Unravelling molecular mechanisms involved in resistance priming against downy mildew (Plasmopara viticola) in grapevine (Vitis vinifera L.).

Downy mildew (DM; Plasmopara viticola) is amongst the most severe fungal diseases in viticulture and the reason for the majority of fungicide applications. To reduce synthetic and copper-based fungicides, there is an urgent need for natural alternatives, which are being increasingly tested by the industry and the research community. However, their mode of action remains unclear. Therefore, our study aimed to investigate the transcriptomic changes induced by oregano essential oil vapour (OEOV) in DM-infected grapevines. OEOV was applied at different time points before and after DM infection to differentiate between a priming effect and a direct effect. Both pre-DM treatment with OEOV and post-infection treatment resulted in a significant reduction in DM sporulation. RNA-seq, followed by differential gene expression and weighted gene co-expression network analysis, identified co-expressed gene modules associated with secondary metabolism, pathogen recognition and response. Surprisingly, the molecular mechanisms underlying the efficiency of OEOV against DM appear to be independent of stilbene synthesis, and instead involve genes from a putative signalling pathway that has yet to be characterized. This study enhances our understanding of the molecular regulation of innate plant immunity and provides new insights into the mode of action of alternative natural antifungal agents.

Nuances of Responses to Two Sources of Grapevine Leafroll Disease on Pinot Noir Grown in the Field for 17 Years.

Grapevine leafroll disease (GLD) is one of the most economically damaging virus diseases in grapevine, with grapevine leafroll-associated virus 1 (GLRaV-1) and grapevine leafroll-associated virus 3 (GLRaV-3) as the main contributors. This study complements a previously published transcriptomic analysis and compared the impact of two different forms of GLD to a symptomless control treatment: a mildly symptomatic form infected with GLRaV-1 and a severe form with exceptionally early leafroll symptoms (up to six weeks before veraison) infected with GLRaV-1 and GLRaV-3. Vine physiology and fruit composition in 17-year-old Pinot noir vines were measured and a gradient of vigor, yield, and berry quality (sugar content and berry weight) was observed between treatments. Virome composition, confirmed by individual RT-PCR, was compared with biological indexing. Three divergent viromes were recovered, containing between four to seven viruses and two viroids. They included the first detection of grapevine asteroid mosaic-associated virus in Switzerland. This virus did not cause obvious symptoms on the indicators used in biological indexing. Moreover, the presence of grapevine virus B (GVB) did not cause the expected corky bark symptoms on the indicators, thus underlining the important limitations of the biological indexing. Transmission of GLRaV-3 alone or in combination with GVB by Planococcus comstocki mealybug did not reproduce the strong symptoms observed on the donor plant infected with a severe form of GLD. This result raises questions about the contribution of each virus to the symptomatology of the plant.

Modifications of Grapevine Berry Composition Induced by Main Viral and Fungal Pathogens in a Climate Change Scenario.

The grapevine is subject to high number of fungal and viral diseases, which are responsible for important economic losses in the global wine sector every year. These pathogens deteriorate grapevine berry quality either directly via the modulation of fruit metabolic pathways and the production of endogenous compounds associated with bad taste and/or flavor, or indirectly via their impact on vine physiology. The most common and devastating fungal diseases in viticulture are gray mold, downy mildew (DM), and powdery mildew (PM), caused, respectively by Botrytis cinerea, Plasmopara viticola, and Erysiphe necator. Whereas B. cinerea mainly infects and deteriorates the ripening fruit directly, deteriorations by DM and PM are mostly indirect via a reduction of photosynthetic leaf area. Nevertheless, mildews can also infect berries at certain developmental stages and directly alter fruit quality via the biosynthesis of unpleasant flavor compounds that impair ultimate wine quality. The grapevine is furthermore host of a wide range of viruses that reduce vine longevity, productivity and berry quality in different ways. The most widespread virus-related diseases, that are known nowadays, are Grapevine Leafroll Disease (GLRD), Grapevine Fanleaf Disease (GFLD), and the more recently characterized grapevine red blotch disease (GRBD). Future climatic conditions are creating a more favorable environment for the proliferation of most virus-insect vectors, so the spread of virus-related diseases is expected to increase in most wine-growing regions. However, the impact of climate change on the evolution of fungal disease pressure will be variable and depending on region and pathogen, with mildews remaining certainly the major phytosanitary threat in most regions because their development rate is to a large extent temperature-driven. This paper aims to provide a review of published literature on most important grapevine fungal and viral pathogens and their impact on grape berry physiology and quality. Our overview of the published literature highlights gaps in our understanding of plant-pathogen interactions, which are valuable for conceiving future research programs dealing with the different pathogens and their impacts on grapevine berry quality and metabolism.

Sucrose Metabolism and Transport in Grapevines, with Emphasis on Berries and Leaves, and Insights Gained from a Cross-Species Comparison.

In grapevines, as in other plants, sucrose and its constituents glucose and fructose are fundamentally important and carry out a multitude of roles. The aims of this review are three-fold. First, to provide a summary of the metabolism and transport of sucrose in grapevines, together with new insights and interpretations. Second, to stress the importance of considering the compartmentation of metabolism. Third, to outline the key role of acid invertase in osmoregulation associated with sucrose metabolism and transport in plants.

Grape Berry Secondary Metabolites and Their Modulation by Abiotic Factors in a Climate Change Scenario-A Review.

Temperature, water, solar radiation, and atmospheric CO2 concentration are the main abiotic factors that are changing in the course of global warming. These abiotic factors govern the synthesis and degradation of primary (sugars, amino acids, organic acids, etc.) and secondary (phenolic and volatile flavor compounds and their precursors) metabolites directly, via the regulation of their biosynthetic pathways, or indirectly, via their effects on vine physiology and phenology. Several hundred secondary metabolites have been identified in the grape berry. Their biosynthesis and degradation have been characterized and have been shown to occur during different developmental stages of the berry. The understanding of how the different abiotic factors modulate secondary metabolism and thus berry quality is of crucial importance for breeders and growers to develop plant material and viticultural practices to maintain high-quality fruit and wine production in the context of global warming. Here, we review the main secondary metabolites of the grape berry, their biosynthesis, and how their accumulation and degradation is influenced by abiotic factors. The first part of the review provides an update on structure, biosynthesis, and degradation of phenolic compounds (flavonoids and non-flavonoids) and major aroma compounds (terpenes, thiols, methoxypyrazines, and C13 norisoprenoids). The second part gives an update on the influence of abiotic factors, such as water availability, temperature, radiation, and CO2 concentration, on berry secondary metabolism. At the end of the paper, we raise some critical questions regarding intracluster berry heterogeneity and dilution effects and how the sampling strategy can impact the outcome of studies on the grapevine berry response to abiotic factors.

Biosynthesis and Cellular Functions of Tartaric Acid in Grapevines.

Tartaric acid (TA) is an obscure end point to the catabolism of ascorbic acid (Asc). Here, it is proposed as a "specialized primary metabolite", originating from carbohydrate metabolism but with restricted distribution within the plant kingdom and lack of known function in primary metabolic pathways. Grapes fall into the list of high TA-accumulators, with biosynthesis occurring in both leaf and berry. Very little is known of the TA biosynthetic pathway enzymes in any plant species, although recently some progress has been made in this space. New technologies in grapevine research such as the development of global co-expression network analysis tools and genome-wide association studies, should enable more rapid progress. There is also a lack of information regarding roles for this organic acid in plant metabolism. Therefore this review aims to briefly summarize current knowledge about the key intermediates and enzymes of TA biosynthesis in grapes and the regulation of its precursor, ascorbate, followed by speculative discussion around the potential roles of TA based on current knowledge of Asc metabolism, TA biosynthetic enzymes and other aspects of fruit metabolism.

Single berry reconstitution prior to RNA-sequencing reveals novel insights into transcriptomic remodeling by leafroll virus infections in grapevines.

Leafroll viruses are among the most devastating pathogens in viticulture and are responsible for major economic losses in the wine industry. However, the molecular interactions underlying the effects on fruit quality deterioration are not well understood. The few molecular studies conducted on berries from infected vines, associated quality decreases with the repression of key genes in sugar transport and anthocyanin biosynthesis. Sampling protocols in these studies did however not account for berry heterogeneity and potential virus induced phenological shifts, which could have biased the molecular information. In the present study, we adopted an innovative individual berry sampling protocol to produce homogeneous batches for RNA extraction, thereby circumventing berry heterogeneity and compensating for virus induced phenological shifts. This way a characterization of the transcriptomic modulation by viral infections was possible and explain why our results differ significantly from previously reported repression of anthocyanin biosynthesis and sugar metabolism. The present study provides new insights into the berry transcriptome modulation by leafroll infection, highlighting the virus induced upregulation of plant innate immunity as well as an increased responsiveness of the early ripening berry to biotic stressors. The study furthermore emphasizes the importance of sampling protocols in physiological studies on grapevine berry metabolism.

Oregano essential oil vapour prevents Plasmopara viticola infection in grapevine (Vitis Vinifera) and primes plant immunity mechanisms.

The reduction of synthetic fungicides in agriculture is necessary to guarantee a sustainable production that protects the environment and consumers' health. Downy mildew caused by the oomycete Plasmopara viticola is the major pathogen in viticulture worldwide and responsible for up to 60% of pesticide treatments. Alternatives to reduce fungicides are thus utterly needed to ensure sustainable vineyard-ecosystems, consumer health and public acceptance. Essential oils (EOs) are amongst the most promising natural plant protection alternatives and have shown their antibacterial, antiviral and antifungal properties on several agricultural crops. However, the efficiency of EOs highly depends on timing, application method and the molecular interactions between the host, the pathogen and EO. Despite proven EO efficiency, the underlying processes are still not understood and remain a black box. The objectives of the present study were: a) to evaluate whether a continuous fumigation of a particular EO can control downy mildew in order to circumvent the drawbacks of direct application, b) to decipher molecular mechanisms that could be triggered in the host and the pathogen by EO application and c) to try to differentiate whether essential oils directly repress the oomycete or act as plant resistance primers. To achieve this a custom-made climatic chamber was constructed that enabled a continuous fumigation of potted vines with different EOs during long-term experiments. The grapevine (Vitis vinifera) cv Chasselas was chosen in reason of its high susceptibility to Plasmopara viticola. Grapevine cuttings were infected with P. viticola and subsequently exposed to continuous fumigation of different EOs at different concentrations, during 2 application time spans (24 hours and 10 days). Experiments were stopped when infection symptoms were clearly observed on the leaves of the control plants. Plant physiology (photosynthesis and growth rate parameters) were recorded and leaves were sampled at different time points for subsequent RNA extraction and transcriptomics analysis. Strikingly, the Oregano vulgare EO vapour treatment during 24h post-infection proved to be sufficient to reduce downy mildew development by 95%. Total RNA was extracted from leaves of 24h and 10d treatments and used for whole transcriptome shotgun sequencing (RNA-seq). Sequenced reads were then mapped onto the V. vinifera and P. viticola genomes. Less than 1% of reads could be mapped onto the P. viticola genome from treated samples, whereas up to 30% reads from the controls mapped onto the P. viticola genome, thereby confirming the visual observation of P. viticola absence in the treated plants. On average, 80% of reads could be mapped onto the V. vinifera genome for differential expression analysis, which yielded 4800 modulated genes. Transcriptomic data clearly showed that the treatment triggered the plant's innate immune system with genes involved in salicylic, jasmonic acid and ethylene synthesis and signaling, activating Pathogenesis-Related-proteins as well as phytoalexin synthesis. These results elucidate EO-host-pathogen interactions for the first time and indicate that the antifungal efficiency of EO is mainly due to the triggering of resistance pathways inside the host plants. This is of major importance for the production and research on biopesticides, plant stimulation products and for resistance-breeding strategies.

Temperature desynchronizes sugar and organic acid metabolism in ripening grapevine fruits and remodels their transcriptome.

BACKGROUND: Fruit composition at harvest is strongly dependent on the temperature during the grapevine developmental cycle. This raises serious concerns regarding the sustainability of viticulture and the socio-economic repercussions of global warming for many regions where the most heat-tolerant varieties are already cultivated. Despite recent progress, the direct and indirect effects of temperature on fruit development are far from being understood. Experimental limitations such as fluctuating environmental conditions, intra-cluster heterogeneity and the annual reproductive cycle introduce unquantifiable biases for gene expression and physiological studies with grapevine. In the present study, DRCF grapevine mutants (microvine) were grown under several temperature regimes in duly-controlled environmental conditions. A singly berry selection increased the accuracy of fruit phenotyping and subsequent gene expression analyses. The physiological and transcriptomic responses of five key stages sampled simultaneously at day and nighttime were studied by RNA-seq analysis. RESULTS: A total of 674 millions reads were sequenced from all experiments. Analysis of differential expression yielded in a total of 10 788 transcripts modulated by temperature. An acceleration of green berry development under higher temperature was correlated with the induction of several candidate genes linked to cell expansion. High temperatures impaired tannin synthesis and degree of galloylation at the transcriptomic levels. The timing of malate breakdown was delayed to mid-ripening in transgressively cool conditions, revealing unsuspected plasticity of berry primary metabolism. Specific ATPases and malate transporters displayed development and temperature-dependent expression patterns, besides less marked but significant regulation of other genes in the malate pathway. CONCLUSION: The present study represents, to our knowledge the first abiotic stress study performed on a fleshy fruits model using RNA-seq for transcriptomic analysis. It confirms that a careful stage selection and a rigorous control of environmental conditions are needed to address the long-term plasticity of berry development with respect to temperature. Original results revealed temperature-dependent regulation of key metabolic processes in the elaboration of berry composition. Malate breakdown no longer appears as an integral part of the veraison program, but as possibly triggered by an imbalance in cytoplasmic sugar, when efficient vacuolar storage is set on with ripening, in usual temperature conditions. Furthermore, variations in heat shock responsive genes that will be very valuable for further research on temperature adaptation of plants have been evidenced.

Identification of stable QTLs for vegetative and reproductive traits in the microvine (Vitis vinifera L.) using the 18 K Infinium chip.

BACKGROUND: The increasing temperature associated with climate change impacts grapevine phenology and development with critical effects on grape yield and composition. Plant breeding has the potential to deliver new cultivars with stable yield and quality under warmer climate conditions, but this requires the identification of stable genetic determinants. This study tested the potentialities of the microvine to boost genetics in grapevine. A mapping population of 129 microvines derived from Picovine x Ugni Blanc flb, was genotyped with the Illumina® 18 K SNP (Single Nucleotide Polymorphism) chip. Forty-three vegetative and reproductive traits were phenotyped outdoors over four cropping cycles, and a subset of 22 traits over two cropping cycles in growth rooms with two contrasted temperatures, in order to map stable QTLs (Quantitative Trait Loci). RESULTS: Ten stable QTLs for berry development and quality or leaf area were identified on the parental maps. A new major QTL explaining up to 44 % of total variance of berry weight was identified on chromosome 7 in Ugni Blanc flb, and co-localized with QTLs for seed number (up to 76 % total variance), major berry acids at green lag phase (up to 35 %), and other yield components (up to 25 %). In addition, a minor QTL for leaf area was found on chromosome 4 of the same parent. In contrast, only minor QTLs for berry acidity and leaf area could be found as moderately stable in Picovine. None of the transporters recently identified as mutated in low acidity apples or Cucurbits were included in the several hundreds of candidate genes underlying the above berry QTLs, which could be reduced to a few dozen candidate genes when a priori pertinent biological functions and organ specific expression were considered. CONCLUSIONS: This study combining the use of microvine and a high throughput genotyping technology was innovative for grapevine genetics. It allowed the identification of 10 stable QTLs, including the first berry acidity QTLs reported so far in a Vitis vinifera intra-specific cross. Robustness of a set of QTLs was assessed with respect to temperature variation.

Day and night heat stress trigger different transcriptomic responses in green and ripening grapevine (vitis vinifera) fruit.

BACKGROUND: Global climate change will noticeably affect plant vegetative and reproductive development. The recent increase in temperatures has already impacted yields and composition of berries in many grapevine-growing regions. Physiological processes underlying temperature response and tolerance of the grapevine fruit have not been extensively investigated. To date, all studies investigating the molecular regulation of fleshly fruit response to abiotic stress were only conducted during the day, overlooking possible critical night-specific variations. The present study explores the night and day transcriptomic response of grapevine fruit to heat stress at several developmental stages. Short heat stresses (2 h) were applied at day and night to vines bearing clusters sequentially ordered according to the developmental stages along their vertical axes. The recently proposed microvine model (DRCF-Dwarf Rapid Cycling and Continuous Flowering) was grown in climatic chambers in order to circumvent common constraints and biases inevitable in field experiments with perennial macrovines. Post-véraison berry heterogeneity within clusters was avoided by constituting homogenous batches following organic acids and sugars measurements of individual berries. A whole genome transcriptomic approach was subsequently conducted using NimbleGen 090818 Vitis 12X (30 K) microarrays. RESULTS: Present work reveals significant differences in heat stress responsive pathways according to day or night treatment, in particular regarding genes associated with acidity and phenylpropanoid metabolism. Precise distinction of ripening stages led to stage-specific detection of malic acid and anthocyanin-related transcripts modulated by heat stress. Important changes in cell wall modification related processes as well as indications for heat-induced delay of ripening and sugar accumulation were observed at véraison, an effect that was reversed at later stages. CONCLUSIONS: This first day - night study on heat stress adaption of the grapevine berry shows that the transcriptome of fleshy fruits is differentially affected by abiotic stress at night. The present results emphasize the necessity of including different developmental stages and especially several daytime points in transcriptomic studies.

Validation and application of an improved method for the rapid determination of proline in grape berries.

A rapid and sensitive method is presented for the determination of proline in grape berries. Following acidification with formic acid, proline is derivatized by heating at 100 °C for 15 min with 3% ninhydrin in dimethyl sulfoxide, and the absorbance, which is stable for at least 60 min, is read at 520 nm. The method was statistically validated in the concentration range from 2.5 to 15 mg/L, giving a repeatability and intermediate precision of generally <3%; linearity was determined using the lack of fit test. Results obtained with this method concurred (r = 0.99) with those obtained for the same samples on an amino acid analyzer. In terms of sample preparation, a simple dilution (5-20-fold) is required, and sugars, primary amino acids, and anthocyanins were demonstrated not to interfere, as the latter are bleached by ninhydrin under the experimental conditions. The method was applied to the study of proline accumulation in the fruits of microvines grown in phytotrons, and it was established that proline accumulation and concentrations closely resemble those of field-grown macrovines.

Is transcriptomic regulation of berry development more important at night than during the day?

Diurnal changes in gene expression occur in all living organisms and have been studied on model plants such as Arabidopsis thaliana. To our knowledge the impact of the nycthemeral cycle on the genetic program of fleshly fruit development has been hitherto overlooked. In order to circumvent environmental changes throughout fruit development, young and ripening berries were sampled simultaneously on continuously flowering microvines acclimated to controlled circadian light and temperature changes. Gene expression profiles along fruit development were monitored during both day and night with whole genome microarrays (Nimblegen® vitis 12x), yielding a total number of 9273 developmentally modulated probesets. All day-detected transcripts were modulated at night, whereas 1843 genes were night-specific. Very similar developmental patterns of gene expression were observed using independent hierarchical clustering of day and night data, whereas functional categories of allocated transcripts varied according to time of day. Many transcripts within pathways, known to be up-regulated during ripening, in particular those linked to secondary metabolism exhibited a clearer developmental regulation at night than during the day. Functional enrichment analysis also indicated that diurnally modulated genes considerably varied during fruit development, with a shift from cellular organization and photosynthesis in green berries to secondary metabolism and stress-related genes in ripening berries. These results reveal critical changes in gene expression during night development that differ from daytime development, which have not been observed in other transcriptomic studies on fruit development thus far.