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A 2000-bp 5′-flanking region of VvPAL-like was isolated from 'Summer Black' grapevine by PCR amplification, named pVvPAL-like. To gain a better understanding of the expression and regulatory mechanism of VvPAL-like, a chimeric expression unit consisting of the β-glucuronidase (GUS) reporter gene under the control of a 2000-bp fragment of the VvPAL-like promoter was transformed into tobacco via Agrobacterium tumefaciens. Histochemical staining showed that the full-length promoter directs efficient expression of the reporter gene in cotyledons and hypocotyls, stigma, style, anthers, pollen, ovary, trichomes, and vascular bundles of transgenic plants. A series of 5′ progressive deletions of the promoter revealed the presence of a negative regulatory region (−424 to −292) in the VvPAL-like promoter. Exposure of the transgenic tobacco plants to various abiotic stresses demonstrated that the full-length construct could be induced by light, copper (Cu), abscisic acid (ABA), indole-3-acetic (IAA), methyl jasmonate (MeJA) ( N-1-naphthylphthalamic acid), ethylene, and drought. Furthermore, the ethylene-responsive region was found to be located in the −1461/−930 fragment, while the element(s) for the MeJA-responsive expression may be present in the −424/−292 region in the VvPAL-like promoter. These findings will help us to better understand the molecular mechanisms by which VvPAL-like participates in biosynthesis of flavonoids and stress responses. [ABSTRACT FROM AUTHOR]

• Low-temperature promotes the accumulation of anthocyanins in grape leaves by up-regulating the expression of structural genes that induce anthocyanin synthesis, related transcription factors and LRR-type receptor-like protein kinases. • Under the same low-temperature environment, the more anthocyanin accumulated in the leaves, the less damage they suffered. • The synthesis and accumulation of anthocyanins promoted the adaptation and resistance of grape leaves to low-temperature environmental conditions. Anthocyanins can be induced by environmental factors such as low- temperature, and play an important role in adapting to abiotic stress. In this study, the leaves of 'Cabernet Gernischt' grapes with different colors under natural low-temperature in autumn were collected and grouped into Green (Gre), Light-red (L-Red), Red (Red), and Dark-red (D-Red). Results showed that the total anthocyanin content and anthocyanin components were increased in turn from Gre to D-red. Under the same low-temperature, Gre was substantially damaged, L-Red was mildly damaged, Red and D-Red had no visible damage. RNA-seq data showed that a total of 3666 DEGs were screened out of all 28,306 genes detected, of which 1936 DEGs were upregulated and 1730 DEGs were downregulated in D-Red vs Gre. Transcriptomics and metabolomics showed that DEGs were significantly enriched in GO terms, such as in defense response, defense response to oomycetes, and response to reactive oxygen species, hydrogen peroxide, heat, salt stress, and biotic stimulus pathways. The KEGG analysis showed that DEGs were mainly enriched in secondary metabolism, carbohydrate metabolism, energy metabolism, and lipid metabolism. Anthocyanin synthesis genes may be regulated by transcription factors such as ERF, MYB, NAC, bHLH and various LRR-type receptor-like protein kinase (LRR-RLK) family genes. In conclusion, the accumulation of anthocyanins significantly increased from green to red in grape leaves during autumn low-temperature, improving the cold resistance of grape leaves. [ABSTRACT FROM AUTHOR]

• The difference in the content and composition of anthocyanins in three grape cultivars was investigated. • Transcriptome sequencing analyzes the changes in gene expression levels during the development of different color grape cultivars. • WGCNA reveals modules of co-expressed genes related to anthocyanins and identified hub genes related to anthocyanin synthesis. Anthocyanins as plant pigments are responsible for the color of the grape berry. The major concern among consumers is color, quality, and appearance, therefore, skin color is the most important item that affects consumer purchases. In this study, we used three grape cultivars namely Red globe (bright red), Kyoho (purple-red), and Wink (purple-black), and evaluated color changes in three different developmental stages according to time course including 15 days after the berry color shift from green to red (V1), 25 days after the berry color shift from green to red (V2), and 35 days after the berry color shift from green to red (V3). The dynamic changes in anthocyanin content and components were detected using "High-Performance Liquid Chromatography mass spectrometry/mass spectrometry (HPLC-MS/MS)" technology. Furthermore, we performed transcriptome sequencing and identified the genes involved in the anthocyanin biosynthesis and accumulation in grape berry development. In total, we detected 13 different anthocyanin components in three grape cultivars. Malvidin, petunidin, and delphinidin derivates are the main components of dark colored grape (Kyoho and Wink), while cyanidin and pelargonidin derivates are the sources of bright-colored grape (Red globe). According to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, many DEGs were enrichment in phenylpropanoid biosynthesis and flavonoid biosynthesis. We also found thirty-nine DEGs to be involved in the anthocyanin metabolism and accumulation. Furthermore, using weighted gene correlation analysis (WGCNA), three modules (blue, magenta, and tan) and nineteen hub genes were highly correlated with anthocyanin variation. Our research provided new insights for elucidating the anthocyanin metabolic pathway and related genes, signal perception and transduction during grape berries development at different stages. [ABSTRACT FROM AUTHOR]

• The more mature leaves have deeper sinuses in 'summer black'. • Significant differences in photosynthesis and physiological traits of different nodal leaves. • Net photosynthetic rate (A) was significantly positively correlated with leaf shape. • Expression trend of aquaporins, magnesium transporters was similar to physiological changes Fruiting shoots are comprised of leaves, fruits, and branches, which determine the grape yield and the quality of produce. Fruiting shoots are also the important structural unit for studying the relationship between the source and sink. The number of leaves, leaf area, photosynthetic activities, and density are directly or indirectly affecting the ability of the fruiting shoot to fix CO 2 and provide a carbon source for the fruit growth and development. 'Summer Black' is used as the main test material and observed certain differences in appearance, microstructure, photosynthetic pigments, relative water content (RWC), organic acids, carbohydrates, mineral elements, and photosynthetic capacity at different nodal leaves. The net photosynthetic rate (A) was significantly positively correlated with leaf shape parameters (the ratio between branch point to lobe tip and distal sinus to petiolar junction), leaf structure thickness, RWC, carotenoids, soluble sugars, and organic acids, but it was significantly negatively correlated with intercellular CO 2 concentration (C i), Chlorophyll b and phosphorus content. The variations in magnesium, water, glucose, and fructose content in leaves matched the expression patterns of aquaporin, magnesium transporter, and sugar metabolism-related genes in various nodal leaves. In addition, the physiological traits and photosynthetic capacity at different nodal leaves of 'Shine Muscat' and 'Kyoho' were similar to those of 'Summer Black'. The results of this study will provide a reference for the differences in leaf sink–source effects at different nodes and provide a basis for reasonably removing leaves. [ABSTRACT FROM AUTHOR]

Elevated CO 2 storage causes overconsumption of reducing sugar and lower energy level in postharvest strawberry fruit, reducing the acceptance of this fruit. In order to develop an approach to improve the impaired quality, we applied additional fumigation of SO 2 in combination with elevated CO 2 storage to postharvest strawberry fruit in the present study. Results exhibited that SO 2 fumigation increased contents of sucrose, glucose, fructose, and adenosine triphosphate (ATP) by 11%, 16%, 19%, and 11% respectively on day 8 as well as energy charge. Meanwhile, the content of alcohol and aldehyde decreased. Furthermore, enzyme activities and transcriptional profiles analysis showed inhibition in glycolysis and alcohol fermentation while the tricarboxylic acid (TCA) cycle and electron transfer chain (ETC) were activated by 19–80%. These results indicated that SO 2 fumigation maintained sugar contents by inhibiting glycolysis and rerouted the direction of pyruvate by activating aerobic respiration and restricting anaerobic. In addition, the activation of aerobic respiration improved the energy level of postharvest strawberry fruit. • Additional SO2 kept higher sugar contents in strawberry under elevated CO2 storage. • Additional SO2 kept higher energy level in strawberry under elevated CO2 storage. • Additional SO2 promoted aerobic respiration but inhibited anaerobic respiration. [ABSTRACT FROM AUTHOR]

• Ethephon treatment strongly stimulated anthocyanin accumulation in the grape. • Ethephon inhibited auxin synthesis and induced its own and ABA synthesis. • Ethephon accelerated glucose and fructose accumulation and sweet taste sensation. • Overexpression VvACO4 and VvEIL3 fruit accelerated fruit ripening and ethylene production. 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. [ABSTRACT FROM AUTHOR]

Abstract: In plants, microRNAs (miRNAs) play significant roles in post-transcriptional gene regulation and have been found to control many genes involved in different biological and metabolic processes. Extensive studies were carried out to discover miRNAs and analyze their functions in model plant species, such as in Arabidopsis and rice that have been reported. In this research, we used bioinformatics to predict microRNAs in an important strawberry rootstock cultivar to discover and validate precise sequences of microRNAs in strawberry. By adopting a range of filtering criteria, we obtained 59 potential miRNAs belonging to 40 miRNA families from the Fragaria vesca genome. Using two specific 5′ and 3′ miRNA RACE PCR reactions and a sequence-directed cloning method, we accurately determined 34 precise sequences of candidate miRNAs, while six other sequences exhibited some minor divergence in their termini nucleotides, and 19 miRNAs that could not be cloned owing to expression abundance may be too low or these mirRNAs predicted could not be existing in strawberry. Potential target genes were further predicted for the miRNAs above. The expression of the 16 miRNAs unreported and having exact sequences and their targets by experiment could be detected in different tissues of strawberry ranging from roots, stems, leaves, flowers and fruits by qRT-PCR and some of them showed differential expression in various tissues. The functional analysis of 16 miRNAs and their targets was carried out. Finally, we conclude that there are 34 mirRNAs in strawberry and their targets play vital roles not only in growth and development, but also in diverse physiological processes. These results show that regulatory miRNAs exist in agronomically important strawberry and might have an important function in strawberry growth and development. [Copyright &y& Elsevier]

• Exogenous NO could prolong the shelf life of table grapes. • The optimum concentration of exogenous NO was 400 μM that significantly increased the activity of antioxidant enzymes POD, SOD and CAT. • NO inhibited the mycelial growth of B. cinerea in the grape fruit. • NO induced the upregulated 2,226 and downregulated 1,249 genes by RNA-Seq, and a total of 300 metabolites by metabolome. The application of exogenous nitric oxide (NO) is convenient and safe approach to preserve fruits, though, the hidden genetic mechanism in treated grape berries is to be explored. In this study, the results depicted that the application of exogenous NO improved the shelf life of grape berries compared with the control group. The SNP treatment(400 μM) significantly increased the antioxidant enzymes activities including: POD, SOD and CAT. NO inhibited the mycelial growth of Botrytis cinerea (B. cinerea) in the grape berry. RNA-Seq data showed that NO upregulated 2,226 and downregulated 1,249 DEGs, and 300 metabolomes were identified including: NOT.vs.NOC_pos annotated 202 metabolites, NOT.vs.NOC_neg annotated 98 metabolites. The RNA-Seq data is coherent with the metabolome that STSs and CHSs expression levels were significantly increased against disease resistance and antioxidant-associated stilbene synthase pathway, and the associated metabolites content were also significantly increased. These changes led to an increase in the content of secondary metabolites such as resveratrol, which ultimately improved the shelf-life of the grapes. [ABSTRACT FROM AUTHOR]

• VvERF17 was an ethylene response factor, the expression of which was strongly correlates with chlorophyll degradation. • Transgenic expression of VvERF17 modulated chlorophyll content and related genes' expression. • VvERF17 could activate the expression of major chlorophyll catabolic genes by directly binding to the ERF binding sites in their promoters. • VvERF17 mediates chlorophyll degradation by transcriptional activation of NOL , PPH , PAO , and RCCR. Chlorophylls in the skin contribute to grape (Vitis vinifera) berry color and quality. Degradation of chlorophyll occurs during berry development in grape, but less is known about the molecular mechanisms of this process. Here, we characterized an ethylene response factor, VvERF17, as a putative regulator of chlorophyll degradation in grape berry. VvERF17 expression was strongly correlates with development-induced and ETH-triggered chlorophyll degradation. Transient overexpression of VvERF17 in tobacco leaves, tomato fruits, and grape leaves led to color break and chlorophyll degradation, which were resulted from the upregulated expression of chlorophyll catabolic genes (CCGs), including Chlorophyll-b-reductase (NOL), pheophytinase (PPH), pheophorbide a oxygenase (PAO), and red Chlorophyll catabolite reductase (RCCR). Inversely, silencing of VvERF17 in grape leaves partially suppressed the expression of CCGs' expression. Major CCGs' expression was closely related to chlorophyll degradation and VvERF17 expression. It was further demonstrated that VvERF17 could activate the expression of major CCGs by directly binding to the ERF binding sites in their promoters. We conclude that VvERF17 mediates chlorophyll degradation by transcriptional activation of NOL , PPH , PAO , and RCCR. These findings elucidate the regulation mechanisms of chlorophyll degradation in grape and enrich our understanding of grape berry color formation. [ABSTRACT FROM AUTHOR]

Main Conclusion: Six grape centromere-specific markers for cytogenetics were mined by combining genetic and immunological assays, and the possible evolution mechanism of centromeric repeats was analyzed. Centromeric histone proteins are functionally conserved; however, centromeric repetitive DNA sequences may represent considerable diversity in related species. Therefore, studying the characteristics and structure of grape centromere repeat sequences contributes to a deeper understanding of the evolutionary process of grape plants, including their origin and mechanisms of polyploidization. Plant centromeric regions are mainly composed of repetitive sequences, including SatDNA and transposable elements (TE). In this research, the characterization of centromere sequences in the whole genome of grapevine (Vitis vinifera L.) has been conducted. Five centromeric tandem repeat sequences (Vv1, Vv2, Vv5, Vv6, and Vv8) and one long terminal repeat (LTR) sequence Vv24 were isolated. These sequences had different centromeric distributions, which indicates that grape centromeric sequences may undergo rapid evolution. The existence of extrachromosomal circular DNA (eccDNA) and gene expression in CenH3 subdomain region may provide various potential mechanisms for the generation of new centromeric regions., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Grape quality is a key factor in determining wine quality, and it depends not only on management skills, but also on the geographic location of the producing area. In China, Shandong is the province with the largest wine production, and 'Cabernet Franc' is widely planted. This study evaluated the 'Cabernet Franc' fruit quality in relation to geographical conditions in five 'Cabernet Franc' producing districts of Shandong province, China, including Dezhou Aodeman Winery (DZ), Tai'an Zhongqingsongshi Winery (TA), Penglai Longhu Winery (PL), Rushan Taiyihu Winery (RS), and Rizhao Taiyangcheng Winery (RZ). At the time of veraison and maturity, fruit was harvested from five areas, and compared for cosmetic and internal fruit quality. The soluble sugar content in the Rizhao area was rich, and the weight and volume of single fruit were relatively large. The titratable acid of the berries in Tai'an area was high. RNA-seq analysis showed that the number of genes in the véraison stage was 19,571-20,750, and the number of genes in the mature stage was 19,176-20,735. The analysis found that areas with multiple high-quality characteristics tended to have more DEGs (differential expressed genes). And the DEGs in different areas were mainly distributed on chromosome 7, and at least on chromosome 15. DEGs in 5 areas were enriched on 855 GO terms and 116 KEGG pathways during berries development. Among them, it was found that the up/down-regulation of DEGs was related to the formation of berry quality, which helps to explain the impact of environment on grape quality components. In summary, this study is helpful to understand the influence of cultivation location on the quality of 'Cabernet Franc' in different production areas in Shandong province, and further provide a reference for the production of high-quality wine grapes and winemaking., (© 2023. The Author(s).)

Autophagy, a conserved degradation and reuse process, plays a crucial role in plant cellular homeostasis during abiotic stress. Although numerous autophagy-related genes ( ATGs ) that regulate abiotic stress have been identified, few functional studies have shown how they confer tolerance to copper (Cu) stress. Here, we cloned a novel Vitis vinifera   ATG6 gene ( VvATG6 ) which was induced by 0.5 and 10 mM Cu stress based on transcriptomic data, and transgenic Arabidopsis thaliana , tobacco ( Nicotiana tabacum ), and grape calli were successfully obtained through Agrobacterium -mediated genetic transformation. The overexpression of VvATG6 enhanced the tolerance of transgenic lines to Cu. After Cu treatment, the lines that overexpressed VvATG6 grew better and increased their production of biomass compared with the wild-type. These changes were accompanied by higher activities of antioxidant enzymes and a lower accumulation of deleterious malondialdehyde and hydrogen peroxide in the transgenic plants. The activities of superoxide dismutase, peroxidase, and catalase were enhanced owing to the elevation of corresponding antioxidant gene expression in the VvATG6 overexpression plants under Cu stress, thereby promoting the clearance of reactive oxygen species (ROS). Simultaneously, there was a decrease in the levels of expression of RbohB and RbohC that are involved in ROS synthesis in transgenic plants under Cu stress. Thus, the accelerated removal of ROS and the inhibition of its synthesis led to a balanced ROS homeostasis environment, which alleviated the damage from Cu. This could benefit from the upregulation of other ATGs that are necessary for the production of autophagosomes under Cu stress. To our knowledge, this study is the first to demonstrate the protective role of VvATG6 in the Cu tolerance of plants., Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-024-01415-y., Competing Interests: Conflict of interestAll authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest or non-financial interest in the subject matter or materials discussed in this manuscript., (© Prof. H.S. Srivastava Foundation for Science and Society 2024. Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.)

Anthocyanins, total phenols, soluble sugar and fruit shape plays a significant role in determining the distinct fruit quality and customer preference. However, for the majority of fruit species, little is known about the transcriptomics and underlying regulatory networks that control the generation of overall quality during fruit growth and ripening. This study incorporated the quality-related transcriptome data from 6 ecological zones across 3 fruit development and maturity phases of Chardonnay cultivars. With the help of this dataset, we were able to build a complex regulatory network that may be used to identify important structural genes and transcription factors that control the anthocyanins, total phenols, soluble sugars and fruit shape in grapes. Overall, our findings set the groundwork to improve grape quality in addition to offering novel views on quality control during grape development and ripening., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Fu, Ren, Kang, Wang, Zhang, Fang and Wu.)

Drought is a common and serious abiotic stress in viticulture, and it is urgent to select effective measures to alleviate it. The new plant growth regulator 5-aminolevulinic acid ( ALA ) has been utilized to alleviate abiotic stresses in agriculture in recent years, which provided a novel idea to mitigate drought stress in viticulture. The leaves of 'Shine Muscat' grapevine ( Vitis vinifera L.) seedlings were treated with drought (Dro), drought plus 5-aminolevulinic acid (ALA, 50 mg/L) (Dro_ALA) and normal watering (Control) to clarify the regulatory network used by ALA to alleviate drought stress in grapevine. Physiological indicators showed that ALA could effectively reduce the accumulation of malondialdehyde (MDA) and increase the activities of peroxidase (POD) and superoxide dismutase (SOD) in grapevine leaves under drought stress. At the end of treatment (day 16), the MDA content in Dro_ALA was reduced by 27.63% compared with that in Dro, while the activities of POD and SOD reached 2.97- and 5.09-fold of those in Dro, respectively. Furthermore, ALA reduces abscisic acid by upregulating CYP707A1 , thus, relieving the closure of stomata under drought. The chlorophyll metabolic pathway and photosynthetic system are the major pathways affected by ALA to alleviate drought. Changes in the genes of chlorophyll synthesis, including CHLH , CHLD , POR , and DVR ; genes related to degradation, such as CLH , SGR , PPH and PAO ; the RCA gene that is related to Rubisco; and the genes AGT1 and GDCSP related to photorespiration form the basis of these pathways. In addition, the antioxidant system and osmotic regulation play important roles that enable ALA to maintain cell homeostasis under drought. The reduction of glutathione, ascorbic acid and betaine after the application of ALA confirmed the alleviation of drought. In summary, this study revealed the mechanism of effects of drought stress on grapevine, and the alleviating effect of ALA, which provides a new concept to alleviate drought stress in grapevine and other plants., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Yang, Xia, Fang, Jia, Wang, Lin, Liu, Ge, Pu, Fang and Shangguan.)

Introduction: Grape rootstocks play critical role in the development of the grape industry over the globe for their higher adaptability to various environments, and the evaluation of their genetic diversity among grape genotypes is necessary to the conservation and utility of genotypes., Methods: To analyze the genetic diversity of grape rootstocks for a better understanding multiple resistance traits, whole-genome re-sequencing of 77 common grape rootstock germplasms was conducted in the present study., Results: About 645 billion genome sequencing data were generated from the 77 grape rootstocks at an average depth of ~15.5×, based on which the phylogenic clusters were generated and the domestication of grapevine rootstocks was explored. The results indicated that the 77 rootstocks originated from five ancestral components. Through phylogenetic, principal components, and identity-by-descent (IBD) analyses, these 77 grape rootstocks were assembled into ten groups. It is noticed that the wild resources of V. amurensis and V. davidii , originating from China and being generally considered to have stronger resistance against biotic and abiotic stresses, were sub-divided from the other populations. Further analysis indicated that a high level of linkage disequilibrium was found among the 77 rootstock genotypes, and a total of 2,805,889 single nucleotide polymorphisms (SNPs) were excavated, GWAS analysis among the grape rootstocks located 631, 13, 9, 2, 810, and 44 SNP loci that were responsible to resistances to phylloxera, root-knot nematodes, salt, drought, cold and waterlogging traits., Discussion: This study generated a significant amount of genomic data from grape rootstocks, thus providing a theoretical basis for further research on the resistance mechanism of grape rootstocks and the breeding of resistant varieties. These findings also reveal that China originated V. amurensis and V. davidii could broaden the genetic background of grapevine rootstocks and be important germplasm used in breeding high stress-resistant grapevine rootstocks., Competing Interests: Author ZZ was employed by Shandong Zhichang Agricultural Science and Technology Development Co. LTD. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Wang, Zhao, Zheng, Liu, Ji, Zhang, Pervaiz, Shangguan and Fang.)

Grapes are subject to a wide range of climatic conditions during their life cycle, but the use of rootstocks can effectively ameliorate the effects of abiotic stress. However, the tolerance mechanism of different grape rootstock varieties varies under various stresses, and systematic research on this aspect is limited. On the basis of previous research, transcriptome sequencing was performed on three tolerant grape rootstock varieties (3309C, 520A, 1103P) and three intolerant grape rootstock varieties (5BB, 101-14, Beta). In total, 56,478,468 clean reads were obtained. One hundred and ten genes only existed in all combinations during P1 with a downregulated trend, and 178 genes existed only in P1 of tolerant grape rootstock varieties. Salt treatment firstly affected the photosynthesis of leaves, and tolerant varieties weakened or even eliminated this effect through their own mechanisms in the later stage. Tolerant varieties mobilized a large number of MFs during the P2 stage, such as hydrolase activity, carboxypeptidase activity, and dioxygenase activity. Carbon metabolism was significantly enriched in P1, while circadian rhythm and flavonoid biosynthesis were only enriched in tolerant varieties. In the intolerant varieties, photosynthesis-related pathways were always the most significantly enriched. There were large differences in the gene expression of the main signal pathways related to salt stress in different varieties. Salt stress affected the expression of genes related to plant abiotic stress, biotic stress, transcription factors, hormones, and secondary metabolism. Tolerant varieties mobilized more bHLH, WRKY, and MYB transcription factors to respond to salt stress than intolerant varieties. In the tolerant rootstocks, SOS was co-expressed. Among these, SOS1 and SOS2 were upregulated, and the SOS3 and SOS5 components were downregulated. The genes of heat shock proteins and the phenylalanine pathway were upregulated in the tolerant varieties. These findings outline a tolerance mechanism model for rootstocks for coping with osmotic stress, providing important information for improving the resistance of grapes under global climate change.

Grapevine downy mildew is the most serious disease of grapevine cultivars that affects the rate of resistance/susceptibility to Plasmopara viticola . In this study, we used the susceptible cultivar "Zitian Seedless" and the resistant cultivar "Kober 5BB" as materials to determine the transcriptome differences and phenotypes of the leaves after inoculation with downy mildew. The differences in microstructures and molecular levels were compared and analyzed. Fluorescence staining and microscopic observations confirmed that hypersensitive cell death occurred around the stomata in "Kober 5BB" infected by downy mildew zoospores. Meanwhile, transcriptomic profiling indicated that there were 11,713 and 6,997 gene expression differences between the resistant and susceptible cultivars at 72 h after inoculation when compared to control (0 h), respectively. The differentially expressed genes of the two cultivars are significantly enriched in different pathways, including response to plant-pathogen interaction, mitogen-activated protein kinase (MAPK) signaling pathway, plant hormone signal transduction, phenylpropanoid, and flavonoid biosynthesis. Furthermore, the results of functional enrichment analysis showed that H 2 O 2 metabolism, cell death, reactive oxygen response, and carbohydrate metabolism are also involved in the defense response of "Kober 5BB," wherein a total of 322 key genes have been identified. The protein interaction network showed that metacaspases (MCAs), vacuolar processing enzymes (VPEs), and Papain-like cysteine proteases (PLCPs) play an important role in the execution of hypersensitive responses (HR). In conclusion, we demonstrated that HR cell death is the key strategy in the process of grape defense against downy mildew, which may be mediated or activated by Caspase-like proteases., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Gong, Kang, Sadeghnezhad, Bao, Ge, Zhuge, Shangguan and Fang.)

Methylation affects different aspects of genetic material stability, gene expression regulation, and histone modification. The previous reports depicted that DNA and histone methylation regulates plant growth and development. In this study, we evaluated the effects of DNA and histone methylation on 'Hongjia' strawberry and 'Lichun' tomato. We investigated the transient transformation system for arginine methyltransferase ( FvPRMT1.5 ) overexpression and interference and assessed the phenotypic appearance and mRNA and protein expression levels. Results depicted that changes in methylation levels caused inhibition of carotenoids and anthocyanins. Furthermore, the profiling of aroma components was altered in response to 5-azacytidine. DNA hypomethylation induced the expression levels of genes involved in photosynthesis, flavonoid biosynthesis, and hormone signal transduction pathways, while the expression levels of related proteins showed a downward trend. Overall, we proposed a model that reveals the possible regulatory effects of DNA and histone methylation during fruit ripening.

To determine the effect of different temperature on strawberry after harvest, physiological indicator analysis and proteomics analysis were conducted on ripened strawberry ("Sweet Charlie") fruit stored at 4, 23, and 37°C for 10 or 20 days. Results showed that 4°C maintained a better visual quality of strawberry, and the weight loss and firmness remained stable within 3 days. Low temperature negatively affected anthocyanin but positively affected soluble sugars. Though anthocyanin content was higher with increasing temperature, anthocyanin synthesis related proteins were downregulated. Higher indole-acetic acid (IAA) content in seeds and lower abscisic acid (ABA) content were found in berry at 4°C. Antioxidant related proteins were upregulated during storage, showing a significant up-regulation of peroxidase (POD) at 4°C, and ascorbate-glutathione (AsA-GSH) cycle related proteins and heat shock proteins (HSPs) at 37°C. In addition, overexpressed sugar phosphate/phosphate translocator, 1-aminocyclopropane-1-carboxylate oxidase, and aquaporin PIP2-2 had a positive effect in response to low temperature stress for containing higher protopectin content and POD activity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Lv, Zheng, Song, Pervaiz, Dong, Zhang, Jia and Fang.)

Cuticular wax covering the surface of fleshy fruit is closely related to fruit glossiness, development, and post-harvest storage quality. However, the information about formation characteristics and molecular mechanisms of cuticular wax in grape berry is limited. In this study, crystal morphology, chemical composition, and gene expression of cuticular wax in grape berry were comprehensively investigated. Morphological analysis revealed high density of irregular lamellar crystal structures, which were correlated with the glaucous appearances of grape berry. Compositional analysis showed that the dominant wax compounds were triterpenoids, while the most diverse were alkanes. The amounts of triterpenoids declined sharply after véraison, while those of other compounds maintained nearly constant throughout the berry development. The amounts of each wax compounds varied among different cultivars and showed no correlation with berry skin colors. Moreover, the expression profiles of related genes were in accordance with the accumulation of wax compounds. Further investigation revealed the contribution of cuticular wax to the water preservation capacity during storage. These findings not only facilitate a better understanding of the characteristics of cuticular wax, but also shed light on the molecular basis of wax biosynthesis in grape., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Zhang, Zhang, Lu, Ou-yang, Zhu-ge, Tian, Jia and Fang.)

Flavonoids in grapes contribute the quality of the berry, but the flavonoid diversity and the regulatory networks underlying the variation require a further investigation. In this study, we integrated multi-omics data to systematically explore the global metabolic and transcriptional profiles in the skins and pulps of three grape cultivars. The results revealed large-scale differences involved in the flavonoid metabolic pathway. A total of 133 flavonoids, including flavone and flavone C -glycosides, were identified. Beyond the visible differences of anthocyanins, there was large variation in other sub-branched flavonoids, most of which were positively correlated with anthocyanins in grapes. The expressions of most flavonoid biosynthetic genes and the major regulators MYBA1 were strongly consistent with the changes in flavonoids. Integrative analysis identified two novel transcription factors ( MYB24 and MADS5 ) and two ubiquitin proteins ( RHA2 ) as promising regulatory candidates for flavonoid biosynthesis in grapes. Further verification in various grape accessions indicated that five major genes including flavonol 3'5'-hydroxylase ( F3'5'H ), UDP-glucose:flavonoid 3-O-glycosyl-transferase , anthocyanin O-methyltransferase , acyltransferase ( 3AT ), and glutathione S-transferase ( GST4 ) controlled flavonoid variation in grape berries. These findings provide valuable information for understanding the mechanism of flavonoid biosynthesis in grape berries and the further development of grape health products.

Glycoside hydrolase (GH, EC 3.2.1) is a group of enzymes that hydrolyzes glycosidic bonds and play a role in the hydrolysis and synthesis of sugars in living organisms. Vitis vinifera is an important fruit crop and it harbors GH17 gene family however, their function in grapes has not been systematically investigated. In this study, a total of 870 GH17 genes were identified from 14 plant species and their structural domain, sequence alignment, phylogenetic tree, collinear analysis, with the expression profiles of VviGH17 gene family was performed. The promoter analysis of VviGH17 gene showed the presence of cis -acting elements, which are responsive to plant growth and development. In addition, elements for plant hormones were found that are triggered in response to abiotic/biological stress. Transcriptomic data led to the identification of several VviGH17 genes, which are associated with bud dormancy and in response to abiotic stress. Transcript analysis was carried out for some of the selected VviGH17 genes RT-qPCR. VviGH17-16 and VviGH17-30 genes were differentially expressed during bud dormancy, fruit development and different abiotic stresses. Moreover, VviGH17-37 and VviGH17-44 were differentially expressed at fruit development, in response to abiotic stress. In addition, subcellular localization predicts that the VviGH17-16 , VviGH17-30 , and VviGH17-37 genes were located in the cell membrane, while VviGH17-44 gene was located in the vacuole. In conclusion, our study led to the identification of several GH17s and their probable role in development and stress., Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-021-01014-1., Competing Interests: Conflict of interestAll authors declared that there is no conflict of interest., (© Prof. H.S. Srivastava Foundation for Science and Society 2021.)

'Kyoho' grapevine ( Vitis vinifera ) treated by calcium ions solution has been proved as an effective treatment to extend grape quality during storage to reduce disease, but its molecular mechanism was not clear yet. In the current work, grape berries were treated with different concentration of Calcium chloride (CaCl 2 ) solution, and their effects on antioxidant enzyme activity and transcriptome and metabolome in fruit were investigated. CaCl 2 treatments reduced weight loss and inhibited the decrement of flesh firmness. 80 mM CaCl 2 significantly increased the activity of antioxidant enzymes POD, SOD and CAT, which was the optimum experimental concentration. The study showed that the expression level of heat shock transcription factor and UBX which involved in endoplasmic reticulum stress and degradation pathway increased significantly. Moreover, the corresponding metabolites, such as heat shock protein and organic acid, also increased significantly. The misfolded proteins are transported to the cytosol for degradation, so that the preservation ability of grape is improved., Competing Interests: Conflict of interestThe authors declare that they have no conflict of interests., (© Association of Food Scientists & Technologists (India) 2020.)

Grape ( Vitis vinifera L.) is one of the most widely cultivated fruit crops globally. Fruit cracking during fruit growth and development severely affects yield and quality, resulting in significant economic losses. Currently, calcium fertilizer application is used to prevent berry cracking. However, the mechanisms by which calcium fertilizer treatment reduces berry cracking are poorly understood. To explore this, transcriptomics and metabolomics were used to identify the differentially expressed genes (DEGs) and differentially abundant metabolites in V. vinifera '90-1'. We found that secondary metabolic pathways were enriched during the veraison and maturity stages, including the flavonoid biosynthesis pathway. Enrichment analysis indicated that most of the DEGs were enriched in the functional category of flavonoid biosynthesis. As secondary metabolites are largely antioxidants, the spraying of calcium fertilizers may improve the antioxidant capacity of the berries by regulating genes related to the flavonoid metabolism pathway, thus reducing the occurrence of berry cracking., Competing Interests: The authors declare no conflict of interest., (© 2021 The Author(s).)

Main Conclusion: Transcriptome and physiological analysis showed that exogenous arginine can delay the ripening process of postharvest strawberry fruit. Arginine (Arg) plays an important role in the growth and development of plants, but its growth and development regulatory mechanisms in strawberry fruit are unknown. In this study, we found that the content of Arg decreased after the onset of fruit coloration and exogenous Arg inhibited fruit coloration. We comprehensively analyzed the transcriptome of 'Sweet Charlie' strawberry fruit with or without Arg treatment and identified a large number of differential genes and metabolites. Based on the transcriptome data, we also found that Arg inhibited ripening, which coincided with changes in several physiological parameters and their corresponding gene transcripts, including firmness, anthocyanin content, sugar content, Arg content, indole-acetic acid (IAA) content, abscisic acid (ABA) content, and ethylene emissions. We also found that Arg induced the expression of heat-shock proteins (HSPs) and antioxidant enzyme genes, which improved strawberry stress resistance. This study elucidated the molecular mechanism by which exogenous Arg delays strawberry fruit ripening, providing some genetic information to help guide the future improvement and cultivation of strawberry.

Chitinases (Chi), an important resistance-related protein, act against fungal pathogens by catalyzing the fungal cell wall, whereas are involved in different biological pathways in grape. In this study, we found 42 Chi family genes in Vitis vinifera L. (VvChis) and evaluated their expression levels after Botrytis infection, stress hormones like ethylene (ETH) and methyl-jasmonate (MeJA), and abiotic stresses like salinity and temperature changes in ripened fruits. VvChis were categorized into five groups including A, B, C, D, and E belonged to glycoside hydrolase family 18 and 19 (GH18 and GH19) according to genes structure, which expression analysis showed distinct temporal and spatial expression patterns changed in different tissues and various development stages. Different responsive elements to biotic and abiotic stresses were determined in the promoter regions of VvChis, specially elicitor-responsive element that was conserved among all VvChis genes. The expression levels of VvChis in groups A, B, and E increased after Botrytis cinerea infection in leaves and berries. Meanwhile, VvChis in glycoside hydrolase family 18 (GH18) were up-regulated under MeJA and ETH treatment, although the induction of VvChis by low temperature was more significant than high temperature. The expression of VvChis was also positively correlated with the concentration of NaCl treatment. Furthermore, differential gene-overexpression of VvChi5, VvChi17, VvChi22, VvChi26, and VvChi31 in strawberry and tomato fruits demonstrated the involvement of various isoforms in resistance to Botrytis infection through antioxidant system and lignin accumulation, which led to a reduction of damage. Among different isoforms of VvChis, we confirmed the interaction of Chi17 with Metallothionein (MTL) as oxidative stress protection, which suggests VvChis can modulate oxidative stress during postharvest storage in ripened fruits.

The fruit is the most important economical organ in the grape; accordingly, to investigate the grapevine genomic methylation landscape and examine its functional significance during fruit development, we generated whole genome DNA methylation maps for various developmental stages in the fruit of grapevine. In this study, thirteen DNA methylation-related genes and their expression profiles were identified and analyzed. The methylation levels for mC, mCG, mCHG, and mCHH contexts in 65 days after flowering (65DAF) fruit (véraison stage) were higher than those in 40DAF (green stage) and 90DAF (mature stage) fruits. Relative to methylation in the mC context, methylation levels in the mCHH context were higher than those of mCG and mCHG. The DNA methylation level in the ncRNA regions was significantly higher than that in exon, gene, intron, and mRNA regions. The differentially methylated regions (DMRs) and differentially methylated promoters (DMPs) in 65DAF_vs_40DAF were both higher than those in 90DAF_vs_65DAF and 90DAF_vs_40DAF. Most DMRs (or DMPs) were involved in metabolic processes and cell processes, binding, and catalytic activity. These results indicated that DNA methylation represses gene expression during grape fruit development, and it broadens our understanding of the landscape and function of DNA methylation in grapevine genomes., (© Prof. H.S. Srivastava Foundation for Science and Society 2020.)

Background: The xylem sap of fruit trees ensures the survival during the dormant period, and its flow during the bleeding period is correlated with the start of a new life cycle. Though the simple exploration on ingredients in the sap was carried out in the early years, the specific life activities and physiology functions of the sap during bleeding period have not been reported yet and the bleeding period is still a fruit tree development period worthy of attention., Objectives: In this study, the microbial community composition during bleeding period were revealed by metatranscriptome and transcriptomic data. For the first time, the microorganism genome and grape genome in xylem sap were analyzed on transcriptional level, based on which the main physiological functions of the sap were also determined., Methods: The genomic RNA in the sap was isolated and sequenced. Kyoto Encyclopedia of Gene and Genome (KEGG), Evolutionary genealogy of genes: Non-supervised Orthologous Groups (eggNOG) and Carbohydrate-Active enzymes Database (CAZy) functional annotation were used to analysis the function of micro-organisms in xylem sap. DEGs were analyzed by gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). The genes responsive to biotic and abiotic stresses were finally screened by transcriptome screening, stress data analysis and vitro validation experiments., Results: The analysis exhibited 36,144,564 micro-related clean reads and 244,213 unigene. KEGG, eggNOG and CAZy functional annotation analysis indicated that signal transduction and material metabolism were the most important function of xylem sap. DEGs analysis were mainly about disease resistance, carbon source metabolism and hormone signal transduction, especially in P3 vs P1, enriched in the plant-pathogen interaction pathway. Analysis on grape genome information revealed xylem sap had little RNA with weak life activity. Metabolic pathways, biosynthesis of secondary metabolites, plant hormone signal transduction and plant-pathogen interaction were the four pathways with the largest number of enriched genes. Moreover, 16 genes responsive to biotic and abiotic stresses were screened out., Conclusion: Promoting plant growth and resisting pathogens were the most important function of xylem sap during the bleeding period, and the function of microbial community were closely related to microorganisms growth and disease resistance. The 16 stress-related genes might be used for the future grape resistance research.

Small interfering RNAs (siRNAs) are effectors of regulatory pathways underlying plant development, metabolism, and stress- and nutrient-signaling regulatory networks. The endogenous siRNAs are generally not conserved between plants; consequently, it is necessary and important to identify and characterize siRNAs from various plants. To address the nature and functions of siRNAs, and understand the biological roles of the huge siRNA population in grapevine (Vitis vinifera L.). The high-throughput sequencing technology was used to identify a large set of putative endogenous siRNAs from six grapevine tissues/organs. Subsequently, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was performed to classify the target genes of siRNA. In total, 520,519 candidate siRNAs were identified and their expression profiles exhibited typical temporal characters during grapevine development. In addition, we identified two grapevine trans-acting siRNA (TAS) gene homologs (VvTAS3 and VvTAS4) and the derived trans-acting siRNAs (tasiRNAs) that could target grapevine auxin response factor (ARF) and myeloblastosis (MYB) genes. Furthermore, the GO and KEGG analysis of target genes showed that most of them covered a broad range of functional categories, especially involving in disease-resistance process. The large-scale and completely genome-wide level identification and characterization of grapevine endogenous siRNAs from the diverse tissues by high throughput technology revealed the nature and functions of siRNAs in grapevine.

Main Conclusion: Grapevine autophagy-related genes (ARGs) include 35 members that have unique evolutionary backgrounds and expression patterns, with some of them responding to abiotic stresses, including copper stress. Autophagy is one of the most crucial self-regulating phenomena in livings organisms, including animals, plants, yeasts, etc. In the genomes of plants, like Arabidopsis, rice, tobacco, and barley, more than 30 autophagy-related genes (ARGs) have been found. These ARGs are involved in plant development, programed cell death, and the stress response process. In plants, and particularly in grapevine, high copper stress results from the application of the Bordeaux mixture, a widely used fungicide. However, the function of autophagy in plant tolerance to copper stress is unknown. Accordingly, in this study, a genome-wide analysis was performed to identify Vitis vinifera ARGs (VvARGs), and 35 VvARGs were detected. A gene family analysis revealed that the tandem and segmental duplication events played significant roles in the VvARG gene family expansion. Moreover, there was more intense signature of purifying selection for the comparison between grape and rice than between grape and Arabidopsis. In response to copper treatment, both the autophagosome number and malondialdehyde concentration increased during the initial 4 h post-treatment, and reached maximal values at 24 h. An expression analysis indicated that most VvARGs responded to copper stress at 4 h post-treatment, and some VvARGs (e.g., VvATG6, VvATG8i, and VvATG18h) exhibited responses to most abiotic stresses. These results provide a detailed overview of the ARGs in grapevine and indicate multiple functions of autophagy in fruit development and abiotic stresses in grapevine. The key ARG (e.g., ATG8i) should be investigated in more detail in grapevine and other plant species.

miRNA prediction algorithms often fail to predict the accurate location of the mature miRNA in a precursor sequence with nucleotide-level precision. miRNAs-rapid amplification of cDNA ends (miR-RACE) is an efficient method to determine the precise sequences of computationally predicted microRNAs (miRNAs). miR-RACE includes the following steps: miRNA-enriched library preparation, two specific 5'- and 3'-miRNA RACE (miR-RACE) PCR reactions, and sequence-directed cloning. The most challenging step is the two gene-specific primers designed for the two RACE reactions. The miR-RACE protocol is rapid and can be executed and completed in 2-3 days.

Computational prediction of microRNAs (miRNAs) is one of the most important approaches in microRNA studies. While validation of the predicted microRNAs' precise sequences is essential for further studies on their biogenesis, evolution, and functions, computational miRNA prediction methods, however, often fail to predict the accurate sequence of the mature miRNA within the precursor at the nucleotide precision level. Here, we depict a highly efficient method for determining the precise sequences of computationally predicted miRNAs. The method combines the generation of miRNA-enriched libraries, with 5'- and 3'-end adaptors being linked to the miRNA molecules, the reverse transcription of small RNAs with an oligo-d(T) anchor primer, two specific 5'- and 3'-miRNA-RACE (miR-RACE) PCR reactions and sequence-directed cloning. The efficiency of this method was demonstrated by the precise sequence validation of computationally predicted miRNAs in citrus, apple, and other fruit crops. Our ongoing research indicates that miR-RACE is also very useful to verify the sequences of putative miRNAs obtained by deep sequencing of small RNA libraries. The protocol of miR-RACE is rapid and can be completed within 2-3 days. miR-RACE should make the bioinformatic prediction of miRNAs more powerful and accurate.

MicroRNAs (miRNAs) are important regulators involved in most biological processes in eukarya. They play critical roles in growth, development, signal transduction, or stress response by controlling gene expression at the posttranscriptional level. Identification and characterization of miRNA-targeted mRNAs is essential for the analysis of miRNA functions. In plants, the perfect complementarity between most miRNAs and their targets enables the accurate predictions of their targets, while slicing of the targeted mRNAs facilitates target validation through RNA Ligase-Mediated (RLM)-Rapid Amplification of cDNA Ends (RACE) method. However, this method only determines the 5'-end of the cleavage product. To more accurately validate the predicted target genes of miRNAs and exactly determine the cleavage sites within the targets, an integrated strategy comprising RLM-RACE, Poly(A) Polymerase-Mediated (PPM)-RACE, and qRT-PCR was developed. The efficiency of this method is illustrated by the precise sequence validation of predicted target mRNAs of miRNAs in grapevine, citrus, peach, and other fruit crops. Our on-going research indicates that RLM-RACE, PPM-RACE, and qRT-PCR are very effective in the verification of sequences of miRNA targets obtained by Degradome sequencing. The protocol for RLM-RACE, PPM-RACE, and qRT-PCR is rapid, effective, cheap, and can be completed within 2-3 days.

In plants, microRNAs (miRNAs) play a critical role in post-transcriptional gene regulation and have been shown to control many genes involved in various biological and metabolic processes. There have been extensive studies to discover miRNAs and analyze their functions in model plant species, such as Arabidopsis and rice. Deep sequencing technologies have facilitated identification of species-specific or lowly expressed as well as conserved or highly expressed miRNAs in plants. In this research, we used Solexa sequencing to discover new miRNAs in cultivated strawberry (Fragaria×ananassa). A total of 23,282 ,309 reads representing 22,500 ,402 distinct sequences were obtained from a short RNA library generated from small RNAs extracted from strawberry fruit tissues. On the basis of sequence similarity and hairpin structure prediction, we found that 156,639 reads representing 153 sequences have good matches to known miRNAs. We also identified 37 novel miRNA candidates. These sequences had not been previously described in other plant species. Potential target genes were predicted for the majority of and novel miRNAs. These results show that regulatory miRNAs exist in the agriculturally important cultivated strawberry and may play an important role in its growth, development and response to disease., (Copyright © Physiologia Plantarum 2012.)

Thirty-one potential miRNAs that belong to 16 miRNA families were discovered from more than 324 000 EST sequences of apple (Malus domestica). In addition, precise sequences, especially terminal nucleotides of the 16 apple miRNAs (mdo-miRNAs) in 16 families were validated by miR-RACE, a newly developed method for the determination of the potential miRNAs predicted computationally. The expression of these 16 microRNAs could be detected in apple young leaf, old leaf, young stem, flower bud, flower and developing fruits by quantitative RT-PCR (qRT-PCR) and some of them showed tissue-specific expression. Fifty-six potential targets were identified for the 16 apple miRNAs, most of which were transcription factors that play important roles in apple development. Twelve target genes were experimentally verified by qRT-PCR, with some exhibiting different expression trends from their corresponding microRNAs, indicating the cleavage mode of miRNAs on their target genes., (Copyright © Physiologia Plantarum 2010.)