ABSTRACT: Six months old seed grown under well-watered greenhouse conditions were used in the current study. The seedlings of the two wild emmer wheat genotypes were vernalized on a moist germination paper (Hofman Manufacturing, Inc, Jefferson, OR, USA) for three weeks in the dark at 4oC, followed by three days acclimation at 24oC. Seedlings were then transplanted into 5L pots containing a mixture of pure quartz sand and peat (4:1 v/v), supplemented with a slow release fertilizer (2 g/L, Osmocote® Standard 14-14-14, Scotts-Sierra Horticulture, Marysville, OH, USA) and by a weekly application of 100 ml/pot of 0.5X Murashigi and Skoog growth solution (Sigma Chemical Co., St Louis, MO, USA). Pots were placed in a screen-house under natural winter conditions (December to February; 5-18°C) in Haifa, Israel (Mt. Carmel; 35o01′ E, 32o45′ N; 480 m above sea level) for 10 weeks and irrigated daily by a drip irrigation system. Three weeks prior to application of terminal drought stress, the pots were transferred to a controlled environment greenhouse (22/18 oC; 12 h day/12 h night) in order to prevent rainfall during the drought experiment. Five pots per genotype served as biological replicates, plants of three pots of each genotype/treatment were used for transcriptome study whereas two additional pots were used for quantitative PCR analysis (altogether five biological replicated of were used for quantitative real time PCR). Three individual plants were grown in each pot, one plant was used for measurements of leaf relative water content (RWC) (Barrs and Weatherley 1968), whereas the other two plants were used for sampling of flag leaf tissue for RNA extraction. Terminal drought stress (D) was applied at inflorescence emergence stage (Zadoks 50-60), (Zadoks et al. 1974), after emergence of 1-2 spikes in all biological replicates of both genotypes. The time from transplanting to inflorescence emergence stage was not significantly different between genotypes (70 days in the S genotype and 72 days in the R genotype). Drought stress application initiated after irrigation with excess amount of water in order to assure that all pots start the experiment at the same soil water capacity. Drought stress was imposed by withholding water for eight days until stress symptoms (i.e. leaf rolling and wilting symptoms) were visible in plants of both genotypes. Stress symptoms were more visible in the S genotype, however, leaf relative water content (RWC), measured after eight days of drought stress was low but was not significantly different between the two genotypes (49.68%±1.48 in the R genotype and 53.34%±1.83 in the S genotype). The well-watered control (C) plants were irrigated daily by ample amount of water. Flag leaf tissues of drought-stressed plants and well-watered control plants were harvested, immediately frozen in liquid nitrogen, and stored at -80ºC for RNA extraction. ****[PLEXdb(http://www.plexdb.org) has submitted this series at GEO on behalf of the original contributor, Krugman Tamar. The equivalent experiment is TA33 at PLEXdb.]
