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…

Influence of cultivar and its drought tolerance on soil microbiome

Soil microbiome plays a crucial role in plant health and resilience, particularly under abiotic stress conditions such as drought.

The Albariño route in Uruguay: A clonal selection process to produce quality wines

In recent years, Uruguay has embraced the Albariño grape variety (Vitis vinifera L.) as one of the most promising for commercial growth. Originally cultivated in Galicia and northern Portugal, Albariño has risen to prominence in the global wine market, driving strong demand and significantly increasing grape prices [1].

Assessment of plant water consumption rates under climate change conditions through an automated modular platform

The impact of climate change is noticeable in the present weather, making water scarcity the most immediate mediator reducing the performance and viability of crops, including grapevine (Vitis vinifera L.). The present study developed a system (hardware, firmware, and software) for the determination of plant water use through changes in weight through a period. The aim is to measure the differences in grapevine water consumption in response to climate change (+4oC and 700 ppm) under controlled conditions. The results reveal a correlation between daily plant consumption rates and reference evapotranspiration (ETo).

Projected changes in vine phenology of two varieties with different thermal requirements cultivated in La Mancha DO (Spain) under climate change scenarios

The aim of this work was to analyze the phenology variability of Tempranillo and Chardonnay cultivars, related to the climatic characteristics in La Mancha Designation of Origin, and their potential changes under climate change scenarios. Phenological dates referred to budbreak, flowering, veraison and harvest were analyzed for the period 2000-2019. The weather conditions at daily time scale, recorded during the same period, were also evaluated. The thermal requirements to reach each of these phenological stages were calculated and expressed as the GDD accumulated from DOY=60. Changes in phenology were projected by 2050 and 2070 taking into account those values and the projected temperatures and precipitation, simulated under two Representative Concentration Pathway (RCP) scenarios –RCP4.5 and RCP8.5– using an ensemble of models. The average phenological dates during the period under study were, April 16th ± 6.6 days and April 5th ± 6.0 days for budbreak, May 31st ± 6.0 days and May 27th ± 5.3 days for flowering, July 26th ± 5.6 days and July 25th ± 5.8 days for veraison, and Ago 23rd ± 10.8 days and Ago 17th ± 9.0 days for harvest, respectively, for Tempranillo and Chardonnay. The projected changes in temperature imply an average change in the maximum growing season (April-August) temperatures of 1.2 and 1.9°C by 2050, and 1.6 and 2.6°C by 2070, under the RCP4.5 and RCP8.5 scenarios, respectively. A reduction in precipitation is predicted, which vary between 15% for 2050 under RCP4.5 scenario and up to 30% by 2070 under RCP8.5. The advance of the phenological dates for 2050, could be of 6, 7, 7, and 8 days for Tempranillo and 4, 6, 6 and 9 days for Chardonnay, respectively for budbreak, flowering, veraison and harvest under the RCP4.5 scenario. Under the RCP8.5 emission scenario, the advance could be up to 30% higher.

Effect of nanofiltration on the chemical composition and wine quality

In Enology the conventional processes of filtration for clarification and stabilization are giving place to alternative membrane processes, including nanofiltration (NF). Furthermore, the increased alcohol content in wines recorded in recent years became an important issue for all the main wine producing countries. Among techniques available to the wine industry to reduce the ethanol content, NF is certainly one of the newest. This study is focused on the evaluation of NF influence on wine physical-chemical composition, including mineral content, which in accordance to our best knowledge is a novelty.