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 induce the conditional expression of the transgene in transgenic
microvine plants. This result was critical to the success of the research outcomes of the
objective 2. 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
is currently optimizing the volatile and aroma analysis on mature fruits of the microvines.