Determining the role of Auxin-Response Factor 4 in the timing of ripening initiation in Vitis vinifera

The timing of ripening initiation is a major trait for wine grape production. Rapidly evolving climactic conditions will affect the ripening process of major cultivars in several growing regions of U.S, which may result in loss of fruit and wine quality. While the development of improved viticulture practices to mitigate effects of climate change is critical, the use of complementary approaches must be investigated. The identification of regulatory genes controlling the timing of ripening initiation is a research avenue to consider for molecular breeding programs in order to identify clones/cultivars more suitable to evolving climatic conditions. Innovative molecular practices in the field such as Spray Induced Gene Silencing have recently received scientific attention and its potential for rapid industrial application can be seen as alternate solution to traditional and molecular breeding. Yet, all these translational tools need scientific validation up front to characterize the cause-to-effect relationship between the gene and/or several genes and the trait of interest (fruit composition, disease resistance, etc.). The current research project aims to validate the contribution of a regulatory protein, VitviARF4, to the ripening initiation in grape berry. Three objectives were designed to achieve this goal; 1) the characterization of VitviARF4 via genetic engineering and the identification of its potential partners during the ripening process, 2) the identification of ripening-related genes that are targeted by VitviARF4, and 3) the evaluation of fruit composition on genetically engineered berries.

After the establishment of the microvine model at OSU to conduct the genetic engineering experiments, our research efforts were focused this year on several milestones of the project. For the objective 1, we conducted all the microvine transformations (four in total, control, two over-expression, and one knock – down) to either turn on or off the expression of VitviARF4. At least 20 independent transformed plants were selected per transformation event. We also confirmed protein-protein interactions of VitviARF4 with other proteins that play a major role in various physiological process of fruit ripening (sugar, brassinosteroids, ethylene, and epigenetic mechanism). For the objective 2, we demonstrated that we can control the expression of the transgene in transgenic microvines during the development of the plants. This result was critical to the success of the research outcomes of the objective 2. We have generated about 15 to 20 GFP positive transformed lines per construct and few of them were transferred to the greenhouse for being characterized. In parallel, we also conducted a Spray Induced Gene Silencing (SIGS) experiment on pre-véraison berries of V6 microvine. Our preliminary findings seem to support the delaying effect of VitviARF4 on the timing of berry ripening. We observed a faster ripening on berries treated with dsRNA aiming at silencing the expression of the endogenous VitviARF4. However, this experiment is currently repeated to confirm this first results. If confirmed, it might lead to a potential direct application of the SIGS technology in the field to manipulate trait. For the objective 3, we developed a
new analytical method to measure organic, amino, and phenolic acids, different types of carbohydrates, polyols, and three classes of flavonoids (anthocyanins, flavonols, and monomer and dimer of tannins). We have built an in-house library of 95 analytes that were tested against berry extracts from pericarp samples collected at different stages of grape berry ripening. We are currently testing the method on mature berries of the microvine and we have identified around 30 analytes covering the major families of compounds existing in grape berry. Dr. Tomasino has optimized the volatile and aroma analyses on mature fruits of regular microvines.