OENO IVAS 2019 banner
IVES 9 IVES Conference Series 9 OENO IVAS 9 OENO IVAS 2019 9 Grape and wine microorganisms: diversity and adaptation 9 Use of antisense RNA technology to modulate gene expression in Œnococcus oeni

Use of antisense RNA technology to modulate gene expression in Œnococcus oeni

Abstract

Œnococcus oeni is a wine-associated lactic acid bacterium performs the malolactic fermentation, which improves the taste and aromatic complexity of many wine. Although, wine exhibits harsh and challenging conditions (low pH, low temperature, nutrient-poor and presence of ethanol), O. oeni possesses a remarkable adaptability to those physiochemical conditions.

Mechanisms for responding to environmental changes are universally present in living beings and are essential for coping with the stress and for adapting to the new conditions. O. oeni tolerance to low pH and ethanol make its an interesting bacteria model for investigating stress response mechanism in lactic acid bacteria. However, lack of appropriate techniques to manipulate O. oeni genome has long delay molecular study of this fastidious bacterium. To get around the lack of genetic tool for gene replacement, we focused our work on gene inactivation by using antisense RNA approach to modulate gene expression. With the goal to understanding the function of O. oeni hsp genes in vivo, we produce antisense RNA targeting genes encoding: a small Hsp (hsp18)1, the master regulator of stress response (ctsR)2 and two caseinolytic protease L members of the HSP100 chaperone family (clpL1, clpL2). Thereby, we highlighted that in vivo inhibition of the expression of some of these genes strongly affects the survival of O. oeni in stress conditions.

This study presents an elegant approach providing access to an in vivo study of gene function in O. oeni.

References:

1. Darsonval, M., Msadek, T., Alexandre, H. & Grandvalet, C. The Antisense RNA Approach: a New Application for In Vivo Investigation of the Stress Response of Oenococcus oeni, a Wine-Associated Lactic Acid Bacterium. Appl. Environ. Microbiol. 82, 18–26 (2016).

2. Darsonval, M., Julliat, F., Msadek, T., Alexandre, H. & Grandvalet, C. CtsR, the Master Regulator of Stress-Response in Oenococcus oeni, Is a Heat Sensor Interacting With ClpL1. Front Microbiol 9, (2018).

DOI:

Publication date: June 10, 2020

Issue: OENO IVAS 2019

Type: Article

Authors

Cosette Grandvalet, Frédérique Julliat, Maud Darsonval, Tarek Msadek, Hervé Alexandre

UMR A 02.102 Procédés Alimentaires et Microbiologiques, AgroSup Dijon – Université de Bourgogne Franche-Comté, Dijon, FRANCE.
Unité de Biologie des Bactéries Pathogènes àGram Positif, Institut Pasteur, Paris, FRANCE.
CNRS ERL 6002, Paris, FRANCE.

Contact the author

Keywords

Oenococcus oeni, lactic acid bacteria, antisense RNA , stress response 

Tags

IVES Conference Series | OENO IVAS 2019

Citation

Related articles…

Long-term drought resilience of traditional red grapevine varieties from a semi-arid region

In recent decades, the scarcity of water resources in agriculture in certain areas has been aggravated by climate change, which has caused an increase in temperatures, changes in rainfall patterns, as well as an increase in the frequency of extreme phenomena such as droughts and heat waves. Although the vine is considered a drought-tolerant specie, it has to satisfy important water requirements to complete its cycle, which coincides with the hottest and driest months. Achieving sustainable viticulture in this scenario requires high levels of efficiency in the use of water, a scarce resource whose use is expected to be severely restricted in the near future. In this regard, the use of drought-tolerant varieties that are able to maintain grape yield and quality could be an effective strategy to face this change. During three consecutive seasons (2018-2020) the behavior in rainfed regime of 13 traditional red grapevine varieties of the Spain central region was studied. These varieties were cultivated in a collection at Centro de Investigación de la Vid y el Vino de Castilla-La Mancha (IVICAM-IRIAF) located in Tomelloso (Castilla-La Mancha, Spain). Yield components (yield, mean bunch and berry weight, pruning weight), physicochemical parameters of the musts (brix degree, total acidity, pH) and some physiological parameters related with water stress during ripening period (δ13C, δ18O) were analysed. The application of different statistical techniques to the results showed the existence of significant differences between varieties in their response to stressful conditions. A few varieties highlighted for their high ability to adapt to drought, being able to maintain high yields due to their efficiency in the use of water. In addition, it was possible quantify to what extent climate can be a determinant in the δ18O of musts under severe water stress conditions.

What are the optimal ranges and thresholds for berry solar radiation for flavonoid biosynthesis?

In wine grape production, canopy management practices are applied to control the source-sink balance and improve the cluster microclimate to enhance berry composition. The aim of this study was to identify the optimal ranges of berry solar radiation exposure (exposure) for upregulation of flavonoid biosynthesis and thresholds for their degradation, to evaluate how canopy management practices such as leaf removal, shoot thinning, and a combination of both affect the grapevine (Vitis vinifera L. cv. Cabernet Sauvignon) yield components, berry composition, and flavonoid profile under context of climate change. First experiment assessed changes in the grape flavonoid content driven by four degrees of exposure. In the second experiment, individual grape berries subjected to different exposures were collected from two cultivars (Cabernet Sauvignon and Petit Verdot). The third experiment consisted of an experiment with three canopy management treatments (i) LR (removal of 5 to 6 basal leaves), (ii) ST (thinned to 24 shoots per vine), and (iii) LRST (a combination of LR and ST) and an untreated control (UNT). Berry composition, flavonoid content and profiles, and 3-isobutyl 2-methoxypyrazine were monitored during berry ripening. Although increasing canopy porosity through canopy management practices can be helpful for other purposes, this may not be the case of flavonoid compounds when a certain proportion of kaempferol was achieved. Our results revealed different sensitivities to degradation within the flavonoid groups, flavonols being the only monitored group that was upregulated by solar radiation. Within different canopy management practices, the main effects were due to the ST. Under environmental conditions given in this trial, ST and LRST hastened fruit maturity; however, a clear improvement of the flavonoid compounds (i.e., greater anthocyanin) was not observed at harvest. Methoxypyrazine berry content decreased with canopy management practices studied. Although some berry traits were improved (i.e. 2.5° Brix increase in berry total soluble solids) due to canopy management practices (ST), this resulted in a four-fold increase in labor operations cost, two-fold decrease in yield with a 10-fold increase in anthocyanin production cost per hectare that should be assessed together as the climate continues to get hot.

‘Cabernet Sauvignon’ (Vitis vinifera L.) berry skin flavonol and anthocyanin composition is affected by trellis systems and applied water amounts

Trellis systems are selected in wine grape vineyards to mainly maximize vineyard yield and maintain berry quality. This study was conducted in 2020 and 2021 to evaluate six commonly utilized trellis systems including a vertical shoot positioning (VSP), two relaxed VSPs (VSP60 and VSP80), a single high wire (SH), a high quadrilateral (HQ), and a guyot (GY), combined with three levels of irrigation regimes based on different crop evapotranspiration (ETc) replacements, including a 25% ETc, 50% ETc, and 100% ETc. The results indicated SH yielded the most fruits and accumulated the most total soluble solids (TSS) at harvest in 2020, however, it showed the lowest TSS in the second season. In 2020, SH and HQ showed higher concentrations in most of the anthocyanin derivatives compared to the VSPs. Similar comparisons were noticed in 2021 as well. SH and HQ also accumulated more flavonols in both years compared to other trellis systems. Overall, this study provides information on the efficacy of trellis systems on grapevine yield and berry flavonoid accumulation in a currently warming climate.

Low-cost sensors as a support tool to monitor soil-plant heat exchanges in a Mediterranean vineyard

Mediterranean viticulture is increasingly exposed to more frequent extreme conditions such as heat waves. These extreme events co-occur with low soil water content, high air vapor pressure deficit and high solar radiant energy fluxes and result in leaf and berry sunburn, lower yield, and berry quality, which is a major constraint for the sustainability of the sector. Grape growers must find ways to proper and effectively manage heat waves and extreme canopy and berry temperatures. Irrigation to keep soil moisture levels and enable adequate plant turgor, and convective and evaporative cooling emerged as a key tool to overcome this major challenge. The effects of irrigation on soil and plant water status are easily quantifiable but the impact of irrigation on soil and canopy temperature and on heat convection from soil to cluster zone remain less characterized. Therefore, a more detailed quantification of vineyard heat fluxes is highly relevant to better understand and implement strategies to limit the effects of extreme weather events on grapevine leaf and berry physiology and vineyards performance. Low-cost sensor technologies emerge as an opportunity to improve monitoring and support decision making in viticulture. However, validation of low-cost sensors is mandatory for practical applicability. A two-year study was carried in a vineyard in Alentejo, south of Portugal, using low-cost thermal cameras (FLIR One, 80×60 pixels and FLIR C5, 160×120 pixels, 8-14 µm, FLIR systems, USA) and pocket thermohygrometers (Extech RHT30, EXTECH instruments, USA) to monitor grapevine and soil temperatures. Preliminary results show that low-cost cameras can detect severe water stress and support the evaluation of vertical canopy temperature variability, providing information on soil surface temperature. All these thermal parameters can be relevant for soil and crop management and be used in decision support systems.

VINIoT – Precision viticulture service

The project VINIoT pursues the creation of a new technological vineyard monitoring service, which will allow companies in the wine sector in the SUDOE space to monitor plantations in real time and remotely at various levels of precision. The system is based on spectral images and an IoT architecture that allows assessing parameters of interest viticulture and the collection of data at a precise scale (level of grape, plant, plot or vineyard) will be designed. In France, three subjects were specifically developed: evaluation of maturity, of water stress, and detection of flavescence dorée. For the evaluation of maturity, it has been decided first to work at the berry scale in the laboratory, then at the bunch scale and finally in the vineyard. The acquisition of the spectral hyperstal image as well as the reference analyzes to measure the maturity, were carried out in the laboratory after harvesting the berries in a maturity monitoring context. This work focuses on a case study to predict sugar content of three different grape varieties: Syrah, Fer Servadou and Mauzac. A robust method called Roboost-PLSR, developed in the framework of this work (Courand et al., 2022), to improve prediction model performance was applied on spectra after the acquirement of hyperspectral images. Regarding the evaluation of water stress, to work with a significant variability in terms of water status, it has been worked first with potted plants under 2 different water regimes. The facilities have allowed the supervision of irrigation and micro-climatic conditions. The regression models on agronomic variables (stomatal conductance, water potential, …) are studied. To detect flavescence dorée, the experimental plan has consisted of work at leaf scale in the laboratory first, and then in the field. To detect the disease from hyper-spectral imaging, a combination of multivariate curve resolution-alternating least squares (MCR-ALS) and factorial discriminant analysis (FDA) was proposed. This strategy proved the potential towards the discrimination of healthy and infected leaves by flavescence dorée based on the use of hyperspectral images (Mas Garcia et al., 2021).