Regulatory Mechanism of Proanthocyanidins in Grape Peels Using vvi-miR828a and Its Target Gene VvMYBPA1.

Anthocyanins and proanthocyanidins are considered to be essential secondary metabolites in grapes and are used to regulate metabolic processes, while miRNAs are involved in their synthesis of anthocyanins and proanthocyanidins to regulate metabolic processes. The present research work was carried out to investigate the underlying regulatory mechanism of target genes in the grape cultivars 'Italia' and 'Benitaka'. miRNA and transnscriptomic sequencing technology were employed to characterize both the profiles of miRNAs and the transcripts of grape peels at 10 and 11 weeks post flowering (10 wpf and 11 wpf). The results revealed that the expression level of vvi-miR828a in 'Italia' at 10 and 11 wpf was significantly higher than that in 'Benitaka'. miRNA-seq analysis predicted MYBPA1 to be the target gene of vvi-miR828a. In transcriptome analysis, the expression level of the VvMYBPA1 gene in 'Benitaka' was significantly higher than that in 'Italia'; in addition, the TPM values (expression levels) of VvMYBPA1 and miR828a also showed an evident negative correlation. The determination of the proanthocyanidin (PA) content in 'Italia' and 'Benitaka' peels at 11 wpf demonstrated that the PA content of 'Benitaka' was significantly higher than that of 'Italia'. The outcomes of RT-qRCR analysis exhibited that the expression levels of the VdPAL, VdCHS, VdCHI, VdDFR, VdMYB5b, VdANR, and VdMYBPA1 genes related anthocyanin and proanthocyanidin pathways were reduced, while the expression levels of all of the above genes were increased after the transient expression of the VvMYBPA1 vector into grape leaves. The results of the transient overexpression experiment of vvi-miR828a before the veraison period of strawberry fruits showed that vvi-miR828a can significantly slow down the coloration of strawberries. The vvi-miR828a negatively regulates the accumulation of proanthocyanidins in grape fruits by inhibiting the expression of VvMYBPA1.

Metabolomics of Vitis davidii Foëx. grapes from southern China: Flavonoids and volatiles reveal the flavor profiles of five spine grape varieties.

The spine grapes (Vitis davidii Foëx.) are wild grape species that grow in southern China, and can be used for table grapes, juicing and winemaking. To systematically investigate the flavor profiles of spine grapes, flavonoids and volatile compounds were detected in five spine grape varieties (Seputao, Ziqiu, Miputao, Tianputao and Baiputao) using HPLC-QqQ-MS/MS and GC-MS. The content of flavonoids highly depended on the variety, such as the total concentrations of anthocyanins (91.43-328.85 mg/kg FW) and flavonols (33.90 to 83.16 mg/kg FW). The volatile compounds with higher odor active value were selected to describe the aroma of spine grapes. Hexanal, (E)-2-hexenal and (E, Z)-2,6-nonadienal contributed to the higher herbaceous flavor to Baiputao and Ziqiu. ß-Damascenone and (E)-2-nonenal gave Baiputao a flavor with more floral, fruity and earthy. Their characteristic flavor compounds were subsequently revealed using multivariate statistical analysis. The results helped producers to further develop and utilize the spine grapes.

Comparative transcriptome analysis of genes involved in paradormant bud release response in 'Summer Black' grape.

Grapevines possess a hierarchy of buds, and the fruitful winter bud forms the foundation of the two-crop-a-year cultivation system, yielding biannual harvests. Throughout its developmental stages, the winter bud sequentially undergoes paradormancy, endodormancy, and ecodormancy to ensure survival in challenging environmental conditions. Releasing the endodormancy of winter bud results in the first crop yield, while breaking the paradormancy of winter bud allows for the second crop harvest. Hydrogen cyanamide serves as an agent to break endodormancy, which counteracting the inhibitory effects of ABA, while H2O2 and ethylene function as signaling molecules in the process of endodormancy release. In the context of breaking paradormancy, common agronomic practices include short pruning and hydrogen cyanamide treatment. However, the mechanism of hydrogen cyanamide contributes to this process remains unknown. This study confirms that hydrogen cyanamide treatment significantly improved both the speed and uniformity of bud sprouting, while short pruning proved to be an effective method for releasing paradormancy until August. This observation highlights the role of apical dominance as a primary inhibitory factor in suppressing the sprouting of paradormant winter bud. Comparative transcriptome analysis revealed that the sixth node winter bud convert to apical tissue following short pruning and established a polar auxin transport canal through the upregulated expression of VvPIN3 and VvTIR1. Moreover, short pruning induced the generation of reactive oxygen species, and wounding, ethylene, and H2O2 collectively acted as stimulating signals and amplified effects through the MAPK cascade. In contrast, hydrogen cyanamide treatment directly disrupted mitochondrial function, resulting in ROS production and an extended efficacy of the growth hormone signaling pathway induction.

Integrated Transcriptome and Metabolome Analysis Revealed the Causal Agent of Primary Bud Necrosis in 'Summer Black' Grape.

Primary bud necrosis of grape buds is a physiological disorder that leads to decreased berry yield and has a catastrophic impact on the double cropping system in sub-tropical areas. The pathogenic mechanisms and potential solutions remain unknown. In this study, the progression and irreversibility patterns of primary bud necrosis in 'Summer Black' were examined via staining and transmission electron microscopy observation. Primary bud necrosis was initiated at 60 days after bud break and was characterized by plasmolysis, mitochondrial swelling, and severe damage to other organelles. To reveal the underlying regulatory networks, winter buds were collected during primary bud necrosis progression for integrated transcriptome and metabolome analysis. The accumulation of reactive oxygen species and subsequent signaling cascades disrupted the regulation systems for cellular protein quality. ROS cascade reactions were related to mitochondrial stress that can lead to mitochondrial dysfunction, lipid peroxidation causing damage to membrane structure, and endoplasmic reticulum stress leading to misfolded protein aggregates. All these factors ultimately resulted in primary bud necrosis. Visible tissue browning was associated with the oxidation and decreased levels of flavonoids during primary bud necrosis, while the products of polyunsaturated fatty acids and stilbenes exhibited an increasing trend, leading to a shift in carbon flow from flavonoids to stilbene. Increased ethylene may be closely related to primary bud necrosis, while auxin accelerated cell growth and alleviated necrosis by co-chaperone VvP23-regulated redistribution of auxin in meristem cells. Altogether, this study provides important clues for further study on primary bud necrosis.

Pectate Lyase Gene VvPL1 Plays a Role in Fruit Cracking of Table Grapes.

Fruit cracking seriously affects the commercial value of table grapes. To explore whether cell wall disassembly influences grape berry cracking, first, the differences in the cell wall metabolism were compared between cracking-resistant "Shennongjinhuanghou" (SN) and cracking-susceptible "Xiangfei" (XF) varieties. Our results showed that cell wall disassembly events were extremely different between "SN" and "XF." The cracking-resistant "SN" had a higher pectinmethylesterase activity in the early stage and lower polygalacturonase, ß-galactosidase, pectate lyase, and cellulase activities from veraison, cooperatively yielding higher ionically bound pectin, covalently bound pectin, hemicellulose, and lower water-soluble pectin, leading to a stronger skin break force and elasticity and conferring "SN" with higher cracking resistance. Furthermore, the function of the VvPL1 gene in fruit cracking was verified by heterologously transforming tomatoes. The transgenic experiment showed that overexpressed fruits had a higher activity of pectate lyase from the breaking stage and a lower level of covalently bound pectin, ionically bound pectin, cellulose, and hemicellulose and a higher level of water-soluble pectin at the red ripe stage, which resulted in a significantly reduced skin break force and flesh firmness and increased fruit cracking incidences. In conclusion, our results demonstrated that the cracking susceptibility of the grape berry is closely related to cell wall disassembly events and VvPL1 plays an important role in fruit cracking.

Pochonia chlamydosporia Isolate PC-170-Induced Expression of Marker Genes for Defense Pathways in Tomatoes Challenged by Different Pathogens.

Pochonia chlamydosporia is a fungal parasite of nematode eggs. Studies have shown that some strains of Pochonia chlamydosporia can promote plant growth and induce plants' systemic resistance to root-knot nematodes by colonizing in their roots. This study aimed to verify the effect of the PC-170 strain on tomato growth and systemic resistance. Split-root experiments were conducted to observe the systemic resistance induced by PC-170. To explore the defense pathway that was excited due to the colonization by PC-170, we tested the expression of marker genes for defense pathways, and used mutant lines to verify the role of plant defense pathways. Our results showed that PC-170 can colonize roots, and promotes growth. We found a role for jasmonic acid (JA) in modulating tomato colonization by PC-170. PC-170 can activate tomato defense responses to reduce susceptibility to infection by the root-knot nematode Meloidogyne incognita, and induced resistance to some pathogens in tomatoes. The marker genes of the defense pathway were significantly induced after PC-170 colonization. However, salicylic acid (SA)- and jasmonic acid (JA)-dependent defenses in roots were variable with the invasion of different pathogens. Defense pathways play different roles at different points in time. SA- and JA-dependent defense pathways were shown to cross-communicate. Different phytohormones have been involved in tomato plants' responses against different pathogens. Our study confirmed that adaptive JA signaling is necessary to regulate PC-170 colonization and induce systemic resistance in tomatoes.

Effect of calcium on relieving berry cracking in grape (Vitis vinifera L.) 'Xiangfei'.

Fruit cracking is a physiological disorder in many plant species that leads to severe economic losses. The aim of this study was to investigate the effect of calcium on fruit cracking and explore the underlying mechanisms. We studied the effect of exogenous calcium on grape berry cracking, calcium absorbance and distribution, and cell wall metabolism in the cracking-susceptible cultivar 'Xiangfei'. Calcium significantly reduced the frequency of fruit cracking, increased the break force of the berry skin, and stimulated storage of calcium. In addition, calcium increased the content of protopectin and inhibited the increase in content of water-soluble pectin, by regulating the transcription and activities of enzymes associated with cell wall metabolism. Taken together, the results indicated that dipping grape berries in calcium solution is effective in preventing fruit cracking by stimulating calcium uptake, inhibiting cell wall disassembly, and promoting cell wall strengthening.

Transcriptome analysis of metabolisms related to fruit cracking during ripening of a cracking-susceptible grape berry cv. Xiangfei (Vitis vinifera L.).

BACKGROUND: Grape is an economically valuable fruit around the world. However, some cultivars are prone to fruit cracking during ripening, leading to severe losses. OBJECTIVE: We aimed to find important metabolisms related to fruit cracking during ripening process. METHODS: RNA-Sequence and analysis was applied to the pericarp of cracking-susceptible 'Xiang Fei' at 1 (W1), 2 (W2) and 3 weeks (W3) after veraison on Illumina HiSeq xten; RESULTS: Compared with W1, the berry cracking rate increased significantly in W2 and W3. Through transcriptomic analysis, a total of 22,609 genes were expressed in the grape pericarp, among which 805 and 2758 genes were significantly differentially regulated in W1-vs.-W2 and W1-vs.-W3 comparison, respectively. Besides, 304 and 354 genes were up- and down-regulated in both comparisons. The significantly enriched GO terms of both W1-W2 and W1-W3 are related to cell wall and wax biosynthesis. And lipid metabolism, which are involved in the top 20 enriched KEGG pathways of both comparisons, was related to wax biosynthesis. Further, GO enrichment analysis of differentially expressed genes (DEGs) with same regulatory changes also indicated that the continuously up-regulated DEGs are significantly enriched in cell wall component biosynthesis and hydrolase. CONCLUSION: These findings suggested that genes related to cell wall metabolism and cuticle biosynthesis may play important roles in regulating grape berry cracking. Our results provide a reference for further studies on the molecular mechanism underlying fruit cracking.

Quantification of Cl-PAHs and their parent compounds in fish by improved ASE method and stable isotope dilution GC-MS.

This study aimed at developing a simple and accurate method for determination of emerging chlorinated polycyclic aromatic hydrocarbons (Cl-PAHs) in fish by stable isotope dilution gas chromatography tandem mass spectrometry. Fish samples were extracted by improved accelerated solvent extraction (ASE) method. Matrix effects were observed, and matrix-matched calibration was verified with good intra-day and inter day precisions (lower than 16.1% and 15.1% respectively). Method detection limits were 0.10-5.62 ng g-1 (dry weight) with satisfactory linearity, and recoveries ranged from 50% to 150%, with relative standard deviation values less than 18.5% at different concentration levels. This improved ASE method was proved to be suitable for analyzing Cl-PAHs in fish samples, with good analytical selectivity, linearity, recovery and precision. Furthermore, the composition analysis revealed that chlorinated compounds of phenanthrene, pyrene and acenaphthene were dominated in Cl-PAHs contaminants. The correlationship between the pollution of Cl-PAHs and their corresponding parent structures in fish samples was also analyzed in detail.

Genome-wide identification of Dicer-like, Argonaute and RNA-dependent RNA polymerase gene families and their expression analyses in response to viral infection and abiotic stresses in Solanum lycopersicum.

Dicer, Argonaute and RNA-dependent RNA polymerase form the core components to trigger RNA silencing. Although tomato (Solanum lycopersicum) is a dicotyledon model plant, no systematic analysis and expression profiling of these genes in tomato has been undertaken previously. In this study, seven Dicer-like (SlDCLs), 15 Argonaute (SlAGOs) and six RNA-dependent RNA polymerase (SlRDRs) genes were identified in tomato. These genes were categorized into four subgroups based on phylogenetic analyses. Comprehensive analyses of gene structure, genomic localization and similarity among these genes were performed. Their expression patterns were investigated by means of expression models in different tissues and organs using online data and semi-quantitative RT-PCR. Many of the candidate genes were up-regulated in response to Tomato yellow leaf curl virus infection and abiotic stresses. The expression models of tandem gene duplications among SlDCL2s indicated the DCL2 family plays an important role in the evolution of tomato.