terclim by ICS banner
IVES 9 IVES Conference Series 9 Open-GPB 9 Open-GPB-2024 9 Flash - New biotechnological tools 9 Enhancing hydric stress tolerance by editing the VviMYB60 promoter with CRISPR/Cas9 

Enhancing hydric stress tolerance by editing the VviMYB60 promoter with CRISPR/Cas9 

Abstract

Climate change presents increasing challenges to viticulture, particularly with rising water stress contributing significantly to yield losses and damages. The identification of the MYB60 transcription factor, which regulates stomatal opening and closing in Arabidopsis thaliana and Vitis vinifera, offers potential solutions. Notably, knockout studies in Arabidopsis have shown reduced stomatal opening and increased drought tolerance in myb60 mutants. Additionally, the grapevine ortholog, VviMYB60, can restore the wild-type phenotype of Arabidopsis myb60 mutants. Further investigation of the Arabidopsis promoter region has revealed that mutations in DOF motifs lead to reduced expression of AtMYB60.

Utilizing the advancements in CRISPR/Cas9 genome editing, this project aims to modify the VviMYB60 promoter region to lower gene expression, thereby reducing stomatal opening in grapevines. Binary vectors for genome editing were constructed to target two specific regions of the VviMYB60 promoter. Agrobacterium-mediated transformation was performed on Chardonnay embryogenic calli, resulting in the successful regeneration of plants under selection conditions. Sanger sequencing analysis of the targeted region confirmed the occurrence of genetic edits in four of the six lines analyzed so far.

To further characterize the edited lines, next-generation sequencing will be utilized, providing a more comprehensive understanding of the mutations, as well as gene expression of VviMYB60 will be evaluated to confirm that the editing reduces its expression. Morphological and physiological parameters will be measured after acclimatation in greenhouse and finally these edited lines will undergo drought tolerance testing to assess their performance.

DOI:

Publication date: June 13, 2024

Issue: Open GPB 2024

Type: Article

Authors

Manuela Campa1*, Loredana Moffa2, Luca Nerva2, Walter Chitarra2, Johan Burger1

1 Genetics Department, Faculty of Agrisciences, Stellenbosch University, 7600 Stellenbosch, South Africa
2 Research Centre for Viticulture and Enology, Council for Agricultural Research and Economics (CREA-VE), Via XXVIII Aprile 26, 31015 Conegliano (Italy)

Contact the author*

Keywords

CRISPR/Cas9, VviMYB60, promoter, drought tolerance, stomatal regulation

Tags

IVES Conference Series | Open GPB | Open GPB 2024

Citation

Related articles…

Towards the definition of a terroir of grape dehydration for the production of ‘Passito’ wines in Valpolicella (Italy)

Aim: The aim of this study was to investigate the relationship between the molecular response of grapes during postharvest dehydration and the specific environment of two naturally ventilated rooms (called ‘fruttai’), located in two different sites in Valpolicella

Bioprotective non-Saccharomyces yeast as an alternative to sulfites for the winemaking process

Sulfur dioxide (SO2) is used in winemaking due of its antioxidant, antioxydasic and antiseptic properties. Excessive amount of SO2 can negatively impact wine sensory perception and be detrimental for health. Agri-food industries are more transparent towards consumers concerning addition of sulfites, and oenology is no exception in this clairvoyance. As a consequence, the increase of consumers preference for wine with low or absent of sulfites addition is notorious. In this context, the impact of low/zero sulfites winemaking process on the microbial community should be evaluated. Moreover, microbial agents corresponding to bioprotective cultures represent a growing interest as an alternative to sulfites preservation in the early stages of vinification. However, scientific studies conducted to demonstrate their real effect are almost rare.

Grapevine genotypes differ in xylem vessel occlusion after winter pruning 

Grapevines are continually wounded throughout their cultivation especially during winter pruning. Grapevines respond to wounding by occluding xylem vessels with gels or tyloses to limit pathogen attack and dehydration of the tissues. Although the production of xylem vessel occlusions has been studied in grapevine, to date we have no knowledge of whether different genotypes respond differently. The objective of this study was to characterize the genetic variation in xylem vessel occulsions in five different scion genotypes pruned at different dates.

Vine environment interaction as a method for land viticultural evaluation. An experience in Friuli Venezia Giulia (N-E of Italy)

For a long time environment was known as one of the most important factors to characterize the quality of wines but at the same time it appears very difficult to distinguish inside the “terroir” the role of the single factor. These remarks partially explain why methods for viticultural evaluation are often quite different (Amerine et al., 1944; Antoniazzi et al., 1986; Asselin et al., 1987; Astruc et al., 1980; Bonfils, 1977; Boselli, 1991; Colugnati, 1990; Costantinescu, 1967; Costantini et al., 1987; Dutt et al., 1981; Falcetti et al., 1992; Fregoni et al., 1992; Hidalgo, 1980; Intrieri et al., 1988; Laville, 1990; Morlat et al., 1991; Scienza et al., 1990; Shubert et al., 1987; Turri et al., 1991).

Nitrogen – Lipid Balance in alcoholic fermentations. Example of Champagne musts

Nutrient availability – nitrogen, lipids, vitamins or oxygen – has a major impact on the kinetics of winemaking fermentations. Nitrogen is usually the growth-limiting nutrient and its availability determines the fermentation rate, and therefore the fermentation duration. In some cases, in particular in Champagne, grape musts have high nitrogen concentrations and are sometimes clarified with turbidity below 50 NTU. In these conditions, lipid deficiencies may occur and longer fermentations can be observed. To better understand this situation, a study was realized using a synthetic medium simulating the composition of a Champagne must : 180 g/L of sugar, 360 mg/L of assimilable nitrogen and a lipid content ranging from 1 to 8 mg/L of phytosterols (mainly β-sitosterol).