Jiang Lu

Whole genome wide expression profiles of Vitis amurensis grape responding to downy mildew by using Solexa sequencing technology

BackgroundDowny mildew (DM), caused by pathogen Plasmopara viticola (PV) is the single most damaging disease of grapes (Vitis L.) worldwide. However, the mechanisms of the disease development in grapes are poorly understood. A method for estimating gene expression levels using Solexa sequencing of Type I restriction-endonuclease-generated cDNA fragments was used for deep sequencing the transcriptomes resulting from PV infected leaves of Vitis amurensis Rupr. cv. Zuoshan-1. Our goal is to identify genes that are involved in resistance to grape DM disease.ResultsApproximately 8.5 million (M) 21-nt cDNA tags were sequenced in the cDNA library derived from PV pathogen-infected leaves, and about 7.5 M were sequenced from the cDNA library constructed from the control leaves. When annotated, a total of 15,249 putative genes were identified from the Solexa sequencing tags for the infection (INF) library and 14,549 for the control (CON) library. Comparative analysis between these two cDNA libraries showed about 0.9% of the unique tags increased by at least five-fold, and about 0.6% of the unique tags decreased more than five-fold in infected leaves, while 98.5% of the unique tags showed less than five-fold difference between the two samples. The expression levels of 12 differentially expressed genes were confirmed by Real-time RT-PCR and the trends observed agreed well with the Solexa expression profiles, although the degree of change was lower in amplitude. After pathway enrichment analysis, a set of significantly enriched pathways were identified for the differentially expressed genes (DEGs), which associated with ribosome structure, photosynthesis, amino acid and sugar metabolism.ConclusionsThis study presented a series of candidate genes and pathways that may contribute to DM resistance in grapes, and illustrated that the Solexa-based tag-sequencing approach was a powerful tool for gene expression comparison between control and treated samples.

Influence of growing season on phenolic compounds and antioxidant properties of grape berries from vines grown in subtropical climate.

The influence of growing season (winter vs summer) on the synthesis and accumulation of phenolic compounds and antioxidant properties was studied in five grape cultivars for three consecutive years. Four phenolic compound parameters (total phenols, flavonoids, flavan-3-ols, and anthocyanins) and three antioxidant property parameters [2,2-diphenyl-1-picrylhydrazyl radical scavenging, 2,2-azinobis(3-ethylbenzothiazolinesulfonic acid) radical scavenging, and ferric reducing antioxidant power] were investigated. Results showed that both phenolic compounds and antioxidant properties in the seed and skin of winter berries were significantly (p < 0.05) higher than those of summer berries for all of the cultivars investigated. The anthocyanin profiles of berry skins appeared to be extremely consistent in different years for the same crop, whereas they varied greatly between the two crops within the same year (winter vs summer). Winter berries contained richer glucosides of delphinidin, cyanidin, peonidin, and malvidin than summer berries. These seasonal variations of phenolic compounds and antioxidant properties on grape berries were largely contributed by climatic factors such as temperature, solar radiation, rainfall, and hydrothermic coefficient between different growing seasons.

Antioxidant, Antibacterial, and Antibiofilm Properties of Polyphenols from Muscadine Grape (Vitis rotundifolia Michx.) Pomace against Selected Foodborne Pathogens

Polyphenols are predominantly secondary metabolites in muscadine grapes, playing an important role in the species strong resistance to pests and diseases. This study examined the above property by evaluating the antioxidant, antibacterial, and antibiofilm activities of muscadine polyphenols against selected foodborne pathogens. Results showed that antioxidant activity for different polyphenols varied greatly, ranging from 5 to 11.1 mmol Trolox/g. Antioxidant and antibacterial activities for polyphenols showed a positive correlation. Muscadine polyphenols exhibited a broad spectrum of antibacterial activity against tested foodborne pathogens, especially Staphylococcus aureus (MIC = 67-152 mg/L). Muscadine polyphenols at 4 × MIC caused nearly a 5 log10 CFU/mL drop in cell viability for S. aureus in 6 h with lysis, whereas at 0.5 × MIC they inhibited its biofilm formation and at 16 × MIC they eradicated biofilms. Muscadine polyphenols showed synergy with antibiotics and maximally caused a 6.2 log10 CFU/mL drop in cell viability at subinhibitory concentration.

Comparative transcriptome analysis reveals defense-related genes and pathways against downy mildew in Vitis amurensis grapevine

Downy mildew (DM), caused by oomycete Plasmopara viticola (Pv), can lead to severe damage to Vitis vinifera grapevines. Vitis amurensis has generally been regarded as a DM resistant species. However, when V.xa0amurensis Shuanghong were inoculated with Pv strains ZJ-1-1 and JL-7-2, the former led to obvious DM symptoms (compatible), while the latter did not develop any DM symptoms but exhibited necrosis (incompatible). In order to underlie molecular mechanism in DM resistance, mRNA-seq based expression profiling of Shuanghong was compared at 12, 24, 48 and 72xa0h post inoculation (hpi) with these two strains. Specific genes and their corresponding pathways responsible for incompatible interaction were extracted by comparing with compatible interaction. In the incompatible interaction, 37 resistance (R) genes were more expressed at the early stage of infection (12 hpi). Similarly, genes involved in defense signaling, including MAPK. ROS/NO, SA, JA, ET and ABA pathways, and genes associated with defense-related metabolites synthesis, such as pathogenesis-related genes and phenylpropanoids/stilbenoids/flavonoids biosynthesizing genes, were also activated mainly during the early stages of infection. On the other hand, Ca(2+) signaling and primary metabolism, such as photosynthesis and fatty acid synthesis, were more repressed after JL-7-2 challenge. Further quantification of some key defense-related factors, including phytohormones, phytoalexins and ROS, generally showed much more accumulation during the incompatible interaction, indicating their important roles in DM defense. In addition, a total of 43 and 52 RxLR effectors were detected during JL-7-2 and ZJ-1-1 infection processes, respectively.

Enzyme release of phenolics from muscadine grape (Vitis rotundifolia Michx.) skins and seeds.

Enzyme degradation of plant cell wall polysaccharides can potentially enhance the release of bioactive phenolics. The aim of this study was to evaluate various combinations of solvent and enzyme, enzyme type (cellulase, pectinase, ß-glucosidase), and hydrolysis time (1, 4, 8, 24 h) on the release of muscadine grape skin and seed phenolics, and their antioxidant activities. Results showed that pre-treated muscadine skins and seeds with enzymes decreased total phenolic yield compared with solvent (50% ethanol) alone. Enzyme release of phenolics from skins of different muscadine varieties was significantly different while release from seeds was similar. Enzyme hydrolysis was found to shorten extraction time. Most importantly, enzyme hydrolysis modified the galloylated form of polyphenols to low molecular weight phenolics, releasing phenolic acids (especially gallic acid), and enhancing antioxidant activity.

Functional characterization of a LAHC sucrose transporter isolated from grape berries in yeast

Large amounts of sugar are imported into grape berries from source leaves during ripening, and sucrose transporters play a key role during this process. In this study, a putative grape sucrose transporter gene VvSUC27, primarily expressed in sink tissue, was transformed into a yeast strain to characterize its function as a sucrose transporter. Sucrose was taken up by yeast transformed with VvSUC27 at an optimum pH of 4.0–5.0 and a Km of 8.0–10.5xa0mM, indicating VvSUC27 is a LAHC (low-affinity/high-capacity) sucrose transporter. The ability of sucrose uptake in transformed yeast was activated by monosaccharides and inhibited by maltose and DEPC.

Isolation of a WRKY30 gene from Muscadinia rotundifolia (Michx) and validation of its function under biotic and abiotic stresses

WRKY transcription factors (TFs) play important roles in many plant processes, including responses to biotic and abiotic stresses. In the present study, Muscadinia rotundifolia MrWRKY30 dramatically accumulated in grapevine leaves in response to inoculation of Plasmopara viticola, a pathogen causing grapevine downy mildew disease. Similar responses were also found on grapevines treated with exogenous SA/JA/ET. Ectopic expression of MrWRKY30 in Arabidopsis thaliana “COL0” enhanced its resistance to downy mildew pathogen Peronospora parasitica. Pathogenesis-related (PR) genes, including AtPR1, AtPR4, AtPR5, and AtPDF1.2, were significantly upregulated in transgenic A. thaliana after P. parasitica inoculation. In the mean time, two critical genes in SA and JA signaling pathways, AtEDS5 and AtJAR1, were abundantly expressed as well, indicating that MrWRKY30 may enhance disease resistance of A. thaliana through SA and JA defense system. The transgenic A. thaliana plants also enhanced tolerance to cold stress accompanied with upregulation of AtCBF1, AtCBF3, AtICE1, and AtCOR47. MrWRKY30 might protect A. thaliana from cold damage by activating the AtCBF-mediated signaling pathway to induce the downstream AtCOR47 gene. Interestingly, the transgenic seedlings had a negative effect on salt tolerance. Reverse transcription PCR (RT-PCR) analysis revealed that antioxidant enzyme genes AtAPX (ascorbate peroxidase), AtCAT (catalase), and AtGST (glutathione-S-transferase) were suppressed in transgenic plants, which may lead to reactive oxygen species (ROS)-mediated sensitivity to salt stress.

Linkage of cold acclimation and disease resistance through plant–pathogen interaction pathway in Vitis amurensis grapevine

Low temperatures cause severe damage to none cold hardy grapevines. A preliminary survey with Solexa sequencing technology was used to analyze gene expression profiles of cold hardy Vitis amurensis ‘Zuoshan-1’ after cold acclimation at 4xa0°C for 48xa0h. A total of 16,750 and 18,068 putative genes were annotated for 4xa0°C-treated and control library, respectively. Among them, 393 genes were upregulated for at least 20-fold, while 69 genes were downregulated for at least 20-fold under the 4xa0°C treatment for 48xa0h. A subset of 101 genes from this survey was investigated further using reverse transcription polymerase chain reaction (RT-PCR). Genes associated with signaling events in pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI), including generation of calcium signals (CNGC, CMLs), jasmonic acid signal (JAZ1), oxidative burst (Rboh), and phosphorylation (FLS2, BAK, MEKK1, MKKs) cascades, were upregulated after cold acclimation. Disease resistance genes (RPM1, RPS5, RIN4, PBS1) in the process of effector-triggered immunity (ETI) were also upregulated in the current condition. Defense-related genes (WRKYs, PR1, MIN7) involved in both PTI and ETI processes were abundantly expressed after cold acclimation. Our results indicated that plant–pathogen interaction pathways were linked to the cold acclimation in V. amurensis grapevine. Other biotic- and abiotic-related genes, such as defense (protein phosphatase 2C, U-box domain proteins, NCED1, stilbene synthase), transcription (DREBs, MYBs, ERFs, ZFPs), signal transduction (kinase, calcium, and auxin signaling), transport (ATP-binding cassette (ABC) transporters, auxin:hydrogen symporter), and various metabolism, were also abundantly expressed in the cold acclimation of V. Amurensis ‘Zuoshan-1’ grapevine. This study revealed a series of critical genes and pathways to delineate important biological processes affected by low temperature in ‘Zuoshan-1’.

Response of phytohormones and correlation of SAR signal pathway genes to the different resistance levels of grapevine against Plasmopara viticola infection

Phytohormones play an important role in the process of disease resistance in plants. Here, we investigated which among salicylic acid, jasmonic acid, and abscisic acid performs a key role in plant defense after Plasmopara viticola infection in grapevine. We used grapevines possessing different resistance levels against P.xa0viticola infection to study the relationship between the expression of key genes in the related resistance signaling pathways and the level of resistance. We performed high-performance liquid chromatography-mass spectrometry to estimate the phytohormone contents in grape leaves at different time points after the infection. Furthermore, we performed quantitative analyses of key genes such as EDS1, PAD4, ICS2, PAL, NPR1, TGA1, and PR1 in the systemic acquired resistance pathway by quantitative reverse transcription-polymerase chain reaction. The results showed an increased variation in the SA content, which was maintained at high levels, after P.xa0viticola infection in plant species exhibiting stronger resistance to the pathogen; this finding highlights the importance of SA in plant defense mechanisms. Moreover, EDS1 and PAD4 expression did not show a positive correlation with disease resistance in grape; however, higher expression of other genes that were analyzed was observed in highly resistant grape varieties. Our results provide insights into the role of phytohormone regulation in the induction and maintenance of plant defense response to pathogens.

Fruit quality, nutraceutical and antimicrobial properties of 58 muscadine grape varieties (Vitis rotundifolia Michx.) grown in United States.

Fifty-eight muscadine grape varieties were evaluated for their fruit quality, nutraceutical, and antimicrobial properties during two growing seasons (2012 vs. 2013). Fruit quality was significantly different among muscadine grape varieties, with weight ranged from 2.93 to 22.32g, pH from 3.01 to 3.84, titratable acidity from 0.27% to 0.83%, and °Brix from 10.92 to 23.91. Total phenols for different muscadine juices varied from 0.26 to 1.28mgGA/mL, skins from 10.13 to 30.02mgGA/g DM, and seeds from 22.47 to 72.01mgGA/g DM. Accordingly, the antioxidant activity of grape juices varied from 0.97 to 6.78mmolTrolox/mL, skins from 83.59 to 221.20μmolTrolox/g DM, and seeds from 178.22 to 619.73μmolTrolox/g DM. Study demonstratedgrape seed polyphenols (MIC 54.8-60.1μg/ml) showed stronger antimicrobial activity against S. aureus than skin polyphenols (MIC 70.7-80.2μg/ml). This information could be a valuable asset in the research and extension of muscadine grapes.