Determining the Role of Auxin-Response Factor 4 in the Timing of Ripening Initiation in Vitis Vinifera
The timing of the ripening initiation is a major trait for wine grape production. Rapidly evolving climactic conditions might affect the ripening process of major cultivars in several growing regions of U.S resulting in loss of fruit and wine quality. While the development of improved viticulture practices to mitigate the effects of climate changes is critical, the use of complementary approaches should be investigated. The identification of genetic markers associated with the timing of ripening initiation will be valuable to develop targeted breeding programs aimed to tackle grapevine production in the context of climate changes. Innovative molecular practices in the field such as Spray Induced Gene Silencing have recently paid scientific attention and is seen as a potential industrial application. Yet, all these translational tools need a scientific study up front to validate the relationship between a gene and/or several genes with the trait of interest (fruit composition, disease resistance, etc.). The current research project aims to validate the role of a regulatory protein, VviARF4, on the timing of ripening initiation in grape berry. Three objectives were designed to achieve this goal; 1) the characterization of VviARF4 through genetic engineering and the identification of VviARF4 interactors during the ripening process, 2) the identification of ripening-related genes directly targeted by the activity of VviARF4, and 3) the evaluation of fruit composition on berries for which VviARF4 activity has been modified through the genetic engineering procedure.
Since June 2017 we focused our research efforts on several milestones of the current project. We established the microvine culture at OSU part of the objective 1. We conducted our first attempt for genetic engineering of the microvine. We successfully
generated embryogenic cells from immature floral buds, which is necessary to perform genetic transformation. For the same objective, we finalized our different genetic constructs to induce and silence VviARF4 in the microvine. Using a protein-protein interaction test, we identified 170 potential protein partners to VviARF4 and we are in the process of confirming some of these interactions by additional in vitro assays. Some of these interactors are very interesting because they are associated with ABA, ethylene
and sugar signaling pathway that are known to be important in the ripening process. For the objective 3, we adapted a new analytical method to measure metabolites associated with organic, amino, and phenolic acids, different types of carbohydrates, polyols, and three classes of flavonoids (anthocyanins, flavonols, and monomer and dimer of tannins). For those metabolites, we have built an in-house library of 95 analytes. We tested this library using berry extracts from pericarp samples collected at different stages of grape
berry ripening. We were able to identify around 30 analytes covering major families of compounds existing in grape berry. These include tartrate, malate, glucose, fructose, sucrose, and several polyphenol-related compounds. We will be using this analytical method for the aim 3 of this research proposal.