In the context of genetic engineering more cold tolerant grapes, we have successfully transformed and regenerated V

Functional Genomics of Vitis vinifera

Project Investigators: Grant R. Cramer and John C. Cushman

Abiotic stresses (e.g. cold, heat, salinity, drought) are the major limitation to agricultural productivity and together account for more than half of all production losses. In arid regions of the west, such as the great basin, where crop production is completely dependent on irrigation, greater investments are needed in the development of crops with high water use efficiency and high value-added products. Wine grapes fulfill both of these criteria. Regulated deficit irrigation has been used successfully to grow grapes with less water, an important feature in arid regions such as Nevada. Furthermore, wine grape production under water deficit irrigation regimes has been shown to improve the aroma, flavor and color components of wine by altering metabolite composition, thereby improving wine quality and human health benefits. However, the mechanistic basis for these quality improvements is poorly understood. As a first step toward understanding how water deficits influence the growth of wine grape vines and berries to bring about wine quality improvements following abiotic stress exposure, we have initiated an expressed sequence tag (EST)-based gene discovery program focused solely on stressed vines. We constructed cDNA libraries from mRNA isolated from leaf, root, and berry tissues of Vitis vinifera cv. Chardonnay. Significantly, 42% of the ESTs identified to date have no known function. With our growing database of EST sequence information, we have generated the molecular genetic resources to be able to conduct large-scale gene expression profiling using microarray technology.

Development of cDNA libraries and ESTs: We exposed plants to a variety of abiotic stresses (drought, cold, heat, salt, and flooding; see Table 1 for details) to develop mixed abiotic stress cDNA libraries.

Table 1. Abiotic stress treatments applied to Chardonnay plants used to make cDNA libraries. The water potential of the leaves was measured with a pressure chamber at midday as described (McCutchan and Shackel 1992). Berries were harvested at 7 different developmental stages with maturity measured by the Brix/Titratable Acidity ratio.

Treatment	Organ harvested
Salt: (20 mM Na₂SO₄, 40 mM NaCl, 10 mM CaSO₄) for 2, 24 and 144 hours; note these Cl^- concentrations would be lethal with long-term exposures	Root and Leaf
Drought: potted plants not watered (leaf water potentials ranged from –1.4 to –2.2 MPa)	Root and Leaf
Drought: field plants harvested at 7 different berry developmental stages (preveraison to over-ripeness) with water potentials ranging from –1.0 to –1.7 MPa	Leaf and Berries
Cold: 2 and 24 h at 4°C; incremental decrease in night temperature (2°C per night) to 4°C over 6 days; 6 days of 4°C nights	Root and Leaf
Heat: 20 min at 42°C; incremental rise (2°C per day) to 42°C over 6 days	Root and Leaf
Flooding: Roots (of potted plants) under water for 24h	Root and Leaf