Vitis Amurensis Research Articles

VaAPL1 Promotes Starch Synthesis to Constantly Contribute to Soluble Sugar Accumulation, Improving Low Temperature Tolerance in Arabidopsis and Tomato.

ADP-glucose pyrophosphorylase (AGPase) is a key rate-limiting enzyme involved in starch synthesis. APL1, an AGPase large subunit, plays an important role in the growth and development of grapes; however, its function in withstanding low temperature (LT) remains elusive. Hence, VaAPL1 was cloned from Vitis amurensis (Zuoshan I), and its function was characterized. The gene was highly expressed in the phloem of V. amurensis during winter dormancy (0, −5, and − 10°C). Phylogenetic relationships demonstrated that VaAPL1 was closely genetic related to SlAPL1 (from Solanum lycopersicum), and clustered into I group. Further, VaAPL1 was ectopically expressed in Arabidopsis thaliana (ecotype Columbia, Col) and tomato (“Micro-Tom” tomato) to characterize its function under LT. Compared with Col, the average survival rate of VaAPL1-overexpressing A. thaliana exceeded 75.47% after freezing treatment. Moreover, reactive oxygen species (ROS) content decreased in VaAPL1-overexpressing A. thaliana and tomato plants under LT stress. The activities of AGPase, and starch contents in VaAPL1-overexpressing A. thaliana were higher than in Col after LT stress. The contents of sucrose and glucose were accumulated in overexpressing plants compared with wild-type at 0 h and 24 h after LT stress. Transcriptome sequencing of overexpressing tomato plants revealed involvement in sugar metabolism and the hormone signal pathway, and Ca2+ signaling pathway-related genes were up-regulated. Hence, these results suggest that overexpression of VaAPL1 not only ensured sufficient starch converting into soluble sugars to maintain cell osmotic potential and provided energy, but also indirectly activated signal pathways involved in LT to enhance plant tolerance.

VaMYB44 transcription factor from Chinese wild Vitis amurensis negatively regulates cold tolerance in transgenic Arabidopsis thaliana and V. vinifera.

Heterologous expression of VaMYB44 gene in Arabidopsis and V. vinifera cv. 'Thompson Seedless' increases cold sensitivity, which is mediated by the interaction of VaMYC2 and VaTIFY5A with VaMYB44 MYB transcription factors play critical roles in plant stress response. However, the function of MYB44 under low temperature stress is largely unknown in grapes. Here, we isolated a VaMYB44 gene from Chinese wild Vitis amurensis acc. 'Shuangyou' (cold-resistant). The VaMYB44 is expressed in various organs and has lower expression levels in stems and young leaves. Exposure of the cold-sensitive V. vinifera cv. 'Thompson Seedless' and cold-resistant 'Shuangyou' grapevines to cold stress (-1°C) resulted in differential expression of MYB44 in leaves with the former reaching 14 folds of the latter after 3h of cold stress. Moreover, the expression of VaMYB44 was induced by exogenous ethylene, abscisic acid, and methyl jasmonate in the leaves of 'Shuangyou'. Notably, the subcellular localization assay identified VaMYB44 in the nucleus. Interestingly, heterologous expression of VaMYB44 in Arabidopsis and 'Thompson Seedless' grape increased freezing-induced damage compared to their wild-type counterparts. Accordingly, the transgenic lines had higher malondialdehyde content and electrolyte permeability, and lower activities of superoxide dismutase, peroxidase, and catalase. Moreover, the expression levels of some cold resistance-related genes decreased in transgenic lines. Protein interaction assays identified VaMYC2 and VaTIFY5A as VaMYB44 interacting proteins, and VaMYC2 could bind to the VaMYB44 promoter and promote its transcription. In conclusion, the study reveals VaMYB44 as the negative regulator of cold tolerance in transgenic Arabidopsis and transgenic grapes, and VaMYC2 and VaTIFY5A are involved in the cold sensitivity of plants by interacting with VaMYB44.

Overexpression of grape ABA receptor gene VaPYL4 enhances tolerance to multiple abiotic stresses in Arabidopsis

BackgroundAbscisic acid (ABA) plays a crucial role in abiotic stress responses. The pyrabactin resistance (PYR)/PYR-like (PYL)/regulatory component of ABA receptor (RCAR) proteins that have been characterized as ABA receptors function as the core components in ABA signaling pathway. However, the functions of grape PYL genes in response to different abiotic stresses, particularly cold stress, remain less studied.ResultsIn this study, we investigated the expression profiles of grape PYL genes upon cold treatment and isolated the VaPYL4 gene from Vitis amurensis, a cold-hardy grape species. Overexpression of VaPYL4 gene in grape calli and Arabidopsis resulted in enhanced cold tolerance. Moreover, plant resistance to drought and salt stress was also improved by overexpressing VaPYL4 in Arabidopsis. More importantly, we evaluated the contribution of VaPYL4 to plant growth and development after the treatment with cold, salt and drought stress simultaneously. The transgenic plants showed higher survival rates, earlier flowering phenotype, and heavier fresh weight of seedlings and siliques when compared with wild-type plants. Physiological analyses showed that transgenic plants had much lower content of malondialdehyde (MDA) and higher peroxidase (POD) activity. Stress-responsive genes such as RD29A (Responsive to desiccation 29A), COR15A (Cold responsive 15A) and KIN2 (Kinase 2) were also significantly up-regulated in VaPYL4-overexpressing Arabidopsis plants.ConclusionsOur results show that overexpression of VaPYL4 could improve plant performance upon different abiotic stresses, which therefore provides a useful strategy for engineering future crops to deal with adverse environments.

Optimisation of embryo rescue for cold-resistant seedless grapevine

ABSTRACT Embryo rescue is essential for the breeding of seedless grapes, but current protocols are inefficient and limit practical application. Here, we documented factors influencing embryo rescue efficiency using crosses of a cold-resistant seeded hybrid 00-1-5 (‘Muscat Hamburg’ × Vitis amurensis) as the paternal parent with four stenospermocarpic seedless grape cvs., ‘Flame Seedless’, ‘Qinhong No. 2’, ‘Qinhong No. 10’, and ‘Ruby Seedless’. The results showed that treatment of inflorescences with 30∼50 mg·L−1 IBA 14 days before flowering significantly increased the embryo development, germination, and transition to seedling for progeny of each of the four crosses. ‘Qinhong No. 2’ × 00-1-5 and ‘Qinhong No. 10’ × 00-1-5, with a large number of ovules, were cultured on solid and solid–liquid biphase MM3 media respectively, the embryo development rate on solid medium was significantly better than that on solid–liquid biphase medium. ‘Ruby Seedless’, ‘Qinhong No. 2’ and ‘Qinhong No. 10’ are more suitable for embryo rescue as maternal parent. The optimal medium for transforming deformed seedlings into normal seedlings was 2 × MS medium containing 0.2 mg·L−1 6-BA, 0.1 mg·L−1 IAA, and 1.6 mg·L−1 ZnSO4. In total, 311 embryos were rescued successfully, which were considered as potential cold-resistant seedless hybrids.

Protective Effect and Mechanism of Soybean Insoluble Dietary Fiber on the Color Stability of Malvidin-3-O-glucoside.

Anthocyanins have great health benefits, especially malvidin. Vitis amurensis Rupr are rich in malvidin, and malvidin-3-O-glucoside (Mv3G) monomer is the most abundant. However, natural anthocyanins are unstable, which limits their wide application in the food field. Soybean insoluble dietary fiber (SIDF) has high stability, and it can be used as an inert substrate to construct a stable system, which may improve the stability of anthocyanins. The optimal condition to construct a stable system of SIDF and Mv3G at pH 3.0 was determined by an orthogonal experiment. The results indicated that SIDF effectively improved the stability of Mv3G under different pH values (1.0~7.0), high temperature (100 °C for 100 min), and sunlight (20 ± 2 °C for 30 d) conditions. The absorption peak intensity of the UV–VIS spectrum of SIDF-Mv3G was enhanced, which indicated that there was interaction between SIDF and Mv3G. Fourier transform infrared spectroscopy analyses revealed that the -OH stretching vibration peak of SIDF-Mv3G was changed, which indicated that the interaction between SIDF and Mv3G was due to hydrogen bonding. X-ray diffraction analysis showed that the crystalline morphology of SIDF was opened, which was combined with Mv3G, and SIDF made Mv3G change to a more stable state. Scanning electron microscope analysis showed that SIDF and Mv3G were closely combined to form an inclusion complex. Overall, this study provides valuable information for enhancing the color stability of anthocyanins, which will further expand the application of anthocyanins in the food field.

The Biodiversity of Grapevine Bacterial Endophytes of Vitis amurensis Rupr.

In this paper, the composition profiles of bacterial endophytes in wild-growing Amur grape Vitis amurensis Rupr. grown in the south of the Russian Far East were analyzed using both a cultivation-dependent (sowing bacteria) and a cultivation-independent (next generation sequencing, NGS) approach. Both methods revealed the prevalent endophytes in V. amurensis were represented by Gammaproteobacteria—40.3–75.8%, Alphaproteobacteria—8.6–18.7%, Actinobacteria—9.2–15.4%, and Bacilli—6.1–6.6%. NGS also showed a large proportion of Bacteroidia (12.2%) and a small proportion of other classes (less than 5.7%). In general, NGS revealed a greater variety of classes and genera in the endophytic bacterial community due to a high number of reads (574,207) in comparison with the number of colonies (933) obtained after the cultivation-dependent method. A comparative analysis performed in this study showed that both wild grape V. amurensis from Russia and domesticated cultivars of V. vinifera from Germany and California (USA) exhibit the same basic composition of endophytic bacteria, while the percentages of major taxa and minor taxa showed some differences depending on the plant organ, grape individuals, environmental conditions, and sampling time. Furthermore, the obtained data revealed that lower temperatures and increased precipitation favored the number and diversity of endophytic bacteria in the wild Amur grape. Thus, this study firstly described and analyzed the biodiversity of endophytic bacteria in wild grapevine V. amurensis.

Impact of Different Oak Chips' Aging on the Volatile Compounds and Sensory Characteristics of Vitis amurensis Wines.

In this work, different oak chips were used to age Vitis amurensis wine, and the effects on sensory properties were observed. Twenty-one different oak chips were added to a one-year-old wine made by a traditional technique. The wine was aged for 6 months before analysis by CIELab for color parameters, GC–MS for volatile compounds, and electronic tongue and a tasting panel for sensory properties. The results showed that the addition of any tested oak chip could significantly strengthen the wine’s red color. Among 61 volatile compounds, alcohols presented the highest concentrations (873 to 1401 mg/L), followed by esters (568 to 1039 mg/L) and organic acids (157 to 435 mg/L), while aldehydes and volatile phenols occurred at low concentrations. Different oak species with different toasting levels could affect, to varying degrees, the concentrations of esters, alcohols, and volatile phenols, but to a lesser extent those of aldehydes. Sensory analysis by a tasting panel indicated that non- and moderately roasted oak chips gave the wines higher scores than those with heavy toasting levels. The major mouthfeel descriptors determined by electronic tongue were in good agreement with those from the tasting panel.

Temperature-phase transcriptomics reveals that hormones and sugars in the phloem of grape participate in tolerance during cold acclimation.

Most of the upregulated genes contributed to the accumulation of soluble sugars and ABA in the phloem of 'Vitis amurensis' compared to 'Merlot' during cold acclimation. Extreme cold is one of the dominant abiotic factors affecting grape yield and quality. However, the changes in sugars, phytohormones, and gene expression in the branch phloem of different tolerant grape varieties during cold acclimation remain elusive. The data supported that with decreasing temperature, the contents of fructose, sucrose, and ABA in the phloem of Vitis amurensis (cold-tolerant, T) and 'Merlot' (cold-sensitive, S) increased during cold acclimation, and these indicators were higher in T than in S. Furthermore, the activities of sucrose synthetase, sucrose phosphate synthetase, and acid invertase peaked in the early phase of cold acclimation (approximately 5°C) compared to other phases (approximately 28°C, 0°C, - 5°C and - 10°C). Moreover, the RNA sequencing results helped identify a total of 11,343 differentially expressed genes in the phloem of T and S, among which 4912 were upregulated and 6431 were downregulated. In the abscisic acid pathway, CRTISO, PSPY1-1, CYCP707A4-2, PYL4-1, PYL4-2, P2C08, SAPK2, TARAB1, and DBF3 were more highly expressed in T than in S. In the starch and sucrose metabolism pathway, HXK1, PGMP, GLGL1, SUS6, VCINV, BGL11, SSY1, GPS, BAM1 and BAM3 were also more highly expressed in T than in S. Moreover, the genes related to oxidative phosphorylation, such as NDHF, ND4, ND1, NAD7, NAD2, ATPB, YMF19, ATP9, PMA1 and AHA8, were upregulated in T. These results will be beneficial for understanding the potential differences in tolerance across two different cold-tolerant grapes with respect to sugar metabolism and gene expression.

Heterologous expression of the cysteine proteinase gene VaCP17 from the cold-adapted grapevine Vitis amurensis increases cold tolerance in Arabidopsis

Cysteine proteinases (thiol) carry out diverse and critical functions in plants through their ability to hydrolyze peptide bonds in target proteins. Here, we cloned a cysteine proteinase gene designated VaCP17 from a highly cold-resistant wild Vitis amurensis accession ‘Shuangyou’, and then its potential function in cold resistance was investigated. The results showed that the CDS of VaCP17 is 1404 bp, encoding 467 amino acids, the VaCP17 protein localized to the cell membrane. Expression of CP17 was highly distinctive among different structures of ‘Shuangyou’ and the cold-sensitive Vitis vinifera cultivar ‘Red Globe’, with the highest expression in the stem of ‘Shuangyou’ and the flower of ‘Red Globe’. Arabidopsis plants constitutively expressing a VaCP17-GREEN FLUORESCENT PROTEIN fusion (35S::VaCP17-GFP) showed increased survival after transient exposure to freezing (-6 °C), and showed lower electrolyte leakage and MDA content, higher soluble sugar content and SOD, POD and CAT activities, as compared with non-transgenic Arabidopsis controls. The expression of nine cold-resistance related genes (CBF1, CBF2, CBF3, RD29A, COR15A, KIN1, NCED3, AOC1 and JAZ10) in 35S::VaCP17-GFP plants was increased under cold treatment at 4 °C, relative to control plants. Using a yeast two-hybrid system, we identified VaNAC72, VaCAM7 and VaDi19 as potential interactors of VaCP17, and their interactions were demonstrated by a bimolecular fluorescence complementation assay. In conclusion, we revealed that VaCP17 can enhance cold resistance by influencing physiology and biochemistry and the expression of cold resistance related genes under cold stress.

Enhancement of Stilbene Biosynthesis in Grape-Cell Cultures by Natural Products Based on Endophytes of the Wild Grape Species Vitis amurensis RUPR.

Enhancement of Stilbene Biosynthesis in Grape-Cell Cultures by Natural Products Based on Endophytes of the Wild Grape Species Vitis amurensis RUPR.

Effect of Calmodulin-like Gene (CML) Overexpression on Stilbene Biosynthesis in Cell Cultures of Vitis amurensis Rupr.

Stilbenes are plant phenolics known to rapidly accumulate in grapevine and other plants in response to injury or pathogen attack and to exhibit a great variety of healing beneficial effects. It has previously been shown that several calmodulin-like protein (CML) genes were highly up-regulated in cell cultures of wild-growing grapevine Vitis amurensis Rupr. in response to stilbene-modulating conditions, such as stress hormones, UV-C, and stilbene precursors. Both CML functions and stilbene biosynthesis regulation are still poorly understood. In this study, we investigated the effect of overexpression of five VaCML genes on stilbene and biomass accumulation in the transformed cell cultures of V. amurensis. We obtained 16 transgenic cell lines transformed with the VaCML52, VaCML65, VaCML86, VaCML93, and VaCML95 genes (3–4 independent lines per gene) under the control of the double CaMV 35S promoter. HPLC-MS analysis showed that overexpression of the VaCML65 led to a considerable and consistent increase in the content of stilbenes of 3.8–23.7 times in all transformed lines in comparison with the control calli, while biomass accumulation was not affected. Transformation of the V. amurensis cells with other analyzed VaCML genes did not lead to a consistent and considerable effect on stilbene biosynthesis in the cell lines. The results indicate that the VaCML65 gene is implicated in the signaling pathway regulating stilbene biosynthesis as a strong positive regulator and can be useful in viticulture and winemaking for obtaining grape cultivars with a high content of stilbenes and stress resistance.

Comparative Metabolomics Analysis of Dormancy Buds During Cold Accumulation between Cold-Sensitive Grapevine (Vitis Vinifera) and Cold-Hardy Grapevine (Vitis Amurensis)

Comparative Metabolomics Analysis of Dormancy Buds During Cold Accumulation between Cold-Sensitive Grapevine (Vitis Vinifera) and Cold-Hardy Grapevine (Vitis Amurensis)

VaAPRT3 Gene is Associated With Sex Determination in Vitis amurensis.

In the past decade, progress has been made in sex determination mechanism in Vitis. However, genes responsible for sexual differentiation and its mechanism in V. amurensis remain unknown. Here, we identify a sex determination candidate gene coding adenine phosphoribosyl transferase 3 (VaAPRT3) in V. amurensis. Cloning and sequencing of the VaAPRT3 gene allowed us to develop a molecular marker able to discriminate female individuals from males or hermaphrodites based on a 22-bp InDel. Gene expression and endogenous cytokinin content analysis revealed that the VaAPRT3 gene is involved in sex determination or, to be precise, in female organ differentiation, through regulating cytokinin metabolism in V. amurensis. This study enlarged the understanding of sex determination mechanism in the genus Vitis, and the sex marker could be used as a helpful tool for sexual identification in breeding programs as well as in investigation and collection of V. amurensis germplasms.

An Amur grape VaHsfC1 is involved in multiple abiotic stresses

Plant heat stress transcription factors (Hsfs) belong to a very large gene family and play dominant roles in response to heat stress. Here, we report the functional analysis of VaHsfC1, a class C Hsf gene from Vitis amurensis, and involved in multiple abiotic stresses. The structure of VaHsfC1 contains a typical HSF domain for DNA-binding at N terminus. Phylogenetic analysis revealed that VaHsfC1 belongs to the C1 family of Hsfs. VaHsfC1 was highly induced by heat, cold, exogenous abscisic acid (ABA) and salinity, respectively. Unusually, VaHsfC1was preferentially localized in nucleus, but less in the cytosol. Also, no transcriptional activation was detected for the protein in yeast cells. Ectopic VaHsfC1 expression in Arabidopsis enhanced heat tolerance characterized by the increased survival rate, higher chlorophyll content, reduced electrolyte leakage, and activated expressions of ascorbate peroxidase (Apx2) and heat shock protein (HSP) expression compared to the WT. Interestingly, overexpression of VaHsfC1 in Arabidopsis could also increase the cold tolerance, as reveled from a series changes at the physiological and biochemical steps, as well as at the transcript levels of CORs in the CBF-dependent responsive pathway. Additionally, the growth of VaHsfC1-overexpressing Arabidopsis showed enhanced seed ABA hypersensitivity during germination and cotyledon development, and a significant reduction in stomatal aperture was also observed in overexpression lines. Furthermore, VaHsfC1-overexpressing lines displayed salt-hypersensitive phenotype with a lower rates both in germination and cotyledon greening, and the inhibition of root growth. Our findings demonstrated that the complex nature of VaHsfC1 in mediating multiple abiotic stress responses, as a key component to further understanding of the ‘crosstalk’ mechanism of multiple abiotic stress response in plants.

Ultrastructural Observations of Botrytis cinerea and Physical Changes in Resistant and Susceptible Grapevines.

The necrotrophic fungus Botrytis cinerea is a major threat to grapevine cultivation worldwide. Here, a highly resistant Chinese wild grapevine, Vitis amurensis 'Shuangyou' (SY), and the susceptible V. vinifera 'Red Globe' (RG) were selected for study, and their pathogenic infection and biochemical responses to B. cinerea were evaluated. The results revealed more trichomes on and a thicker cuticle for leaves of SY than RG under scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Both SEM and TEM also showed that conidial germination, appressorium formation, and hyphal development of B. cinerea were delayed on the leaves of resistant SY. Fewer infected hyphae were also observed in leaves of resistant SY when compared with susceptible RG. The infected leaves of resistant SY harbored higher levels of cellulase and pectinase activity during the early infection stages of B. cinerea at 4 h postinoculation (hpi), and higher glucanase and chitinase activity were maintained in the inoculated leaves of SY from 4 through 18 hpi. Lignin was deposited in the infected leaves of susceptible RG but not in resistant SY. Taken together, these results provide insights into the ultrastructural characterizations and physical changes in resistant and susceptible grapevines.

Overexpression of the VaERD15 gene increases cold tolerance in transgenic grapevine

In plants, the Early Responsive to Dehydration (ERD) genes are rapidly activated in response to drought and other abiotic stresses. Here, we characterize the VaERD15 gene from the cold-resistant grapevine Vitis amurensis accession 'Shuangyou', and its counterpart VvERD15 from the cold-sensitive V. vinifera cultivar 'Red Globe'. To evaluate a role for VaERD15 in promoting cold tolerance, we expressed the VaERD15 in transgenic V. vinifera 'Red Globe' and evaluated its influence on type and cold stress-related physiological and biochemical indices. We also assessed the response of the ERD15 promoter and its deletion fragments from 'Shuangyou' and 'Red Globe' to low temperature, ABA, SA and PEG. Additionally, several proteins interacting with VaERD15 were identified and the expression profiles for the respective genes were evaluated in response to low temperature. We found that the VaERD15 protein contains a PAM2 domain and localizes in the nucleus and cell membrane. Under low-temperature stress, the expression of ERD15 in 'Shuangyou' was greater than that in 'Red Globe’. Transgenic 35S::VaERD15 plants showed greater cold tolerance and had lower electrical conductivity and malondialdehyde content than non-transgenic controls, while the contents of proline, soluble sugar, soluble protein and the activities of SOD, POD and CAT were higher than those in non-transgenic controls. The ERD15 promoter and its deletions could respond to low temperature, ABA, SA and PEG, but the degree of response to the treatments varied. Proteins interacting with VaERD15 were identified as xyloglucan endotransglucosylase/hydrolase protein 7, glutamine synthetase, 40S ribosomal protein 23 and disulfide-isomerase LQY1, and the corresponding genes responded to cold temperatures to varying degrees.

Effect of stabilization malvids anthocyanins on the gut microbiota in mice with oxidative stress.

Malvids anthocyanins have been proven to have a significant antioxidant activity. However, natural anthocyanins are unstable as they are easily affected by temperature, light, and pH. They can produce copigmentation with caffeic acids, leading to the improvement of color stability. The objective of this research was to survey the anti-oxidative stress functional role of stabilization malvids anthocyanins (SMA) in vivo. Changes on the superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), total antioxidant capacity (T-AOC) and malondialdehyde (MDA) in the serum and liver of oxidatively damaged mice of SMA were investigated. The effects of SMA on the diversity of gut microbiota in mice with oxidatively damage were also evaluated. Compared to oxidative damaged mice, SMA increased the activities of SOD, GSH-Px, CAT and T-AOC but decreased the levels of MDA in the serum and liver. SMA significantly changed the composition and diversity of the gut microbiota. Specifically, SMA increased the relative abundance of the phylum Firmicutes and decreased the relative abundance of the phyla Bacteroidetes. At the genus level, SMA significantly increased the relative abundance of Lactobacillus, but decreased the relative abundance of Bacteroides. In addition, SMA also reversed carbohydrate metabolism and amino acid metabolism to normal levels. It indicates that SMA could protect the body from oxidative damage and be used as a potential functional food to prevent diseases related to oxidative stress. PRACTICAL APPLICATIONS: Anthocyanins provide protective effects against harmful effect of oxidative stress. Natural anthocyanins are safer and nutritious as compared to synthetic pigments. However, their stability is poor. The previous research done by this group showed that the anthocyanins content of variety of Vitis amurensis Rupr was as high as 180mg/(100g·FW), and the content of malvids anthocyanidin in its ingredients was the highest of all. Malvids anthocyanin and caffeic acid are bonded to produce stabilized malvids anthocyanins (SMA) high hydrostatic pressure technology, which has better stability. Our results indicate that SMA could increased the activities of antioxidant enzymes and altered the composition and diversity of the gut microbiota in mice with oxidative damage. The study will help to deepen the understanding of antioxidative stress mechanism of SMA and lay a foundation for the application of natural anthocyanidin in health aspect.

Quality Characteristics and Anthocyanin Profiles of Different Vitis amurensis Grape Cultivars and Hybrids from Chinese Germplasm

To evaluate the important Vitis amurensis germplasm, the quality characteristics and anthocyanin profiles of the ripe berries of 20 V. amurensis grapes and 11 interspecific hybrids in two consecutive years were analysed. Compared with the V. vinifera grapes, V. amurensis grapes had small berries with low total soluble solids and high titratable acids, and were richer in phenolic compounds except for flanan-3-ols in their skins but had lower phenolic contents in their seeds and showed lower antioxidant activities. An outstanding feature of the V. amurensis grapes was their abundant anthocyanin contents, which was 8.18-fold higher than the three wine grapes of V. vinifera. The anthocyanin composition of V. amurensis was characterized by an extremely high proportion of diglucoside anthocyanins (91.71%) and low acylated anthocyanins (0.04%). Interestingly, a new type of speculated 3,5,7-O-triglucoside anthocyanins was first identified and only detected in V. amurensis grapes and hybrids. Based on the total phenolic and anthocyanin characteristics, V. amurensis grapes were set apart from V. vinifera cultivars and the interspecific hybrids, for the same qualities, fell between them, as assessed by principal component analysis.

Identification of Stabilization of Malvid Anthocyanins and Antioxidant Stress Activation via the AMPK/SIRT1 Signaling Pathway.

Vitis amurensis Rupr. “Beibinghong” is abundant in anthocyanins, including malvidin (Mv), malvidin-3-glucoside (Mv3G), and malvidin-3,5-diglucoside (Mv35 G). Anthocyanins offer nutritional and pharmacological effects, but their stability is poor. Interaction of malvid anthocyanins with caffeic acid through ultrahigh pressure technology produces stable anthocyanin derivatives. This study aims to identify the structure of stable mallow-like anthocyanins and to determine the effect of these stable anthocyanins on human umbilical vein endothelial cells (HUVECs) with H2O2-induced oxidative damage and the signaling pathway involved. The products of malvid anthocyanins and caffeic acid bonding were identified and analyzed using ultra-high performance liquid chromatography-quadrupole-Orbitrap mass spectrometry (UPLC-Q-Orbitrap MS/MS). The bonding products were malvidin-3-O-guaiacol (Mv3C), malvidin-3-O-(6″-O-caffeoyl)-glucoside (Mv3CG), and malvidin-3-O-(6″-O-caffeoyl)-5-diglucoside (Mv3C5G). An oxidative stress injury model in HUVECs was established using H2O2 and treated with Mv, Mv3G, Mv35 G, Mv3C, Mv3CG, and Mv3C5G at different concentrations (10, 50, and 100 μmol/L). Results showed that the above compound concentrations can significantly increase cell proliferation rate and reduce intracellular reactive oxygen species at 100 μmol/L. The effects of the most active products Mv and Mv3C on the AMP-activated protein (AMPK)/silencing information regulator-1 (SIRT1) pathway were analyzed. Results showed that Mv and Mv3C significantly increased SOD activity in the cells and significantly upregulated the expression of SIRT1 mRNA, SIRT1, and p-AMPK protein. However, they did not significantly change the expression of AMPK protein. After the silent intervention of siRNA in SIRT1 gene expression, the upregulation of SIRT1 and p-AMPK protein by Mv and Mv3C was significantly inhibited. These results indicate that stabilization malvid anthocyanins exerts an antioxidant activity via the AMPK/SIRT1 signaling pathway.

VaCRK2 Mediates Gray Mold Resistance in Vitis amurensis by Activating the Jasmonate Signaling Pathway

Cysteine-rich receptor-like kinases (CRKs) are ubiquitous plant receptor-like kinases, which play a significant role in plant disease resistance. Gray mold is an economically important disease of grapes caused by Botrytis cinerea. However, CRK genes and their function in gray mold disease resistance in grapes have not been elucidated. This study aimed to identify and characterize CRKs in grapes and determine their role in gray mold resistance. Four CRKs were identified in Vitis amurensis and named VaCRK1–VaCRK4 according to their genomic distribution. The four VaCRKs were ectopically expressed in Arabidopsis thaliana to study their function in defense response against B. cinerea. Heterologous expression of VaCRK2 in A. thaliana conferred resistance to B. cinerea. VaCRK2 expression in gray mold-resistant grape cultivar increased significantly after B. cinerea inoculation and methyl jasmonate treatment. Furthermore, the expression of jasmonic acid (JA) signaling pathway-related genes in VaCRK2 overexpression lines of A. thaliana was significantly increased after B. cinerea inoculation, leading to the upregulation of pathogenesis-related (PR) genes and reactive oxygen species (ROS) accumulation. Overall, these results suggest that VaCRK2 confers resistance to B. cinerea by activating PR gene expression and oxidative burst through the JA signaling pathway.