*4.6. Plant Materials, Growth Conditions, and Stress Treatments*

Young roots, stems, and fully expanded leaves, as well as flowers and mature fruit (70 mm, red peel, 150 days after bloom), were collected from apple plants that were five years old after bud grafting. The scion was *Malus domestica* "Golden Delicious", and the rootstock was *M*. *hupehensis*. Samples used for examining the effects of water deficits were harvested from plants three months after bud grafting was performed with "Golden Delicious" scions and *M. hupehensis* rootstocks. These grafted plants were grown in pots (height, 320 mm; diameter, 300 mm) in a greenhouse and treatments began when the plants were approximately 500-mm tall. To induce a water deficit, irrigation was withheld from certain plants for up to 8 days while the designated control plants continued to receive normally scheduled irrigation [52]. Our sampling schedule involved harvesting mature leaves at the middle nodes on days 0, 4, and 8 of the deficit period. All of the tissues were frozen immediately in liquid N<sup>2</sup> and stored at −80 ◦C.

Seedlings of *Arabidopsis thaliana* L. (Heyn), cv. Columbia ("Col"), were used for genetic transformations and assays of osmotic and drought tolerance. They were cultured in a growth chamber under a 16-h photoperiod at 23 ◦C. For the drought tolerance assay, water was withheld from four-week-old plants for 20 days before they were rewatered. Survival rates were scored 2 days after rewatering began. Well-watered plants were used as the negative control. For the osmotic stress assay, five-day-old seedlings grown on MS agar plates were vertically plated on an MS agar medium supplemented with 0, 200, or 300 mM of mannitol. Their root lengths, fresh weights, relative electrolyte leakage (REL), and concentrations of chlorophyll, malondialdehyde (MDA), and proline were measured 11 days after that transfer. All of the experiments were repeated three times.
