Soil salinity severely restricts grapevine growth and development. Here, we integrated whole-genome bisulfite sequencing (WGBS) and RNA sequencing (RNA-seq) to investigate DNA methylation changes and their relationship with gene expression under salt stress in Vitis vinifera L. ‘Shine Muscat’ grapevine. Salt stress altered genome-wide DNA methylation patterns, reducing methylation levels in CG, CHG, and CHH contexts following NaCl treatment. We identified 8606 differentially methylated regions (DMRs) and 3106 DMR-associated genes (DMGs) under salt stress. RNA-seq analysis revealed 2691 differentially expressed genes (DEGs), including multiple stress-related transcription factors (e.g., MYB, NAC, WRKY) and hormone-related genes strongly induced by salinity. Integrative analysis identified 171 genes that were both differentially methylated and differentially expressed, primarily enriched in metabolic pathways, fructose and mannose metabolism, and fatty acid biosynthesis. Notably, several key stress-responsive genes (e.g., VvNAC72, VvBAK1, VvMYBS3) showed coordinated changes between methylation status and transcript abundance. Collectively, this study provides a comprehensive integrative analysis of DNA methylation and transcriptome reprogramming in ‘Shine Muscat’ grapevine under salt stress, revealing potential epigenetic mechanisms involved in transcriptional regulation and salt adaptation. The identified candidate genes provide valuable targets for further functional validation and genetic improvement of grapevine salt tolerance.

MET1 encodes DNA methyltransferase 1, which is increasingly being identified as a major regulator of abiotic stress responses and adaptation. To explore the function of DNA methyltransferase 1 in salt stress, we cloned a grape (Vitis vinifera L.) MET1 subfamily gene, VvMET2b, and overexpressed it into A. thaliana. The phenotypic analysis of transgenic Arabidopsis revealed that VvMET2b improved seed germination and seedling survival under NaCl treatment. Detailed methylome analysis revealed that VvMET2b increased the global methylation level of transgenic plants and altered the quantity of differentially methylated regions (DMRs) and DNA methylation types. Comprehensive transcriptome analyses indicated that many transcription factors, such as NACs, MYBs, and WRKYs were differentially expressed in VvMET2bOE plants under salt stress. VvMET2b overexpression induced the expression of cytokinin negative regulator type-A ARRs, the transmembrane transporter KAT1, inhibited the expression of MYB6, and the up-regulated expression of auxin-related genes Aux/IAAs and down-regulated expression of GH3s, expansin EXPA17 and tonoplast aquaporin TIP2 were mitigated. VvMET2b altered DNA methylation level of MYB6, TIP2 and EXPA17 and thereby may regulate the expression of these genes. Taken together, VvMET2b may regulate seed salt tolerance through DNA methylation changes and certain key gene expression.

Protected cultivation is an effective measure for high-end grape production. Nevertheless, the long-time application of plastic film negatively influences the light environment, and results in a certain decrease in berry quality. In this study, six different light treatments, including white (W), red (R), blue (B), and three different combinations with different ratios of red and blue light (1:1, 4:1, 1:4, respectively), were applied to monitor the quality and sensory properties of 'Queen Nina' grapes. Compared to the control group (without supplemental light), all light treatments significantly increased the size and weight of berries, as well as improved their sugar, anthocyanins, flavonoids, and volatile organic compounds (VOCs) content, whereas all light treatments decreased the levels of chlorophylls and organic acids. Furthermore, the R1B4 treatment improved the content of cyanidin-3-O-glucoside (Cy) and peonidin-3-O-glucoside (Pn), which are the dominant anthocyanin compounds in red grape berry. Additionally, esters, accounting for more than 42% of the VOCs, are the main volatile compounds in 'Queen Nina' grape, and R1B4 treatment was the most favorable treatment for VOCs accumulation. The combination of red and blue light at the 1:4 ratio (R1B4) obtained the highest composite and sensory scores and had the most positive impact on berry coloration, sugars, anthocyanins, flavonoids, and VOCs accumulation, followed by the blue light treatment. In summary, the present results highlight the effective strategy of R1B4 light treatment to increase the berry quality of 'Queen Nina' grape berries.

Although abscisic acid (ABA) plays versatile roles at the fruit development and ripening in the non-climacteric grapevine (Vitis vinifera), the ABA-regulated molecular mechanisms underlying the ripening of non-climacteric grape berry are still insufficient. Here, exogenous ABA application increased endogenous ABA contents by triggering its own biosynthetic and signaling pathway, especially VvNCEDs. Transient overexpression of VvNCED2 in tomato accelerated fruit ripening and ABA accumulation. ABA treatment also significantly affected the abundance of genes participated in ethylene and auxin signaling processes and their hormones cross-regulation ultimately activated the outbreak of ripening on grape berry. Furthermore, ABA obviously improved several ripening-associated procedures, including color development, sugar accumulation and berry softening, by coordinating a lot of genes elaborated in biosynthetic pathways of anthocyanin, sugar and cell wall, respectively. ABA also extensively mediated the transcription levels of several transcription factor family (e.g. MYB, ERF, bHLH, bZIP and WRKY), indicating a involvement of ABA-mediated transcriptional regulation during berry ripening. Our results of the ABA cascade not only help us better understanding the regulation mechanism of berry ripening but also contribute to other areas of viticultural interest.

Grapevine (Vitis vinifera) is an important and popular perennial fruit tree cultivated worldwide. Grapevine ripening is affected by flowering time, and although members of the MADS-box protein family play vital roles in regulating flowering in plants, the functions of MADS-box proteins in grapevine remain largely unknown. AGAMOUS-LIKE 11 (VvAGL11), a MADS-box gene in grapevine, was reported to be a regulator of seed morphogenesis. In this study, heterologous overexpression of VvAGL11 was found to significantly promote flowering in Arabidopsis, suggesting that its active expression in grapevine may induce early flowering and ripening. Transcriptome analysis showed that VvAGL11 overexpression affected the expression of genes involved in stress responses, hormonal signaling responses, and flowering regulation. Notably, VvAGL11 significantly increased the expression of key flowering genes such as FLOWERING LOCUS T (FT), APETALA3 (AP3), and SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 5 (SPL5), which might have contributed to the early flowering in Arabidopsis. In summary, we characterized a novel flowering regulator, VvAGL11, which could be a potential target for early ripening breeding in grapevine.

Ethylene is one crucial phytohormone modulating plants' organ development and ripening process, especially in fruits, but its action modes and discrepancies in non-climacteric grape and climacteric peach in these processes remain elusive. This work is focused on the action mode divergences of ethylene during the modulation of the organ development and ripening process in climacteric/non-climacteric plants. We characterized the key enzyme genes in the ethylene synthesis pathway, VvACO1 and PpACO1, and uncovered that their sequence structures are highly conserved, although their promoters exhibit important divergences in the numbers and types of the cis-elements responsive to hormones, implying various responses to hormone signals. Subsequently, we found the two have similar expression modes in vegetative organ development but inverse patterns in reproductive ones, especially in fruits. Then, VvACO1 and PpACO1 were further validated in promoting fruit ripening functions through their transient over-expression/RNAi-expression in tomatoes, of which the former possesses a weaker role than the latter in the fruit ripening process. Our findings illuminated the divergence in the action patterns and function traits of the key VvACO1/PpACO1 genes in the tissue development of climacteric/non-climacteric plants, and they have implications for further gaining insight into the interaction mechanism of ethylene signaling during the modulation of the organ development and ripening process in climacteric/non-climacteric plants.

Petrocosmea qinlingensis is a protected wild plant endemic in China, inhabiting low-light limestone cliffs but the complete chloroplast genome has not been reported. In this study, we first sequenced and assembled the complete chloroplast genome of P. qinlingensis. The total size of this genome was 153,865 bp, including a large single-copy (LSC) region (84,737 bp), a small single-copy (SSC) region (18,244 bp), and two inverted repeats (IRs) regions (25,442 bp). This genome encoded 111 uniquegenes, consisted of 77 protein-coding genes, four ribosomal RNA genes, and 30 transfer RNA genes. Phylogenomic analysis based on the chloroplast protein-coding genes and showed that the genus Petrocosmea was the closest relative to Raphiocarpus. Our results will support further phylogeographic, population genetic studies of this species.

This study generated whole genome DNA methylation maps to characterize DNA methylomes of grape (cv. ‘Cabernet Franc’) skins and examine their functional significance during grape skin coloration. We sampled grape skin tissues at three key stages (the early stage of grape berry swelling, the late stage of grape berry swelling and the veraison) during which the color of grape berries changed from green to red. DNA methylation levels of grape skins at the three stages were higher in transposable element regions than in the genic regions, and the CG and CHG DNA methylation levels of the genic region were higher than the CHH DNA methylation levels. We identified differentially methylated regions (DMRs) in S2_vs_S1 and S3_vs_S1. The results indicated that DMRs predominantly occurred within the CHH context during grape skin coloration. Many gene ontology (GO)-enriched DMR-related genes were involved in “nucleotide binding,” “catalytic activity” and “ribonucleotide binding” terms; however, many KEGG-enriched DMR-related genes were involved in the “flavonoid biosynthesis” pathway. Our results could provide an important foundation for future research on the development mechanism of grape berries.

BACKGROUND:Plastids originated from an ancient endosymbiotic event and evolved into the photosynthetic organelles in plant cells. They absorb light energy and carbon dioxide, converting them into chemical energy and oxygen, which are crucial for plant development and adaptation. However, little is known about the plastid genome to light adaptation. Petrocosmea, a member of the Gesneriaceae family, comprises approximately 70 species with diverse light environment, serve as an ideal subject for studying plastomes adapt to light. RESULTS:In this study, we selected ten representative species of Petrocosmea from diverse light environments, assembled their plastid genomes, and conducted a comparative genomic analysis. We found that the plastid genome of Petrocosmea is highly conserved in both structure and gene content. The phylogenetic relationships reconstructed based on the plastid genes were divided into five clades, which is consistent with the results of previous studies. The vast majority of plastid protein-coding genes were under purifying selection, with only the rps8 and rps16 genes identified under positive selection in different light environments. Notably, significant differences of evolutionary rate were observed in NADH dehydrogenase, ATPase ribosome, and RNA polymerase between Clade A and the other clades. Additionally, we identified ycf1 and several intergenic regions (trnH-psbA, trnK-rps16, rpoB-trnC, petA-psbJ, ccsA-trnL, rps16-trnQ, and trnS-trnG) as candidate barcodes for this emerging ornamental horticulture. CONCLUSION:We newly assembled ten plastid genomes of Petrocosmea and identified several hypervariable regions, providing genetic resources and candidate markers for this promising emerging ornamental horticulture. Furthermore, our study suggested that rps8 and rps16 were under positive selection and that the evolutionary patterns of NADH dehydrogenase, ATPase ribosome, and RNA polymerase were related to the diversity light environment in Petrocosmea. This revealed an evolutionary scenario for light adaptation of the plastid genome in plants.

AbstractTo date, there is no high-quality sequence for genomes of the East Asian grape species, hindering biological and breeding research efforts to improve grape cultivars. This study presents a ∼522 Mb of theVitis amurensis(Va) genome sequence containing 27,635 coding genes. Phylogenetic analysis indicated thatV. riparia(Vr) may firstly split from the other two species,Va,V. Vinifera(Vv; Pinot Noir: PN40024 and Cabernet Sauvignon). Much divergent gene reservation among three grape duplicated gene sets suggests that the core eudicot common hexaploidy (ECH), 130 million years ago (Mya), has still played a non-negligible role in grape species divergence and biological innovation. Prominent accumulation of sequence variants might have improved cold resistance inVa, resulting in a more robust cold resistance gene regulatory network than those inVvandVr. In contrast,Vapreserved much fewer NBS disease resistance genes than the other grapes. Notably, multi-omics analysis identified one trans-cinnamate 4-monooxygenase gene positively correlated to the resveratrol accumulated duringVaberry development. A selective sweep analysis revealed a hypotheticalVasex-determination region (SDR). Besides, a PPR-containing protein-coding gene in the hypothetical SDR may be related with sex determination inVa. The content and arrangement order of genes in the putative SDR of femaleVawere similar to the SDR of femaleVv. However, the putative SDR of femaleValost one Flavin-containing monooxygenases (FMO) and contained one extra uncharacterized protein-coding gene. These findings will improve the understanding ofVitisbiology and contribute to the improvement of grape breeding.

Ribosome biogenesis is a fundamental process in eukaryotic cells. NOTCHLESS (NLE) is involved in 60S ribosome biogenesis in yeast, but its role in Arabidopsis (A. thaliana) remains exclusive. Here, we found that Arabidopsis NLE (AtNLE) is highly conservative in phylogeny, which encoding a WD40-repeat protein. AtNLE is expressed in actively dividing tissues. AtNLE-GFP is localized in the nucleus. AtNLE physically interacts with the MIDAS domain of AtMDN1, a protein involved in the biogenesis of the 60S ribosomal subunit in Arabidopsis. The underexpressing mutant nle-2 shows short roots and reduced cell number in the root meristem. In addition, the null mutant nle-1 is embryo lethal, and defective embryos are arrested at the early globular stage. This work suggests that AtNLE interacts with AtMDN1, and AtNLE functions in root and embryo development.

In this study, four wine grape polysaccharides were extracted and optimized by using an efficient ultrasound-assisted extraction. A three-level, three-factor Box Behnken Design (BBD) combining with response surface methodology (RSM) was employed to optimize the extraction conditions including ultrasonic power, ultrasonic time and liquid-to-solid ratio. Furthermore, their physicochemical structures, antioxidant and liver protective activity were investigated and compared. Results revealed that the functional groups and monosaccharide compositions of these grape polysaccharides collected from different varieties were similar. Nevertheless, their molecular weights, molar ratios of monosaccharide compositions and surface morphological features were different. And the antioxidant activities of these polysaccharides were screened by free radical scavenging test. 'Beichun' (BC) and 'Benni fuji' (BF) polysaccharides possessed better antioxidant function. Further, the in vivo evaluation indicated that the polysaccharides of BC and BF have a protective effect against myocardial I/R injury in mice by inhibiting myocardial necroptosis mediated by mitochondrial ROS generation. Therefore, BC and BF grapes have potential applications in the medical and food industries.

In plant cells, multiple paralogs from ribosomal protein (RP) families are always synchronously expressed, which is likely contributing to ribosome heterogeneity or functional specialization. However, previous studies have shown that most RP mutants share common phenotypes. Consequently, it is difficult to distinguish whether the phenotypes of the mutants have resulted from the loss of specific genes or a global ribosome deficiency. Here, to investigate the role of a specific RP gene, we employed a gene overexpression strategy. We found that Arabidopsis lines overexpressing RPL16D (L16D-OEs) display short and curled rosette leaves. Microscopic observations reveal that both the cell size and cell arrangement are affected in L16D-OEs. The severity of the defect is positively correlated with RPL16D dosage. By combining transcriptomic and proteomic profiling, we found that overexpressing RPL16D decreases the expression of genes involved in plant growth, but increases the expression of genes involved in immune response. Overall, our results suggest that RPL16D is involved in the balance between plant growth and immune response.

Although grapevine (Vitis vinifera L.) is generally classified as a non-climacteric fruit, the regulatory mechanisms of ethylene in the ripening of non-climacteric fruit are still poorly understood. In this study, exogenous ethephon (ETH) strongly stimulated fruit color and anthocyanin accumulation, which was consistent with the increased expression of anthocyanin structural, regulatory, and transport genes. ETH application increased ABA content and decreased IAA content by coordinating ABA and auxin biosynthesis regulatory network. ETH treatment also accelerated sugar (glucose and fructose) accumulation by enhancing the gene expression involved in sugar transport and sucrose cleavage. ETH treatment blocked the synthesis of cellulose and accelerated the degradation of pectin, which was strongly associated with berry softening. To further confirm the function of ethylene biosynthesis and signaling genes, transient overexpression of VvACO4 and VvEIL3 were performed in both in tomato and strawberry fruits. These findings of the ethylene cascade add to our understanding of ethylene in non-climacteric berry ripening regulation and revealed a complex involvement of ethylene and its interplay with phytohormones during grapevine berry ripening.

Core Ideas First study of PpEIN3 by transgenic experiments to verify its function in the maturity process PpEIN3 is a positive regulator of ethylene signal transduction pathway to promote fruits ripening Ethylene is one of the most important phytohormone in plants and plays a critical role during growth, development, maturity, and aging. The framework of the ethylene signaling pathway is well reported. Nevertheless, studies on Ethylene Insensitive 3 (EIN3), the downstream regulator of the ethylene signaling pathway, need to be investigated, especially in peach [ Prunus persica (L.) Batsch]. In this study, we cloned PpEIN3 from peach and characterized it in tomato ( Solanum lycopersicum L.). Our results depicted that the open‐reading frame of PpEIN3 was 1875 bp, encoding a protein with 624 amino acid residues that contained a conserved EIN3 domain, a highly conserved N‐terminal region, and seven DNA‐binding sites. PpEIN3 showed very close association with homologous EIN genes from apple ( Malus domestica Borkh.) and grapevine ( Vitis vinifera L.). All investigated EIN proteins shared similar domains and structures. The PpEIN3 promoter possessed several motifs related to hormones that affect fruit development and ripening. Spatial–temporal expression analysis revealed that PpEIN3 was expressed at high levels in the late stage of fruit development vs. the early stage. In transgenic tomato, PpEIIN3 showed overexpression and the key ethylene biosynthesis genes SlACO1 , SlACS1 , and SlSAMS1 were upregulated and promoted early maturation in fruit. By contrast, PpEIIN3 silencing delayed ripening and reduced SlEIN3 expression in tomato. The results confirmed that PpEIN3 is a positive regulator of the ethylene signal transduction pathway, which promoted fruit ripening. Our findings provide valuable insight to the roles in ethylene signal components in the modulation of peach fruit ripening.

Background CD19-chimericantigen receptor (CAR) modified T cells (CD19-CAR T cells) have been well documented to possess potent anti-tumor properties against CD19-expressingleukemia cells. As a traditional medicine, metformin has been widely used to treat type II diabetes mellitus and more recently has become a candidate for the treatment of cancer. However, no report has revealed the direct effect of metformin on CD19-CAR T cell biological function and its underling mechanisms. Purpose The purpose of this research was to explore the effect of metformin on CD19-CAR T cell biological function and the mechanisms involved. Methods CD19-CAR T cells proliferation, apoptosis and cytotoxicity were mainly tested by CCK-8 assay, flow cytometry and ELISA. The detection of mechanism primarily used western blot. Bioluminescence imaging is the main application technology of animal studies. Results In the current study, it was found that metformin inhibited CD19-CAR T cell proliferation and cytotoxicity and induced apoptosis. Furthermore, our study revealed that metformin activated AMPK and suppressed mTOR and HIF1α expression. By using an AMPK inhibitor, compound C, we demonstrated the crucial roles of AMPK in CD19-CAR T cells when they were treated with metformin. Finally, we verified that metformin suppressed the cytotoxicity of CD19-CAR T cell in vivo. Conclusion Taken together, these results indicated that metformin may play an important role in modulating CD19-CAR T cell biological functions in an AMPK-dependent and mTOR/HIF1α-independent manner.

Grapevine berry development is a complex and genetically controlled process, with many morphological, biochemical and physiological changes occurring during the maturation process. Research carried out on grapevine berry development has been mainly concerned with wine grape, while barely focusing on table grape. ‘Fujiminori’ is an important table grapevine cultivar, which is cultivated in most provinces of China. In order to uncover the dynamic networks involved in anthocyanin biosynthesis, cell wall development, lipid metabolism and starch-sugar metabolism in ‘Fujiminori’ fruit, we employed RNA-sequencing (RNA-seq) and analyzed the whole transcriptome of grape berry during development at the expanding period (40 days after full bloom, 40DAF), véraison period (65DAF), and mature period (90DAF). The sequencing depth in each sample was greater than 12×, and the expression level of nearly half of the expressed genes were greater than 1. Moreover, greater than 64% of the clean reads were aligned to the Vitis vinifera reference genome, and 5,620, 3,381, and 5,196 differentially expressed genes (DEGs) were identified between different fruit stages, respectively. Results of the analysis of DEGs showed that the most significant changes in various processes occurred from the expanding stage to the véraison stage. The expression patterns of F3’H and F3’5’H were crucial in determining red or blue color of the fruit skin. The dynamic networks of cell wall development, lipid metabolism and starch-sugar metabolism were also constructed. A total of 4,934 SSR loci were also identified from 4,337 grapevine genes, which may be helpful for the development of phylogenetic analysis in grapevine and other fruit trees. Our work provides the foundation for developmental research of grapevine fruit as well as other non-climacteric fruits.

Carotenoids not only play indispensable roles in plant growth and development but also enhance nutritional value and health benefits for humans. In this study, total carotenoids progressively decreased during fruit ripening. Fifty-four genes involving in mevalonate (MVA), 2-C-methyl-D-erythritol 4-phosphate (MEP), carotenoid biosynthesis and catabolism pathway were identified. The expression levels of most of the carotenoid metabolism related genes kept changing during fruit ripening generating a metabolic flux toward carotenoid synthesis. Down regulation of VvDXS, VvDXR, VvGGPPS and VvPSY and a dramatic increase in the transcription levels of VvCCD might be responsible for the reduction of carotenoids content. The visible correlation between carotenoid content and gene expression profiles suggested that transcriptional regulation of carotenoid biosynthesis pathway genes is a key mechanism of carotenoid accumulation. In addition, the decline of carotenoids was also accompanied with the reduction of chlorophyll content. The reduction of chlorophyll content might be due to the obstruction in chlorophyll synthesis and acceleration of chlorophyll degradation. These results will be helpful for better understanding of carotenoid biosynthesis in grapevine fruit and contribute to the development of conventional and transgenic grapevine cultivars for further enrichment of carotenoid content.

Ethylene, which plays important roles in regulating plants’ life cycles, is biologically active in trace amounts, and its effects are of great commercial importance. The large-scale identification and comparison of the genes, which are involved in ethylene biosynthesis and signaling in multiple plants has not been reported. In this study, some key enzymes that involved in ethylene biosynthesis and signaling pathway in two non-climacteric fruits and three climacteric fruits have been identified through the comparison of gene copy number and related ESTs. The total EST number of the ethylene biosynthesis and signaling pathway related genes in grape and apple was more than that in peach. However, the ratios of the EST number in fruit to those in all the other tissues of the related genes in peach were more than that in apple and grape. We verified 27 genes in pear, 18 genes in apple, 23 genes in strawberry, 16 genes in peach and 23 genes in grape. The result showed that the transcript amounts of different members in the same gene family will be different in the expression and function of fruit ripening process. The difference between non-climacteric fruits and climacteric fruits was that the former could produce significant levels of ethylene during the ripening of fruits. Ethylene was produced during the early stages of fruit developments, which may indicate that the mechanism of ethylene perception occurs in these fruits prior to ripening.

GRAS domain genes are a group of important plant-specific transcription factors that have been reported to be involved in plant development. In order to know the roles of GRAS genes in grapevine, a widely cultivated fruit crop, the study on grapevine GRAS (VvGRAS) was carried out, and from which, 43 were identified from 12× assemble grapevine genomic sequences. Further, the genomic structures, synteny, phylogeny, expression profiles in different tissues of these genes, and their roles in response to stress were investigated. Among the genes, two potential target genes (VvSCL15 and VvSCL22) for VvmiR171 were experimentally verified by PPM-RACE and RLM-RACE, in that not only the cleavage sites of miR171 on the target mRNA were mapped but also the cleaved fragments and their expressing patterns were detected. Transgenic Arabidopsis plants over expression VvSCL15 showed lower tolerance to drought and salt treatments.