Zhenchang Liang

Genome-wide identification of WRKY family genes and their response to cold stress in Vitis vinifera

BackgroundWRKY transcription factors are one of the largest families of transcriptional regulators in plants. WRKY genes are not only found to play significant roles in biotic and abiotic stress response, but also regulate growth and development. Grapevine (Vitis vinifera) production is largely limited by stressful climate conditions such as cold stress and the role of WRKY genes in the survival of grapevine under these conditions remains unknown.ResultsWe identified a total of 59 VvWRKYs from the V. vinifera genome, belonging to four subgroups according to conserved WRKY domains and zinc-finger structure. The majority of VvWRKYs were expressed in more than one tissue among the 7 tissues examined which included young leaves, mature leaves, tendril, stem apex, root, young fruits and ripe fruits. Publicly available microarray data suggested that a subset of VvWRKYs was activated in response to diverse stresses. Quantitative real-time PCR (qRT-PCR) results demonstrated that the expression levels of 36 VvWRKYs are changed following cold exposure. Comparative analysis was performed on data from publicly available microarray experiments, previous global transcriptome analysis studies, and qRT-PCR. We identified 15 VvWRKYs in at least two of these databases which may relate to cold stress. Among them, the transcription of three genes can be induced by exogenous ABA application, suggesting that they can be involved in an ABA-dependent signaling pathway in response to cold stress.ConclusionsWe identified 59 VvWRKYs from the V. vinifera genome and 15 of them showed cold stress-induced expression patterns. These genes represented candidate genes for future functional analysis of VvWRKYs involved in the low temperature-related signal pathways in grape.

Polyphenolic profiles detected in the ripe berries of Vitis vinifera germplasm

Polyphenolic profiles in the berry samples of 344 European grape (Vitis vinifera) cultivars were evaluated for two consecutive years. These cultivars represent a diverse collection of V. vinifera germplasm maintained at the USDA-Agricultural Research Service Vitis Clonal Repository in Davis of California, USA. A total of 36 polyphenolic compounds, including 16 anthocyanins, 6 flavonols, 6 flavanols, 6 hydroxycinnamic acids and 2 hydroxybenzoic acids, were identified via HPLC-MS and quantified by HPLC-DAD. The mean contents for anthocyanins, flavanols, flavonols, hydroxycinnamic acids and hydroxybenzoic acids were 0.946 (coloured cultivars), 0.147, 0.043, 0.195 and 0.016mgg(-1) FW, respectively. On average, wine grapes had higher concentrations than had table grapes for all of these compounds except hydroxycinnamic acids. Berry colours affected the total contents of anthocyanins, but not others. Positive correlations (0.151-0.535) were found among these groups of compounds. As expected, these groups of compounds were all negatively correlated with berry weight.

CRISPR/Cas9-mediated efficient targeted mutagenesis in Chardonnay ( Vitis vinifera L.)

The type II clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 system (CRISPR/Cas9) has been successfully applied to edit target genes in multiple plant species. However, it remains unknown whether this system can be used for genome editing in grape. In this study, we described genome editing and targeted gene mutation in ‘Chardonnay’ suspension cells and plants via the CRISPR/Cas9 system. Two single guide RNAs (sgRNAs) were designed to target distinct sites of the L-idonate dehydrogenase gene (IdnDH). CEL I endonuclease assay and sequencing results revealed the expected indel mutations at the target site, and a mutation frequency of 100% was observed in the transgenic cell mass (CM) as well as corresponding regenerated plants with expression of sgRNA1/Cas9. The majority of the detected mutations in transgenic CM were 1-bp insertions, followed by 1- to 3-nucleotide deletions. Off-target activities were also evaluated by sequencing the potential off-target sites, and no obvious off-target events were detected. Our results demonstrated that the CRISPR/Cas9 system is an efficient and specific tool for precise genome editing in grape.

CIELAB Coordinates in Response to Berry Skin Anthocyanins and Their Composition in Vitis

Berry skin color OIV index, anthocyanin composition, and content of 78 grape cultivars were surveyed using a CIELAB system and high-performance liquid chromatography (HPLC)-mass spectrometry (MS) coupled with photodiode array detection. There were high correlations between L*, b*, and color, while a* was not a representative parameter. L* and b* values declined as berry skin color OIV became darker, and a* increased as berry skin color OIV became darker in pink and red grape cultivars only. The composition and content of anthocyanins varied widely among the cultivars. Total anthocyanins and types of anthocyanins were significantly correlated with color OIV parameters. Through multiple linear regression analysis, cyanidin derivatives had a positive effect on values of L* and b*. Delphinidin derivatives had positive effects on the value of a*. The CIELAB system gave good results for differentiation of grape berry skin color OIV.

The effects of UV-B radiation on photosynthesis in relation to Photosystem II photochemistry, thermal dissipation and antioxidant defenses in winter wheat (Triticum aestivum L.) seedlings at different growth temperatures

To study the UV-B effect on photosynthesis in winter wheat at different day/night temperatures, biologically effective UV-B radiation at 4.2 (LUVB) and 10.3 (HUVB) kJ m-2 d-1 was provided on the seedlings at 25/20°C or 10/5°C. UV-B radiation inhibited net photosynthesis rate (Pn) by enhanced intensity and decreased temperature without change of intercellular CO2 concentrations (Ci). Decreased maximal quantum yield of Photosystem II (Fv/Fm) and increased minimum fluorescence (Fo) were observed in HUVB at both temperatures and LUVB at 10/5°C. HUVB increased total pool size (VAZ) of xanthophyll cycle pigments, but decreased the de-epoxidation state (DEPS) of these pigments at both temperatures, while LUVB only decreased DEPS at 10/5°C. The activities of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR) and the redox states of ascorbate and glutathione (AsA/DAsA and GSH/GSSG) were enhanced at 25/20°C, while there were increased SOD and CAT, unaltered APX activities and AsA/DHA, as well as decreased GR activity and GSH/GSSG in LUVB and HUVB at 10/5°C. UV-B radiation resulted in higher H2O2 and thiobarbituric acid reactive substance (TBARS) concentrations at 10/5°C than 25/20°C. It appears that low temperature alone did not influence photosynthesis but aggravated UV-B induced photoinhibition, which was associated with PSII photochemistry rather than stomatal limitation. Xanthophyll cycle pigments failed to provide photoprotection through thermal dissipation. The antioxidant system was up-regulated in LUVB and HUVB at 25/20°C, but was impaired at 10/5°C. Low temperature intensified UV-B induced photoinhibition and damage by weakening the antioxidant system.

Inheritance of anthocyanins in berries of Vitis vinifera grapes

Inheritance patterns for table grape anthocyanins were investigated on three cross offspring populations during two successive years. Sixteen anthocyanins were detected, and all were monoglucoside derivatives. The proportion of anthocyanins in the maternal parent determined the proportion of anthocyanins in the offspring. But the absolute content of the maternal parent had no significant effect on progenies. Peonidin 3-O-glucoside and malvidin 3-O-glucoside were the most abundant anthocyanins, not only in the maternal parent but also in the progenies. The presence or absence of anthocyanins in grape skin was inheritance of a quality character controlled by oligogenes, and anthocyanins content was a quantitative character controlled by polygenes. Via principal component (PC) analysis, factors that affected the total content of cross progeny populations were peonidin 3-O-glucoside, malvidin 3-O-glucoside, delphinidin 3-O-glucoside, cyanidin 3-O-glucoside, petunidin 3-O-glucoside, peonidin 3-O-(6-O-coumaryl)-glucoside, and malvidin 3-O-(6-O-coumaryl)-glucoside. Anthocyanins content was a high broad sense heritability character (H2), and H2 was stable in different cross combinations (ranging from 0.65 to 0.98).

Changes of Polyphenols, Sugars, and Organic Acid in 5 Vitis Genotypes during Berry Ripening

UNLABELLED The concentrations of sugars, organic acids, and polyphenols during berry ripening in 5 grape genotypes were analyzed using HPLC and HPLC-MS to determine which were correlated. Veraison is critical stage during grape berry development, and marks the beginning of ripening. Glucose and fructose accumulated sharply in a 1: 1ratio, though the concentration of fructose was slightly higher than that of glucose at maturation. Malic acid and tartaric acid were the dominant organic acids, and they decreased rapidly during berry ripening. The 5 cultivars contained 28 anthocyanins and 8 other polyphenols. All anthocyanins accumulated and were positively correlated with the sugars and negatively correlated with the organic acids. Hydroxycinnamic acids declined and were positively correlated with anthocyanin contents. Changes in flavanols and flavonols different among the 5 genotypes, with flavonols positively correlated with anthocyanin content, but the flavanols procyanidin B1and epicatechin negatively correlated with anthocyanins content. There were obvious differences in patterns of change of polyphenols among 5 grape genotypes. PRACTICAL APPLICATION The results could be used for improving grape berry quality during ripening and developing a comprehensive database of primary and secondary metabolites in the Vitis for grape breeding.

Identification of cold-inducible microRNAs in grapevine

Low temperature is one of the most important environmental factors that limits the geographical distribution and productivity of grapevine. However, the molecular mechanisms on how grapevine responds to cold stress remains to be elucidated. MicroRNAs (miRNAs) are a class of endogenous small non-coding RNAs that play an essential role during plant development and stress responses. Although miRNAs and their targets have been identified in several Vitis species, their participation during cold accumulation in grapevine remains unknown. In this study, two small RNA libraries were generated from micropropagated ‘Muscat Hamburg’ (V. vinifera) plantlets under normal and low temperatures (4°C). A total of 163 known miRNAs and 67 putative novel miRNAs were detected from two small RNA libraries by Solexa sequencing. Forty-four cold-inducible miRNAs were identified through differentially expressed miRNAs (DEMs) analysis; among which, 13 belonged to upregulated DEMs while 31 belonged downregulated DEMs. The expression patterns of the 13 DEMs were verified by real-time RT-PCR analysis. The prediction of the target genes for DEMs indicated that miRNA may regulate transcription factors, including AP2, SBP, MYB, bHLH, GRAS, and bZIP under cold stress. The 5′-RLM RACE were conducted to verify the cleavage site of predicted targets. Seven predicted target genes for four known and three novel vvi-miRNAs showed specific cleavage sites corresponding to their miRNA complementary sequences. The expression pattern of these seven target genes revealed negative correlation with the expression level of the corresponding vvi-miRNAs. Our results indicated that a diverse set of miRNAs in V. vinifera are cold-inducible and may play an important role in cold stress response.

Construction of a high-density genetic map and QTLs mapping for sugars and acids in grape berries

BackgroundQTLs controlling individual sugars and acids (fructose, glucose, malic acid and tartaric acid) in grape berries have not yet been identified. The present study aimed to construct a high-density, high-quality genetic map of a winemaking grape cross with a complex parentage (V. vinifera × V. amurensis) × ((V. labrusca × V. riparia) × V. vinifera), using next-generation restriction site-associated DNA sequencing, and then to identify loci related to phenotypic variability over three years.ResultsIn total, 1 826 SNP-based markers were developed. Of these, 621 markers were assembled into 19 linkage groups (LGs) for the maternal map, 696 for the paternal map, and 1 254 for the integrated map. Markers showed good linear agreement on most chromosomes between our genetic maps and the previously published V. vinifera reference sequence. However marker order was different in some chromosome regions, indicating both conservation and variation within the genome. Despite the identification of a range of QTLs controlling the traits of interest, these QTLs explained a relatively small percentage of the observed phenotypic variance. Although they exhibited a large degree of instability from year to year, QTLs were identified for all traits but tartaric acid and titratable acidity in the three years of the study; however only the QTLs for malic acid and β ratio (tartaric acid-to-malic acid ratio) were stable in two years. QTLs related to sugars were located within ten LGs (01, 02, 03, 04, 07, 09, 11, 14, 17, 18), and those related to acids within three LGs (06, 13, 18). Overlapping QTLs in LG14 were observed for fructose, glucose and total sugar. Malic acid, total acid and β ratio each had several QTLs in LG18, and malic acid also had a QTL in LG06. A set of 10 genes underlying these QTLs may be involved in determining the malic acid content of berries.ConclusionThe genetic map constructed in this study is potentially a high-density, high-quality map, which could be used for QTL detection, genome comparison, and sequence assembly. It may also serve to broaden our understanding of the grape genome.

Antioxidant and Antiproliferative Activities of Twenty-Four Vitis vinifera Grapes

Grapes are rich in phytochemicals with many proven health benefits. Phenolic profiles, antioxidant and antiproliferative activities of twenty-four selected Vitis vinifera grape cultivars were investigated in this study. Large ranges of variation were found in these cultivars for the contents of total phenolics (95.3 to 686.5 mg/100 g) and flavonoids (94.7 to 1055 mg/100 g) and antioxidant activities (oxygen radical absorbance capacity 378.7 to 3386.0 mg of Trolox equivalents/100 g and peroxylradical scavenging capacity14.2 to 557 mg of vitamin C equivalents/100 g), cellular antioxidant activities (3.9 to 139.9 µmol of quercetin equivalents/100 g without PBS wash and 1.4 to 95.8 µmol of quercetin equivalents /100 g with PBS wash) and antiproliferative activities (25 to 82% at the concentrations of 100 mg/mL extracts).The total antioxidant activities were significantly correlated with the total phenolics and flavonoids. However, no significant correlations were found between antiproliferative activities and total phenolics or total flavonoids content. Wine grapes and color grapes showed much higher levels of phytochemicals and antioxidant activities than table grapes and green/yellow grapes. Several germplasm accessions with much high contents of phenolics and flavonoids, and total antioxidant activity were identified. These germplasm can be valuable sources of genes for breeding grape cultivars with better nutritional qualities of wine and table grapes in the future.