Identification of watermelon H3K4 and H3K27 genes and their expression profiles during watermelon fruit development.

BACKGROUND: The ClaH3K4s and ClaH3K27s gene families are subfamilies of the SET family, each with a highly conserved SET structure domain and a PHD structural domain. Both participate in histone protein methylation, which affects the chromosome structure and gene expression, and is essential for fruit growth and development. METHODS AND RESULTS: In order to demonstrate the structure and expression characteristics of ClaH3K4s and ClaH3K27s in watermelon, members of the watermelon H3K4 and H3K27 gene families were identified, and their chromosomal localization, gene structure, and protein structural domains were analyzed. The phylogeny and covariance of the gene families with other species were subsequently determined, and the expression profiles were obtained by performing RNA-Seq and qRT-PCR. The watermelon genome had five H3K4 genes with 3207-8043 bp nucleotide sequence lengths and four H3K27 genes with a 1107-5499 bp nucleotide sequence. Synteny analysis revealed the close relationship between watermelon and cucumber, with the majority of members displaying a one-to-one covariance. Approximately half of the 'Hua-Jing 13 watermelon' ClaH3K4s and ClaH3K27s genes were expressed more in the late fruit development stages, while the changes were minimal for the remaining half. H3K4-2 expression was observed to be slightly greater on day 21 compared to other periods. Moreover, ClaH3K27-1 and ClaH3K27-2 were hardly expressed throughout the developing period, and ClaH3K27-4 exhibited the highest expression. CONCLUSION: These results serve as a basis for further functional characterization of the H3K4 and H3K27 genes in the fruit development of watermelon.

Comparative microbiome analysis reveals the variation in microbial communities between 'Kyoho' grape and its bud mutant variety.

Microbes are an important part of the vineyard ecosystem, which significantly influence the quality of grapes. Previously, we identified a bud mutant variety (named 'Fengzao') from 'Kyoho' grapes. The variation of microbial communities in grape and its bud mutant variety has not been studied yet. So, in this study, with the samples of both 'Fengzao' and 'Kyoho', we conducted high-throughput microbiome sequencing and investigated their microbial communities in different tissues. Obvious differences were observed in the microbial communities between 'Fengzao' and 'Kyoho'. The fruit and the stem are the tissues with relatively higher abundance of microbes, while the leaves contained less microbes. The fruit and the stem of 'Kyoho' and the stem of 'Fengzao' had relatively higher species diversity based on the alpha diversity analysis. Proteobacteria, Enterobacteriaceae and Rhodobacteraceae had significantly high abundance in 'Fengzao'. Firmicutes and Pseudomonas were highly abundant in the stems of 'Kyoho', and family of Spirochaetaceae, Anaplasmataceae, Chlorobiaceae, and Sphingomonadaceae, and genera of Spirochaeta, Sphingomonas, Chlorobaculum and Wolbachia were abundant in the fruits of 'Kyoho'. These identified microbes are main components of the microbial communities, and could be important regulators of grapevine growth and development. This study revealed the differences in the microbial compositions between 'Kyoho' and its bud mutant, and these identified microbes will be significant resources for the future researches on the quality regulation and disease control of grapevines.

Genome-wide characterization of long terminal repeat retrotransposons provides insights into trait evolution of four cucurbit species.

Cucurbits are a diverse plant family that includes economically important crops, such as cucumber, watermelon, melon, and pumpkin. Knowledge of the roles that long terminal repeat retrotransposons (LTR-RTs) have played in diversification of cucurbit species is limited; to add to understanding of the roles of LTR-RTs, we assessed their distributions in four cucurbit species. We identified 381, 578, 1086, and 623 intact LTR-RTs in cucumber (Cucumis sativus L. var. sativus cv. Chinese Long), watermelon (Citrullus lanatus subsp. vulgaris cv. 97103), melon (Cucumis melo cv. DHL92), and Cucurbita (Cucurbita moschata var. Rifu), respectively. Among these LTR-RTs, the Ale clade of the Copia superfamily was the most abundant in all the four cucurbit species. Insertion time and copy number analysis revealed that an LTR-RT burst occurred approximately 2 million years ago in cucumber, watermelon, melon, and Cucurbita, and may have contributed to their genome size variation. Phylogenetic and nucleotide polymorphism analyses suggested that most LTR-RTs were formed after species diversification. Analysis of gene insertions by LTR-RTs revealed that the most frequent insertions were of Ale and Tekay and that genes related to dietary fiber synthesis were the most commonly affected by LTR-RTs in Cucurbita. These results increase our understanding of LTR-RTs and their roles in genome evolution and trait characterization in cucurbits.

Proteome-Wide Identification of Non-histone Lysine Methylation during Grape Berry Ripening.

To gain a comprehensive understanding of non-histone methylation during berry ripening in grape (Vitis vinifera L.), the methylation of non-histone lysine residues was studied using a 4D label-free quantitative proteomics approach. In total, 822 methylation sites in 416 methylated proteins were identified, with xxExxx_K_xxxxxx as the conserved motif. Functional annotation of non-histone proteins with methylated lysine residues indicated that these proteins were mostly associated with "ripening and senescence", "energy metabolism", "oxidation-reduction process", and "stimulus response". Most of the genes encoding proteins subjected to methylation during grape berry ripening showed a significant increase in expression during maturation at least at one developmental stage. The correlation of methylated proteins with QTLs, SNPs, and selective regions associated with fruit quality and development was also investigated. This study reports the first proteomic analysis of non-histone lysine methylation in grape berry and indicates that non-histone methylation plays an important role in grape berry ripening.

DNA methylation reprogramming provides insights into light-induced anthocyanin biosynthesis in red pear.

DNA methylation, an epigenetic mark, is proposed to regulate plant anthocyanin biosynthesis. It well known that light induces anthocyanin accumulation, with bagging treatments commonly used to investigate light-controlled anthocyanin biosynthesis. We studied the DNA methylome landscape during pear skin coloration under various conditions (fruits re-exposed to sunlight after bag removal). The DNA methylation level in gene body/TE and its flanking sequence was generally similar between debagged and bagged treatments, however differentially methylated regions (DMRs) were re-modelled after light-exposure. Both DNA demethylase homologs and the RNA-directed DNA methylation (RdDM) pathways contributed to this re-distribution. A total of 310 DEGs were DMR-associated during light-induced anthocyanin biosynthesis between debagged and bagged treatments. The hypomethylated mCHH context was seen within the promoter of PyUFGT, together with other anthocyanin biosynthesis genes (PyPAL, PyDFR and PyANS). This enhanced transcriptional activation and promoted anthocyanin accumulation after light re-exposure. Unlike previous reports on bud sports, we did not detect DMRs within the MYB10 promoter. Instead, we observed the genome-wide re-distribution of methylation patterns, suggesting different mechanisms underlying methylation patterns of differentially accumulated anthocyanins caused by either bud mutation or environment change. We investigate the dynamic landscape of genome-scale DNA methylation, which is the combined effect of DNA demethylation and RdDM pathway, in the process of light-induced fruit colour formation in pear. This process is regulated by methylation changes on promoter regions of several DEGs. These results provide a DMR-associated DEGs set and new insight into the mechanism of DNA methylation involved in light-induced anthocyanin biosynthesis.

Dissection of the Pearl of Csaba pedigree identifies key genomic segments related to early ripening in grape.

Pearl of Csaba (PC) is a valuable backbone parent for early-ripening grapevine (Vitis vinifera) breeding, from which many excellent early ripening varieties have been bred. However, the genetic basis of the stable inheritance of its early ripening trait remains largely unknown. Here, the pedigree, consisting of 40 varieties derived from PC, was re-sequenced for an average depth of ∼30×. Combined with the resequencing data of 24 other late-ripening varieties, 5,795,881 high-quality single nucleotide polymorphisms (SNPs) were identified following a strict filtering pipeline. The population genetic analysis showed that these varieties could be distinguished clearly, and the pedigree was characterized by lower nucleotide diversity and stronger linkage disequilibrium than the non-pedigree varieties. The conserved haplotypes (CHs) transmitted in the pedigree were obtained via identity-by-descent analysis. Subsequently, the key genomic segments were identified based on the combination analysis of haplotypes, selective signatures, known ripening-related quantitative trait loci (QTLs), and transcriptomic data. The results demonstrated that varieties with a superior haplotype, H1, significantly (one-way ANOVA, P < 0.001) exhibited early grapevine berry development. Further analyses indicated that H1 encompassed VIT_16s0039g00720 encoding a folate/biopterin transporter protein (VvFBT) with a missense mutation. VvFBT was specifically and highly expressed during grapevine berry development, particularly at veraison. Exogenous folate treatment advanced the veraison of "Kyoho". This work uncovered core haplotypes and genomic segments related to the early ripening trait of PC and provided an important reference for the molecular breeding of early-ripening grapevine varieties.

A simple and efficient protocol for transient transformation of sliced grape berries.

Grape is an economically important crop but recalcitrant to Agrobacterium-mediated genetic transformation and in vitro regeneration. Here, we have developed a protocol for transient transformation of grapes by investigating the effects of explant pre-culture and duration of vacuum infiltration on transformation efficiency. Using sliced grape berries of "Shine-Muscat" (Vitis labrusca × Vitis vinifera) between the end of fruit expansion phase and the mature stage as explants, we firstly compared the effect of pre-culture explants into a susceptible state (incubation on Murashige and Skoog (MS) agar plate in the dark at 25 ± 1 °C for 48 h) with no pre-culture and then tested different vacuum infiltration times on transformation efficiency using ß-glucuronidase (GUS) reporter system. Pre-culture increased the susceptibility of explants to the agrobacteria infection and increased transient transformation efficiency as assessed by histochemical GUS activity, with intense blue coloration compared with the faint staining observed in the non-susceptible explants. Using a Circulating Water Vacuum Pump system to facilitate agrobacteria entry into berry cells, we tested vacuum durations of 5, 10, and 15 min and observed that transformation efficiency increased with vacuum duration of infiltration. These results were confirmed by relative gene expression of GUS transgene as assessed by RT-qPCR and GUS activity assay. To further confirm the usefulness of our protocol, we transiently transformed grape berries with the hydrogen peroxide sensor gene VvHPCA3, and this was confirmed by gene expression analysis as well as increased sensitivity of the explants to hydrogen peroxide treatment. Overall, this study has resulted in a simple but efficient transient transformation protocol for grape berries and would be a valuable tool for the rapid testing of gene function and the study of key regulatory networks in this important crop.

Large-scale discovery of non-conventional peptides in grape (Vitis vinifera L.) through peptidogenomics.

Non-conventional peptides (NCPs), which are peptides derived from previously unannotated coding sequences, play important biological roles in plants. In this study, we used peptidogenomic methods that integrated mass spectrometry (MS) peptidomics and a six-frame translation database to extensively identify NCPs in grape. In total, 188 and 2021 non-redundant peptides from the Arabidopsis thaliana and Vitis vinifera L. protein database at Ensembl/URGI and an individualized peptidogenomic database were identified. Unlike conventional peptides, these NCPs derived mainly from intergenic, intronic, upstream ORF, 5'UTR, 3'UTR, and downstream ORF regions. These results show that unannotated regions are translated more broadly than we thought. We also found that most NCPs were derived from regions related to phenotypic variations, LTR retrotransposons, and domestication selection, indicating that the NCPs have an important function in complex biological processes. We also found that the NCPs were developmentally specific and had transient and specific functions in grape berry development. In summary, our study is the first to extensively identify NCPs in grape. It demonstrated that there was a large amount of translation in the genome. These results lay a foundation for studying the functions of NCPs and also provide a reference for the discovery of new functional genes in grape.

Identification of grape H3K4 genes and their expression profiles during grape fruit ripening and postharvest ROS treatment.

Fruit development is modified by different types of epigenetics. Histone methylation is an important way of epigenetic modification. Eight genes related to H3K4 methyltransferase, named VvH3K4s, were identified and isolated from the grape genome based on conserved domain analysis, which could be divided into 3 categories by the phylogenetic relationship. Transcriptome data showed that VvH3K4-5 was obviously up-regulated during fruit ripe, and its expression level was significantly different between 'Kyoho' and 'Fengzao'. The VvH3K4s promoters contains cis-acting elements of in response to stress, indicating that they may be involved in the metabolic pathways regulated by ROS signaling. The subcellular localization experiment and promoter activity analysis experiment on VvH3K4-5 showed that VvH3K4s may be regulated by H2O2. With H2O2 and Hypotaurine treatment, it was found that the expression pattern of most genes was opposite, and the expression level showed different expression trend with the extension of treatment time.

Overexpression of VvPPR1, a DYW-type PPR protein in grape, affects the phenotype of Arabidopsis thaliana leaves.

Pentatricopeptide repeat (PPR) proteins play important roles in plant growth and development. However, little is known about their functions in the leaf morphogenesis of Jingxiu grape (Vitis vinifera L.). Here, we explored the function of VvPPR1, which encodes a DYW-type PPR protein in grape. We showed that VvPPR1 is involved in the regulation of leaf rolling, anthocyanin accumulation, and trichome formation in Arabidopsis thaliana. Analysis of structural characteristics showed that VvPPR1 is a DYW-type PPR gene in the PLS subfamily consisting of 15 PPR motifs. The N-terminal had a targeted chloroplast site, and the C-terminal had a DYW domain. Quantitative PCR analysis revealed that the expression level of VvPPR1 was highest in grape leaves. Subcellular localization revealed that VvPPR1 is localized in the cytoplasm and chloroplast. VvPPR1-overexpressing plants had rolled leaves, high degrees of anthocyanin accumulation, and longer trichomes. The expression levels of genes related to these phenotypes were either significantly up-regulated or down-regulated. These results demonstrate that VvPPR1 is involved in leaf rolling, anthocyanin accumulation, and trichome formation in Arabidopsis; more generally, our findings indicate that VvPPR1 could be a target for improving the cultivation of horticultural crops.

[Synergetic effects of silicon carbide and molecular sieve loaded catalyst on microwave assisted catalytic oxidation of toluene].

Molecular sieve loaded catalyst was prepared by impregnation method, microwave-absorbing material silicon carbide and the catalyst were investigated for catalytic oxidation of toluene by microwave irradiation. Research work examined effects of silicon carbide and molecular sieve loading Cu-V catalyst's mixture ratio as well as mixed approach changes on degradation of toluene, and characteristics of catalyst were measured through scanning electron microscope, specific surface area test and X-ray diffraction analysis. The result showed that the fixed bed reactor had advantages of both thermal storage property and low-temperature catalytic oxidation when 20% silicon carbide was filled at the bottom of the reactor, and this could effectively improve the utilization of microwave energy as well as catalytic oxidation efficiency of toluene. Under microwave power of 75 W and 47 W, complete-combustion temperatures of molecular sieve loaded Cu-V catalyst and Cu-V-Ce catalyst to toluene were 325 degrees C and 160 degrees C, respectively. Characteristics of the catalysts showed that mixture of rare-earth element Ce increased the dispersion of active components in the surface of catalyst, micropore structure of catalyst effectively guaranteed high adsorption capacity for toluene, while amorphous phase of Cu and V oxides increased the activity of catalyst greatly.

[Study on catalytic oxidation of benzene by microwave heating].

The performance in catalytic oxidation of benzene was investigated in two different heating modes, microwave heating and conventional electric furnace heating. The effects of copper (Cu)-manganese (Mn) mass ratio, doping dose of cerium (Ce) and calcination temperature on the catalytic activity of Cu-Mn-Ce/molecular sieve catalyst were also checked in catalytic oxidation of benzene with microwave heating, and the catalysts were subsequently characterized by scanning electron microscope (SEM) and X-ray diffraction (XRD). The results showed that the catalyst had better catalytic activity for the oxidation of benzene under microwave heating than electric furnace heating, and high oxidation efficiency for benzene was reached due to the "local hot spots" and dipole polarization effect of microwave and stable bed reaction temperature. Under the conditions of Cu, Mn and Ce mass ratio 1:1:0.33 and calcination temperature 500 degrees C, the catalyst had the optimal catalytic activity for benzene oxidation, and its light-off temperature and complete combustion temperature were 165 degrees C and 230 degrees C, respectively. It was indicated by characteristics of XRD and SEM that the presence of copper and manganese oxides and Cu1.5Mn1.5O4 with spinel crystal improved the catalytic activity of the catalyst, and the doping of Ce promoted the dispersion and regularization of active components. High calcination temperature led to the sintering of the catalyst surface and agglomeration of active components, which decreased the catalytic activity of the catalyst in the catalytic oxidation

Color Doppler flow imaging diagnosis and treatment selection for erectile dysfunction.

BACKGROUND: Targeted therapy for erectile dysfunction (ED) involves fewer screening tests and provides a variety of treatment choices for patients. Although the advantage of targeted therapy in diagnosis and therapy for ED has been recognized, the rational mode for oriented ED therapy has not been established. This study aimed to investigate targeted diagnosis and therapy for ED. METHODS: A total of 198 patients with ED were included in the study. After intracavernosal vasoactive agent injection was given, color Doppler flow imaging was performed and penile rigidity was classified as Schramek grade 5 (10 minutes duration), grade 4 (10 minutes duration), grade 3 and grade 2, defining four patient groups as group V (143 cases), group IV (23 cases), group III (18 cases), and group II (14 cases). Appropriate and acceptable treatment was recommended to patients according to erection grade. RESULTS: In 198 patients with ED, the peak systolic velocity, end diastolic velocity, and resistance index in the cavernosal artery and dorsal artery and the flow velocity in the deep dorsal vein were not significantly different before injection (P > 0.05). After injection, peak systolic velocity, end diastolic velocity, and resistance index in the cavernosal artery were different among the four groups (P < 0.05). Between each two groups, the difference in resistance index was significant (P < 0.05). The statistical differences in other indexes were not significant (P > 0.05). Selective targeted therapy based on erection grade by color Doppler flow imaging improved the clinical satisfaction rate to 91.91% (182/198). CONCLUSIONS: Based on the routine diagnosis of ED, blood flow indexes in the cavernosal artery are measured by color Doppler flow imaging following minimally invasive intercavernosal injection, which is combined with the Schramek grade of erection. The most appropriate and acceptable treatment is recommended according to the different groups, which improves the clinical satisfaction of treatment for ED and is an ideal model of targeted diagnosis and treatment.

[Value of degree diagnosis with color Doppler flow imaging in the treatment of male erectile dysfunction].

OBJECTIVE: To study the value of degree diagnosis with color Doppler flow imaging (CDFI) in the treatment of male erectile dysfunction (ED). METHODS: After CDFI and intracavernosal vasoactive agent injection (ICI) with papaverine 60 mg and prostaglandin E(1) 10 microg, 198 male ED patients were divided into 4 groups by their penile stiffness based on the Schramek criterion. The therapeutic methods such as antibiotics for prostatitis, sexual behavior therapy, sildenafil, vacuum constriction device (VCD), vena fistula repairing operation, prosthetic implantation and enhanced external counterpulsation (EECP) were recommended to the patients according to their different penile stiffness degree and their different preferences. RESULTS: Prior to ICI, there was no significant difference in the cavernosa artery peak systolic velocities (PSV), end diastolic velocities (EDV), resistant indexes (RI), dorsal artery PSV, EDV, RI and deep dorsal vein velocity (V) (P > 0.05). After ICI, the cavernosa artery PSV, EDV and RI were significantly different (P < 0.05) while other indices were not (P > 0.05). The patients were treated differently according to their preferences. The selective goal-oriented therapy increased the satisfactory rate to 91.91% (182/198). CONCLUSION: Based on the penile stiffness degree with CDFI plus ICI, different therapeutic methods yield a better clinical outcome.

Persistent hiccup associated with intrathecal morphine infusion pump therapy.

Intraspinal drug-delivery therapy has been increasingly used in patients with intractable nonmalignant pain syndromes during the past two decades. Morphine, the only FDA-approved opioid for intrathecal administration, has been the principle agent for such therapy. Although intrathecal morphine infusion can produce profound spinal analgesia, it may also cause some untoward side effects. We describe the first case of persistent hiccup caused by intrathecal morphine infusion therapy.