM leaves and negatively correlated with glucose content, and the transcript levels of VvCWINV, VvHTs, VvTMT1, and VvHXK2. In conclusion, this study indicates that leaf sugar metabolism and transport are related to photosynthesis in Vitis vinifera L., which provides a theoretical basis for improving grape photosynthesis.

Genetic diversity of the cultivated Salvia miltiorrhiza populations revealed by four intergenic spacers.

For better understanding the genetic diversity and phylogeny of the cultivated Salvia miltiorrhiza populations, four intergenic spacer sequences, ETS, psbA-trnH, trnL-trnF, and ycf1-rps15 of the 40 populations collected from China were Polymerase Chain Reaction (PCR) amplified, analyzed both individually and in combination. Haplotype diversity analysis showed that the cultivated S. miltiorrhiza populations had a very rich genetic diversity and an excellent capacity to resist environmental pressure. The best-fit nucleotide substitution models for ETS, psbA-trnH, trnL-trnF, ycf1-rps15, and their combined sequences were HKY+I, T92, T92, T92+G, and T92+G, respectively; the nucleotide conversion frequency in the combined sequences was lower than the transversion, and the relatively high nucleotide substitution frequencies suggests its high genetic variability. Neutral tests showed that the spacer sequences of the populations conform with the neutral evolution model, and there has been no current expansion events occurred. Phylogeny analyses based on both the individual and the combined sequences showed that the 40 populations were clustered in two clades with a very similar topological structure. The discrimination rate of the combined sequence marker is significantly increased to 52.5% (21 populations) over the highest 35% (13 populations) by the single marker of ETS, though still inadequate but a big step forward. Further exploration of more DNA markers is needed. This study for the first time revealed the rich genetic diversity and phylogeny of the currently cultivated S. miltiorrhiza populations in China and provides novel alternative molecular markers for the genetic identification and resources evaluation of the cultivated S. miltiorrhiza populations.

Dynamic changes in monoterpene accumulation and biosynthesis during grape ripening in three Vitis vinifera L. cultivars.

Monoterpenes are important aroma components in grapes and wines. We analyzed the free and bound monoterpene profiles and the transcript levels of terpenoid biosynthesis genes in Vitis Vinifera cvs. Muscat Hamburg, Riesling, and Sauvignon Blanc grapes at five ripening stages. Principal component analyses revealed that the three cultivars had different free monoterpene profiles at harvest and the early stage of ripening. In all cultivars, the total bound monoterpene contents were higher than the free monoterpene contents during grape ripening. The changes in monoterpene profiles in different grape varieties were correlated with the transcript levels of some VviTPS and VviGT genes. In Riesling, the VviGT14 and VviUGT88A1L1 transcript levels were related to geraniol glucoside accumulation. In Muscat Hamburg, the VviPNLGl1, VviPNLGl2, and VviPNLGl4 transcript levels were related to linalool accumulation. Understanding the dynamic changes in monoterpene accumulation and biosynthesis will allow winemakers to devise strategies to improve grape and wine aromas.

Coexpression of Sucrose Synthase and the SWEET Transporter, Which Are Associated With Sugar Hydrolysis and Transport, Respectively, Increases the Hexose Content in Vitis vinifera L. Grape Berries.

The sugar content of grape berries is affected by many factors. To explore the hexose content in different cultivars, the photosynthesis, vegetative, and reproductive biomass, as well as the enzyme activities and expression levels of genes related to sugar metabolism and sugar contents were measured. Samples were collected 70-110 days after anthesis (DAA), from Riesling (RI), Petit Manseng (PM), and Cabernet Sauvignon (CS) berries cultivated in the field. The results indicated that high expression levels of VvSWEET15 and VvSS3 and a high activity of sucrose synthase (SS) are associated with a higher hexose content in the berries of PM than in the berries of the other two cultivars. These genes promoted hexose accumulation in the berries by regulating sugar hydrolysis and transport. The results of this study indicate that active sugar hydrolysis and transport increase the hexose content of PM berries, which provides insights for grape berry quality improvement and breeding projects in wine production. Main Conclusion: The active VvSS3, sucrose synthase (SS), and VvSWEET15 increases the hexose content in Petit Manseng berries, which are associated with sugar hydrolysis and transport.

Isolation and Identification of Endophytic Fungi in Kernels of Coix lachrymal-jobi L. Cultivars.

Coix lachrymal-jobi L. var. ma-yuen Stapf of Gramineae are annual or perennial herbs and an important food-medicine homologous plants of high value in nutrition, health protection, and comprehensive utilization. In recent years, the revival of researches on its roles in food and medicinal applications of this underutilized grass for food security and economic empowerment of rural communities has been seen . In this research, Coix kernel endophytic fungi were isolated and identified by fungal colony morphology observation combined with the PCR-amplified fungal internal transcribed spacer (ITS) sequence analyses. All together six isolates to five species of Coix endophytic fungi and two isolates to the genus level were identified from the kernels of six Coix cultivars: Penicillium expansum, Penicillium polonicum, Cladosporium cladosporioides, Alternaria alternata, Aspergillus flavus, and two genera of Aspergillus and Fusarium. Potential benefits and harms analyses showed that Penicillium expansum, Aspergillus oryzae, and Cladosporium cladosporioides can produce a variety of beneficial composite enzymes and have an extensive application in microbial chemistry, food science, and fermentation, whereas Penicillium, Aspergillus flavus, Alternaria alternate, and Fusarium can produce corresponding toxins harmful to plants, animals, and humans. These results not only provided a basis for the targeted prevention of contamination in the tissue culture of Coix kernels by the addition of specific antibiotics, but also enriched the endophytic fungi resource pool of Gramineae crops and suggested new ideas for the improvement, cultivation, post-harvest seeds/kernels storage, and the development of new natural drugs.

Foliar-sprayed manganese sulfate improves flavonoid content in grape berry skin of Cabernet Sauvignon (Vitis vinifera L.) growing on alkaline soil and wine chromatic characteristics.

Flavonoids are key determinants of grape quality and wine color. Grapevines growing in alkaline soil are prone to manganese deficiency, which can decrease the contents of secondary metabolites, including flavonoids. We determined the effects of a foliar Mn treatment (MnSO4·H2O) of Cabernet Sauvignon grapevines (V. vinifera L.) growing in alkaline soil on the flavonoid contents in grape skin, and the quality of wine. The Mn treatments were applied in 2017 and 2018, and tended to increase the grape sugars, berry weight, and the contents of phenolic compounds from veraison until harvest. The Mn treatments increased the amounts of acetylated, methylated, and total anthocyanins, as well as the total flavonol contents in grape berry skin at harvest. The wines prepared from these grapes had a higher color intensity than those prepared from grapes from control vines. Foliar-applied MnSO4·H2O can promote flavonoid biosynthesis in grape berries, and improve the color of wine.

Fertility restoration of maize CMS-C altered by a single amino acid substitution within the Rf4 bHLH transcription factor.

Type C cytoplasmic male sterility (CMS-C) is the most commonly used form of CMS in maize hybrid seed production. Restorer of fertility 4 (Rf4), the major fertility restorer gene of CMS-C, is located on chromosome 8S. To positionally clone Rf4, a large F3 population derived from a cross between a non-restorer and restorer (n = 5104) was screened for recombinants and then phenotyped for tassel fertility, resulting in a final map-based cloning interval of 12 kb. Within this 12-kb interval, the only likely candidate for Rf4 was GRMZM2G021276, a basic helix-loop-helix (bHLH) transcription factor with tassel-specific expression. The Rf4 gene product contains a nuclear localization signal and is likely to not interact directly with the mitochondria. Sequence analysis of Rf4 revealed four encoded amino acid substitutions between restoring and non-restoring inbreds, however only one substitution, F187Y, was within the highly conserved bHLH domain. The hypothesis that Rf4 restoration is altered by a single amino acid was tested by using clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR associated protein 9 (Cas9) homology directed repair (HDR) to create isogenic lines that varied for the F187Y substitution. In a population of these CRISPR-Cas9 edited plants (n = 780) that was phenotyped for tassel fertility, plants containing F187 were completely fertile, indicating fertility restoration, and plants containing Y187 were sterile, indicating lack of fertility restoration. Structural modeling shows that this amino acid residue 187 is located within the four helix bundle core, a critical region for stabilizing dimer conformation and affecting interaction partner selection.

Combining powers of linkage and association mapping for precise dissection of QTL controlling resistance to gray leaf spot disease in maize (Zea mays L.).

BACKGROUND: Gray Leaf Spot (GLS causal agents Cercospora zeae-maydis and Cercospora zeina) is one of the most important foliar diseases of maize in all areas where the crop is being cultivated. Although in the USA the situation with GLS severity is not as critical as in sub-Saharan Africa or Brazil, the evidence of climate change, increasing corn monoculture as well as the narrow genetic base of North American resistant germplasm can turn the disease into a serious threat to US corn production. The development of GLS resistant cultivars is one way to control the disease. In this study we combined the high QTL detection power of genetic linkage mapping with the high resolution power of genome-wide association study (GWAS) to precisely dissect QTL controlling GLS resistance and identify closely linked molecular markers for robust marker-assisted selection and trait introgression. RESULTS: Using genetic linkage analysis with a small bi-parental mapping population, we identified four GLS resistance QTL on chromosomes 1, 6, 7, and 8, which were validated by GWAS. GWAS enabled us to dramatically increase the resolution within the confidence intervals of the above-mentioned QTL. Particularly, GWAS revealed that QTLGLSchr8, detected by genetic linkage mapping as a locus with major effect, was likely represented by two QTL with smaller effects. Conducted in parallel, GWAS of days-to-silking demonstrated the co-localization of flowering time QTL with GLS resistance QTL on chromosome 7 indicating that either QTLGLSchr7 is a flowering time QTL or it is a GLS resistance QTL that co-segregates with the latter. As a result, this genetic linkage - GWAS hybrid mapping system enabled us to identify one novel GLS resistance QTL (QTLGLSchr8a) and confirm with more refined positions four more previously mapped QTL (QTLGLSchr1, QTLGLSchr6, QTLGLSchr7, and QTLGLSchr8b). Through the novel Single Donor vs. Elite Panel method we were able to identify within QTL confidence intervals SNP markers that would be suitable for marker-assisted selection of gray leaf spot resistant genotypes containing the above-mentioned GLS resistance QTL. CONCLUSION: The application of a genetic linkage - GWAS hybrid mapping system enabled us to dramatically increase the resolution within the confidence interval of GLS resistance QTL by-passing labor- and time-intensive fine mapping. This method appears to have a great potential to accelerate the pace of QTL mapping projects. It is universal and can be used in the QTL mapping projects in any crops.

Development of highly polymorphic SNP markers from the complexity reduced portion of maize [Zea mays L.] genome for use in marker-assisted breeding.

The duplicated and the highly repetitive nature of the maize genome has historically impeded the development of true single nucleotide polymorphism (SNP) markers in this crop. Recent advances in genome complexity reduction methods coupled with sequencing-by-synthesis technologies permit the implementation of efficient genome-wide SNP discovery in maize. In this study, we have applied Complexity Reduction of Polymorphic Sequences technology (Keygene N.V., Wageningen, The Netherlands) for the identification of informative SNPs between two genetically distinct maize inbred lines of North and South American origins. This approach resulted in the discovery of 1,123 putative SNPs representing low and single copy loci. In silico and experimental (Illumina GoldenGate (GG) assay) validation of putative SNPs resulted in mapping of 604 markers, out of which 188 SNPs represented 43 haplotype blocks distributed across all ten chromosomes. We have determined and clearly stated a specific combination of stringent criteria (>0.3 minor allele frequency, >0.8 GenTrainScore and >0.5 Chi_test100 score) necessary for the identification of highly polymorphic and genetically stable SNP markers. Due to these criteria, we identified a subset of 120 high-quality SNP markers to leverage in GG assay-based marker-assisted selection projects. A total of 32 high-quality SNPs represented 21 haplotypes out of 43 identified in this study. The information on the selection criteria of highly polymorphic SNPs in a complex genome such as maize and the public availability of these SNP assays will be of great value for the maize molecular genetics and breeding community.