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…

Metschnikowia pulcherrima: A valuable microbial bioresource from wine for smart agrifood

The yeast Metschnikowia pulcherrima is a microorganism of great biotechnological interest, both for improving winemaking processes and for other applications outside the wine supply chain.

Wine odors: chemicals, physicochemical and perceptive processes involved in their perception

The odors of wines are diverse, complex and dynamic and much research has been devoted to the understanding of their chemical bases. However, while the “basic” chemical part of the problem, namely the identity of the chemicals responsible for the different odor nuances, was satisfactorily solved years ago, there are some relevant questions precluding a clear understanding. These questions are related to the physicochemical interactions determining the effective volatilities of the odorants and, particularly, to the perceptual interactions between different odor molecules affecting in different ways to the final sensory outputs.

Use of glutathione and a selected strain of metschnikowia pulcherrima as alternatives to sulphur dioxide to inhibit natural tyrosinase of grape must and prevent browning

The enzymatic browning of grape must is still a major problem in oenology today [1] being particularly serious when the grapes have been infected by grey rot [2]. Browning is an oxidation process that causes certain foods to turn brown, which often leads to them being rejected by consumers [3]. This is a particular problem in the case of wine, because grape must is very vulnerable to enzymatic browning [4].

Study of the aromatic oxidation markers of Tempranillo long aged wines

The aromatic quality of wines after a long aging period in bottle is one of key points for oenologists. The objective of this work is to determine the main representative aromatic compounds found in long aged wines from D.O.Ca. Rioja. This study was made by 32 wines from 1971 to 2010 vintages. Sotolon, acetaldehyde, phenylacetaldehyde, 1,1,6-trimethyl-1,2-dihydronaptalene (TDN), β-damascenone, Y-decalactone and Y-dodecalactone were determined as the most important oxidation markers by GC-MS analysis. Moreover, sensory analysis using triangular tests were performed from wines with and without the addition of the mentioned compounds. Four different concentrations of each odorant were added, as individual compounds and as mixtures. The additions were ranged from values close to the reference odour thresholds up to high level concentrations. The most identified aroma was sotolon, which is commonly associated to curry and coffee liqueur aromatic notes. Other oxidative compounds were easily detected by panellists, such as Y-decalactone (peach compote), Y-dodecalactone (ripe fruit). The mixtures of the odorants were most easily detected than the individual compounds. It should be noted that acetaldehyde and phenylacetaldehyde were rarely perceived and distinguished.

Aroma composition of young and aged Lugana and Verdicchio

AIM Verdicchio and Lugana are two Italian white wines produced in the Marche and Garda lake regions respectively. They are however obtained using grape varieties sharing the same genetic background, locally known as Verdicchio in Marche and Trebbiano di Soave in Garda. Anecdotal evidence suggests that these two wine types exhibit distinctive aroma features. The aim of this work was to explore the existence of a recognizable odour profile for Lugana and Verdicchio, and whether specific aroma chemical markers could be identified. METHODS 13 commercial wines, 6 Lugana and 7 Verdicchio were used. Sensory analysis was done using sorting task methodology, assessing only odor similarities. A total of 53 volatile compounds were identified and quantified GC-MS analysis. Aging behaviors were also evaluated after an accelerated aging at 40 ° C for 3 months. RESULTS HCA analysis of sorting task data identified indeed two groups: one characterized by floral and minty notes and mostly associated with Lugana wines, the other characterized by spicy and toasted aromas and mostly associated with Verdicchio. From a chemical point of view, major differences between the two wines types were observed for cis-3-hexenol, methionol, phenylethyl alcohol, and geraniol.