Comparative metabolic profiling of Vitis amurensis and Vitis vinifera during cold acclimation

Vitis amurensis is a wild Vitis plant that can withstand extreme cold temperatures.However,the accumulation of metabolites during cold acclimation(CA)in V.amurensis remains largely unknown.In this study,plantlets of V.amurensis and V.vinifera cv.Muscat of Hamburg were treated at 4℃ for 24 and 72 h,and changes of metabolites in leaves were detected by gas chromatography coupled with time-of-flight mass spectrometry.Most of the identified metabolites,including carbohydrates,amino acids,and organic acids,accumulated in the two types of grape after CA.Galactinol,raffinose,fructose,mannose,glycine,and ascorbate were continuously induced by cold in V.amurensis,but not in Muscat of Hamburg.Twelve metabolites,including isoleucine,valine,proline,2-oxoglutarate,and putrescine,increased in V.amurensis during CA.More galactinol,ascorbate,2-oxoglutarate,and putrescine,accumulated in V.amurensis,but not in Muscat of Hamburg,during CA,which may be responsible for the excellent cold tolerance in V.amurensis.The expression levels of the genes encodingβ-amylase(BAMY),galactinol synthase(GolS),and raffinose synthase(RafS)were evaluated by quantitative reverse transcription-PCR.The expression BAMY(VIT_02s0012 g00170)and RafS(VIT_05s0077 g00840)were primarily responsible for the accumulation of maltose and raffinose,respectively.The accumulation of galactinol was attributed to different members of GolS in the two grapes.In conclusion,these results show the inherent differences in metabolites between V.amurensis and V.vinifera under CA.

山葡萄(Vitis amurensis)资源核心种质的初步构建

采用省份分组-花型分组-聚类-分组法构建了山葡萄核心种质,在此基础上,考虑到山葡萄品种在生产上和雄株山葡萄在科学研究上的作用,把未进入核心种质的山葡萄品种和雄株山葡萄也并入核心种质。该核心种质包括48份资源(占资源总数的31%),其中来自吉林省的资源18份(占37.5%),黑龙江省的资源29份(占60.4%),辽宁省的资源1份(占2.1%);两性花资源22份(占45.8%),雌能花资源21份(占43.8%),雄株5份(占10.4%)。除出汁率外,其他性状的符合度达72%以上;除果粒质量外,其他性状的变幅吻合度达79%以上。以上结果表明,所构建的核心种质较好地代表了评价的山葡萄资源。

QTL detection and candidate gene analysis of grape white rot resistance by interspecific grape (Vitis vinifera L. × Vitis davidii Foex.) crossing

Grape white rot,a devastating disease of grapevines caused by Coniella diplodiella(Speg.)Sacc.,leads to significant yield losses in grape.Breeding grape cultivars resistant to white rot is essential to reduce the regular use of chemical treatments.In recent years,Chinese grape species have gained more attention for grape breeding due to their high tolerance to various biotic and abiotic factors along with changing climatic conditions.In this study,we employed whole-genome resequencing(WGR)to genotype the parents of‘Manicure Finger’(Vitis vinifera,female)and‘0940’(Vitis davidii,male),along with 101 F1 mapping population individuals,thereby constructing a linkage genetic map.The linkage map contained 9337 single-nucleotide polymorphism(SNP)markers with an average marker distance of 0.3 cM.After 3 years of phenotypic evaluation of the progeny for white rot resistance,we confirmed one stable quantitative trait locus(QTL)for white rot resistance on chromosome 3,explaining up to 17.9%of the phenotypic variation.For this locus,we used RNA-seq to detect candidate gene expression and identified PR1 as a candidate gene involved in white rot resistance.Finally,we demonstrated that recombinant PR1 protein could inhibit the growth of C.diplodiella and that overexpression of PR1 in susceptible V.vinifera increased grape resistance to the pathogen.

VitisMod:一个葡萄基因共表达数据库

葡萄转录组已在各种组织、发育阶段、生物胁迫、非生物胁迫和其他条件下被测定。目前,仍没有简单实用的网络工具来探索这些宝贵的数据。本文从美国国立生物技术信息中心的基因表达数据库(NCBI GEO)下载1019个基因表达芯片数据,进行权重基因共表达网络分析(WGCNA)。鉴定到41个基因共表达模块。功能富集分析发现这些模块具有不同的功能,并与实验/表型相关。通过模块内连接度筛选枢纽基因,这些基因可能具有重要功能。通过关联推定(Guilt-by-association)原理对模块内功能未知的基因进行功能预测。最后,构建了免费的网络工具VitisMod,为葡萄的基因功能研究提供新资源,网址为:http://bioinformatics.fafu.edu.cn/grape。

Synthesis of Amurensin H, a New Resveratrol Dimer from the Roots of Vitis Amurensis

Amurensin H(1) is a new resveratrol dimer isolated from the roots of Vitis amurensis Rupr. Its structure was determined by spectroscopic methods. II was synthesized from resveratrol with an oxidative coupling reaction as a key step.

A New Oligostilbene from the Roots of Vitis Amurensis

A new resveratrol trimer. amurensin G (1), was isolated from the roots of Vitis amurensis Rupr. Its structure and relative configuration were established on the basis of spectral evidence. especially on HMBC spectrum and NOE difference experiments.

Expression of structural genes related to anthocyanin biosynthesis of Vitis amurensis

This research was designed to assess the changes in anthocyanin content in grape skins of Vitis amurensis and to explore m RNA transcriptions of 11 structural genes（PAL,CHS3, CHI1, F3H2, F30 H, F3050 H, DFR, LDOX, UFGT,OMT and GST） related to anthocyanin biosynthesis during grape berry development, by the use of HPLC-MS/MS and real-time Q-PCR analysis. Accumulation of anthocyanins began at veraison, continued throughout the later berry development and reached a peak at maturity. Veraison is the time when the berries turn from green to purple. Expression of PAL, CHI1, and LDOX were up-regulated from 2 to4 weeks after flowering（WAF）, down-regulated from6 WAF to veraison, whereas DFR was up-regulated at8 WAF, and then up-regulated from veraison to maturity.CHS3, F3050 H, UFGT, GST, and OMT were down-regulated from 2 WAF to veraison, and then up-regulated from veraison to maturity. The transcriptional expressions of the11 structural genes also showed positive correlations with the anthocyanin content from veraison to maturity. Positive correlations were also observed between OMT transcriptional level and the content of methoxyl-anthocyanins, and between F3050 H transcriptional level and the content of delphinidin anthocyanins. F3H2 and F30 H expression was up-regulated at 2 WAF. F3H2 expression was down-regulated from 4 WAF to veraison and then up-regulated again from veraison to maturity. F30 H expression was down-regulated at 4 WAF and then up-regulated again from 6 WAF to maturity. F30 H transcriptional level was correlated positively with the cyanidin anthocyanin concentration from veraison to maturity. These results indicate that the onset of anthocyanin synthesis during berry development coincides with a coordinated increase in the expression of a number of genes in the anthocyanin biosynthetic pathway.

Expressed sequence tags (ESTs) analysis of the ripening Vitis amurensis cv. Shuang Hong berry skins

Vitis amurensis is a valuable resource for wine production. Ripening of the grape berry is the key phase which determines the com- position of wine. To better understand the gene expression that manifest in V. amurensis berry skins during the ripening, cDNA library of V. amurensis berry skins was constructed. A total of 935 high quality ex- pressed sequence tags （ESTs） were obtained from the library. These ESTs represent 636 unigenes, including 108 contigs and 528 singletons. The EST analysis was performed and genes were assigned to functional categories according to their primary BLAST match. Of these 25.35% were involved with metabolism, 6.27% with cell rescue and defense, 6.84% energy, 11.68% protein synthesis, 18.8% protein activity regula- tion, 11.11% cell structure, 7.98% transport, 6.27% transcription and the remaining 5.7% were signal transduction. The generated ESTs were characterized by the gene ontology analysis and were categorized ac- cording to its cellular component, molecular function and biological process. In the cDNA library, some genes are relevant to the biosynthesis of anthocyanins, while some genes are related to grape berry maturation.

Isolation and expression analysis of CBF4 from <i>Vitis amurensis</i>associated with stress

The C-repeat Binding Factor/Dehydration Responsive Element Binding factor (CBF/DREB) responsive pathway is involved in plant response to abiotic stress. In this study, we report the isolation of VaCBF4 (a complete cDNA) and its promoter from Vitis amurensis through rapid amplification of cDNA ends and genome walking techniques, respectively. The CBF4 transcript accumulation of V. amurensis increased under cold, salinity, and abscisic acid (ABA) and salicylic acid (SA) treatments, whereas that of Vitis vinifera showed a different change. The transcript levels of VaCBF4 inthe roots, stems, leaves, and petioles under cold, salinity, and ABA and SA treatments were up-regulated in a timedependent manner. The presence of the cis-elements MBC, ABRE, and as-2-box in the VaCBF4 promoter further confirmed that this promoter is a component of the CBF transduction pathway, which is involved in plant response to multistress.

Isolation and expression characterization of CBF2 in <i>vitis amurensis</i>with stress

The transcription factor VaCBF2, which interacts with C-repeat/DRE and its promoter, was isolated from Vitis amurensis. The VaCBF2 amino acid sequence contained a conserved AP2 domain of 56 amino acids and a potential nuclear localization sequence. The sequence of VaCBF2 showed a high level of homology with other CBF2 family members. Phylogenetic analysis showed that the amino acid sequences may be CBF2 proteins with evolutionary relationship. Quantitative reverse-transcription polymerase chain reaction analysis indicated that the expression of VaCBF2 gene in tissues (roots, stems, leaves, and petioles) was induced by low temperature, high salinity, and application of abscisic acid and salicylic acid in a time-dependent manner but to different extents in the cold-hardy V. amurensis and the less cold-hardy Vitis vinifera. The presence of cis-elements such as MYC and ABRE in VaCBF2 promoter further confirmed that this promoter was a component of the CBF transduction pathway involved in plant response to multiple stresses.

COR-Like Gene Is Involved in Induced-Expression Response to Multiple Abiotic Stresses in Grape Vine (<i>Vitis amurensis</i>) Tissues

Cold stress, which causes dehydration damage to plants, is one of the most common abiotic stresses that limit plant distributions and affect crop growth and development. To improve their cold tolerance, plants often upregulate the expression of some cold-related genes. In this study, a cold-regulated (COR) gene was isolated from Vitis amurensis and designated as VaCOR. RT-PCR analysis demonstrated that VaCOR was expressed at high levels in the roots, stems, leaves, and petioles under low temperature, but it was not detected under normal temperatures. Further analysis revealed that salinity and the application of exogenous abscisic acid and salicylic acid significantly induced VaCOR transcription, with apparent differences in its expression in different organs. The data also showed that COR gene expression was higher in cold-resistant wild V. amurensis than in cold-sensitive Vitis vinifera “Manicure Finger” under low temperature. These results suggest that the VaCOR gene in V. amurensis grapes is involved in multiple stresses and plays a central role in stress-induced and stress-tolerance.

Analysis of Genetic Diversity of Vitis amurensis Rupr.(Vitaceae) Germplasm Resources

[ Objective] This study aimed to explore the genetic diversity of Vitis amurensis Rupr. （Vitaceae） germplasm resources. [ Method ] Out of total 245 pairs of primers, 18 were selected for SSR amplification of 360 V. amurensis experimental materials. [Result] The number of bands amplified by each primer ranged from 4 to 13 with a mean of 9.44. The length of bands ranged from 150 to 1 000 bp, concentrated at 200 -750 bp. The 18 pairs of primers amplified 170 bands totally, of which 167 bands were polymorphic with a polymorphism ratio of 98.2%. The Shannon＇s diversity index （I） is 1. 778 051. With the SSR-PCR am- plification of 360 V. amurensis varieties （ strains）, 5 specific bands were amplified by certain primers in several varieties（strains） accounting for 2.95% of the total bands. [ Conclusion] SSR molecular marker technique was an efficient method to detect the genetic diversity of V. amurensis and thereby is an effective tool for pedigree analysis and variety identification of A. amurensis varieties（strains）.

葡萄(Vitis vinifera L.)试管繁殖中芽增殖能力衰减的原因及其防治方法的研究

以20个葡萄品种试管苗作为实验材料,经四年、四十多代的继代培养反复对比实验发现:在葡萄试管繁殖中,连续恒温(25±1℃)条件下培养或只采用固体培养方法,不适期进行继代培养均会导致芽增殖能力的衰减,根据生物统计分析,当继代培养超过16～17代后,芽增殖能力即随着继代培养次数的增加而逐渐下降,采取变温培养、变换培养方法、适期继代培养或在增殖培养基中附加适量的椰子乳汁等方法均可防治芽增殖能力的哀减,保持高的芽增殖能力。

葡萄(Vitis Vinifera L.)X-射线能谱元素微区污染分析研究

对葡萄果实不同处理的细胞组织多位点进行X -射线能谱微区元素分析。结果 :葡萄不同处理的细胞组织中共发现Mg、Al、Si、P、S、Cl、K、Ca、Fe、Cu十种元素。且不同处理的组织细胞中各元素含量分布有差异 ,未清洗果实表皮处理的Al、Si、S、Ca、Cu含量均显著高于对照 。

Genome and transcriptome analysis of the grapevine(Vitis vinifera L.) WRKY gene family

The plant WRKY gene family represents an ancient and complex class of zinc-finger transcription factors(TFs)that are involved in the regulation of various physiological processes,such as development and senescence,and in plant response to many biotic and abiotic stresses.Despite the growing number of studies on the genomic organisation of WRKY gene family in different species,little information is available about this family in grapevine(Vitis vinifera L.).In the present study,a total number of 59 putative grapevine WRKY transcription factors(VvWRKYs)were identified based on the analysis of various genomic and proteomic grapevine databases.According to their structural and phylogentic features,the identified grapevine WRKY transcription factors were classified into three main groups.In order to shed light into their regulatory roles in growth and development as well as in response to biotic and abiotic stress in grapevine,the VvWRKYs expression profiles were examined in publicly available microarray data.Bioinformatics analysis of these data revealed distinct temporal and spatial expression patterns of VvWRKYs in various tissues,organs and developmental stages,as well as in response to biotic and abiotic stresses.To also extend our analysis to situations not covered by the arrays and to validate our results,the expression profiles of selected VvWRKYs in response to drought stress,Erysiphe necator(powdery mildew)infection,and hormone treatments(salicilic acid and ethylene),were investigated by quantitative real-time reverse transcription PCR(qRT-PCR).The present study provides a foundation for further comparative genomics and functional studies of this important class of transcriptional regulators in grapevine.

The Establishment of an Agrobacterium-Mediated Transformation Platform for the Non-Embryogenic Calli of Vitis vinifera L.

Non-embryogenic calli （NEC） was inevitably and heavily produced when grape embryogenic calli （EC） was induced from explants or during the subculture of EC.A stable and highly efficient NEC transformation platform is required to further sort out and verify key genes which determine/switch the identity of NEC and EC.In this research,a vector pA5 containing a chitinase signal sequence fused to gfp （green fluorescent protein） and an HDEL motive was used to target and immobilize into Agrobacterium strain EHA105 to establish a transformation platform for Vitis vinifera L.cv.Chardonnay NEC.It was determined that NEC 10 d after subculture was the best target tissue;30 min for inoculation followed by 3 d co-cultivation with the addition of 200 μmol L-1 acetosyringone （AS） was optimized as protocol.The use of bacterial densities as 1.0 at OD600 did not result in serious tissue hypersensitive reaction and it had higher efficiency.Kanamycin at 200 mg L-1 was picked for positive expression selection.The stable transformation of NEC was proved by reverse transcription-polymerase chain reaction techniques （RT-PCR） and fluorescent microscopy after three sub-cultures of the selected cell line.Highly efficient genetic transformation protocol of grape NEC was achieved and some of the optimized parameters were different from that reported for EC.This transformation platform could facilitate the verification of candidate somatic embryogenesis （SE） decisive genes,and the successfully transformed NEC with certain genes can also be used as bioreactors for the production of functional products,as NEC not only proliferates fast,but also keeps in a rather stable condition.

Anthocyanin profi les and color properties of red wines made from Vitis davidii and Vitis vinifera grapes

Spine grape(Vitis davidii Foex.)is an important wild grape species native to China.Fifteen red spine grape clones and three red Vitis vinifera grape varieties were used to evaluate the differences in the anthocyanin profiles and color properties of wines made from V.davidii and V.vinifera grapes.Among spine wines,‘Junzi#2’wine had the highest total phenolic and total anthocyanin,‘Xiangzhenzhu’wine had the highest total flavonoids,and‘Junzi#1’wine had the highest total tannin.The anthocyanin compositions of all of the spine wines were dominated by Mv-3,5-diglucoside.The total individual anthocyanin contents in spine wines,except‘Gaoshan#5’,‘Junzi#5055’,‘Junzi#5061’,and‘Junzi#5044’,were signifi cantly higher than in V.vinifera wines.Most of the spine wines had a stronger red intensity and a brighter chroma with a bluer hue than V.vinifera wines.Correlation analysis revealed that the color properties were closely related to the anthocyanin composition.These results suggest that wines made from spine grapes may be useful for the wine industry for their color properties and high individual anthocyanin contents.

The potential role of grape (Vitis vinifera L.) in prevention of threatened abortion via immunomodulatory and anti-inflammatory abilities: a hypothesis

Threatened abortion is a common problem in early pregnancy. This early vaginal bleeding happens in about 25% of pregnant women. The medications including progesterone, uterine muscle relaxant or human chorionic gonadotropin have essential effects in developing pregnancy, but the clinical data are insufficient to prescribe them. In recent decades, medicinal herbs can help us to present new treatments. Grape (Vitis vinifera L.) can protect the fetus from the perspective of Persian medicine. So, we hypothesize about the salutary effects of grape in miscarriage prevention. We found five standard expected mechanisms of grape to prevent threatened abortion: immunomodulatory and anti-inflammatory, anti-oxidant, anti-contraction, hormonal and anti-stress activities. Grape reduces nitric oxide, prostaglandin E2, expression of nuclear factor κB and other pro-inflammatory cytokines like IL (Interleukin)-1β, IL-6, IL-8, and tumor necrosis factor-α. It also elevates anti-inflammatory mediators and expression of peroxisome proliferator-activated receptor-γ. Grape polyphenols have a crucial role in fetus protection with high antioxidant power and other functions such as prevention of stress-triggered abortion with proanthocyanidins, or hormonal effects and inhibition of uterine contractions with resveratrol. So according to these studies, grape probably has effects on the immune and endocrine factors involved in threatened miscarriage.

Somatic embryogenesis and histological analysis from zygotic embryos in Vitis vinifera L.‘Moldova’

We examined the somatic embryogenesis from and histological studies of zygotic embryos of seeds in European Grape ＇Moldova＇ （Vitis vinifera U ＇Moldova＇）. Primary calli were initiated on Nitsch and Nitsch （NN） medium supplemented with 1.0 mg·L^-1 2,4-D and 0.5 mg·L^-1 6-BA. Embryogenic calli were produced upon transfer to a NN medium with 0.5 mg·L^-1 6-BA and 2 mg·L^-1 NAA and somatic embryos were obtained on a half strength MS medium without plant growth regulators. During the somatic embryo germination, an addition of 1.0 mg·L^-1 6-BA in the medium could accelerate somatic embryos to develop into normal plants and increase the conversion rate from 0 to 43.3%. Histological studies of embryogenic calli and somatic embryos demonstrated dynamic changes of proteins and starch grains. The developmental processes of somatic embryos were similar to those of zygotic embryos, including typical epiderma, cotyledon primordium and vascular tissue.

Identification and Characterization of MicroRNAs and Their Targets in Grapevine(Vitis vinifera)

MicroRNAs （miRNAs） are a class of newly identified, small, non-coding RNAs that play vital roles in regulation. Based on miRNAs unique features of expression pattern, evolutionary conservation, secondary structure and genetic requirements for biogenesis, computational predication strategy is adopted to predicate the novel miRNAs. In this research, potential miRNAs and their targets in grapevine （Vitis vinifera） were predicted. We used previously known plant miRNAs against grapevine genome sequence databases to search for potential miRNAs. A total of 81 potential miRNAs were detected following a range of strict filtering criteria. Using these potential miRNA sequences, we could further blast the mRNA database to find the potential targets in this species. Comparative analysis of miRNAs in grapevine and other species reveals that miRNAs exhibit an evolutional conservation, the number and function of miRNAs must have significantly expanded during the evolution of land plants. Furthermore divergence made versatile functions of miRNAs feasible. Cluster of miRNAs likely represents an ancient expression mechanism. Predicted target genes include not only transcription factors but also genes implicated in floral development, signal transduction, diseases and stress response. Till now, little is known about experimental or computational identification of miRNA in grapevine species. Increased knowledge of the biological mechanisms of the grapevine will allow targeted approaches to increase the quality of fruit and reduce the impact of parasites together with stress, which could enable a sustainable, environmentally-sound, farming policv.