Changes in the Levels of Abscisic Acid and Its Metabolites in Excised Leaf Blades of Xanthium strumarium during and after Water Stress

Abstract

The time course of abscisic acid (ABA) accumulation during water stress and of degradation following rehydration was investigated by analyzing the levels of ABA and its metabolites phaseic acid (PA) mulation and of the decline in ABA content after removal of the stress condition. To avoid complications with export from leaves on intact plants, all work was conducted with excised leaf blades. In such a system, the disappearance of ABA during recovery from stress must be accomplished by the formation of one or more ABA metabolites. To determine via which pathway(s) ABA was metabolized after relief of stress, the leaf samples were analyzed for ABA, PA2, and alkali-hydrolyzable conjugated ABA. A reverse-phase HPLC system was employed with which all three compounds could be isolated simultaneously from crude leaf extracts. MATERIALS AND METHODSPLANT MATERIAL Xanthium strumarium L., Chicago strain, was grown in a greenhouse under a photoperiod of 20 h to keep the plants in the vegetative state. Environmental conditions and growing procedures were the same as described earlier (24). During the 24-h period preceding an experiment, the plants were watered at least four times to keep the leaves fully turgid.To determine the effect of wilting on ABA accumulation, the youngest fully expanded leaf blade, hereafter called leaf, was excised from each plant. This leaf had a length along the midrib between 11 and 12 cm, and was called leaf size 4 in previous work (24). Excised leaves were wilted by exposing them to a stream of warm air from a fan until they had lost 10% of their fresh weight, which required up to 30 min. The wilted leaves were kept in plastic bags in darkness at 22 C for the desired periods of time. Nonwilted leaves were either harvested immediately or kept in plastic bags. For recovery from stress, leaves were submerged in distilled H20 for 5 min. Afterwards, they were held vertically for a few seconds to allow H20 to drain from the surface and then returned to plastic bags. Harvested leaves were frozen in liquid N2, pulverized, and stored at -20 C until used.In most experiments, there were three leaves per treatment. In some experiments, only one leaf was used per treatment; in this case, various treatments were replicated three times. EXTRACTION PROCEDUREFrozen samples were extracted three times at 4 C with acetone containing 1% (v/v) glacial acetic acid and 100 mg/l of the antioxidant 2,6-di-tert-butyl-4-methylphenol (19

Ethylene-Induced Flavonol Accumulation in Guard Cells Suppresses Reactive Oxygen Species and Moderates Stomatal Aperture

“…In response to environmental stress, the plant hormone ABA signals stomatal closure (Zeevaart, 1980;Hirayama and Shinozaki, 2007;Cho et al, 2009;Joshi-Saha et al, 2011). This signaling pathway employs H 2 O 2 as a ROS second messenger (McAinsh et al, 1996;Murata et al, 2001;Zhang et al, 2001;Kwak et al, 2003).…”

“…Guard cells therefore must modulate the stomatal aperture in response to the changing environment (Hirayama and Shinozaki, 2007). In response to stimuli that close stomata, such as drought stress, the hormone abscisic acid (ABA) is increased in the cytosol of guard cells (Zeevaart, 1980;Hirayama and Shinozaki (2007)). ABA induces ion movements across the cell membrane expelling K + and Cl 2 ions through ion efflux channels.…”

Effects of Cycloheximide on Abscisic Acid Biosynthesis and Stomatal Aperture in Bean Leaves

“…This increase is due to an increased rate of ABA biosynthesis (5,12) and can be blocked by inhibitors of cytoplasmic protein synthesis such as CH3 and puromycin (2,7,8), as well as inhibitors of transcription such as actinomycin D and cordycepin (2,8). Boggess and Stewart (1) reported that CH caused sunflower leaves to wilt, and Quarrie and Lister (7) reported a similar response in wheat leaves after a few hours of treatment.…”

Cell-Wall Proteins Induced by Water Deficit in Bean (Phaseolus vulgaris L.) Seedlings

“…In addition, increases in the content of the growth regulator ABA have been reported in a number of plant species subjected to water deficit (Hsiao, 1973;Walton et al, 1977;Walton, 1980;Hartung and Davies, 1991). A substantial number of drought effects on plants can be mimicked by application of ABA (Jones and Mansfield, 1972;Hsiao, 1973;Walton et al, 1977;Walton, 1980;Zeevart, 1980;Davies and Mansfield, 1983;Bensen et al, 1988;Creelman et al, 1990;Hartung and Davies, 1991). These and other observations have suggested that ABA may act as an in vivo effector of some metabolic events during the response of the plant to water deficit.…”