Phaseic acid (PA) and dihydrophaseic acid (DPA) are the major metabolites observed when (S)-2-14C-abscisic acid (ABA) is fed to 14-day excised primary bean leaves (Phaseolus vulgaris L. cv. Red Kidney). The distribution of 14C in leaves which were wilted after feeding ABA appears to be the same as that observed in unwilted leaves. A reduction in the relative specific radioactivities of the two metabolites after wilting, compared with the specific radioactivities measured in unwilted plants, indicated that these metabolites continue to be formed endogenously after wilting. Estimates of the endogenous ABA levels showed that they rose from 0.04,ug to approximately 0.5 pug/g fresh weight within 4 hours after the beginning of a 10% wilt and remained at that level during a subsequent 20 hours of wilt. In unwilted leaves, the levels of PA and DPA were 5 times and 20 times higher than that of ABA, respectively. Both PA we undertook the work described in this paper to determine the effects of water stress on the metabolism of ABA in leaves.
MATERIALS AND METHODSSeeds of Phaseolus vulgaris L. cv. Red Kidney were germinated in vermiculite for 3 days, then transferred to a well aerated solution containing 0.5 g/l Hyponex 7:6:19 all purpose plant food (Hydroponic Chemical, Copley, Ohio) to which 2 mg/l FeCl3 were added. The seedlings were grown in a growth chamber at 25 C under 16-hr days with the relative humidity maintained between 65 and 85%. The primary leaves of 14-day seedlings were excised at the base of the petiole under degassed water, and the excised leaves were kept in water overnight in a humid chamber prior to use.The leaves were allowed to take up (S)-2-'4C-ABA (specific radioactivity 5 ,iCi/l,mole) (10), contained in 0.2 ml of 10 mm K phosphate buffer, pH 7, through the cut petiole. The ABA was administered so that the leaves took up approximately 0.25 ,ug/g leaf fresh weight. After uptake of the feeding solution and two subsequent 0.2-ml buffer washes had been completed (about 1 hr), the leaves were transferred to degassed water for a period of several hours. Leaves to be wilted were removed from the water, placed under a fan until a 10% reduction in fresh weight had been attained, wrapped in aluminum foil, and stored at 25 C until they were extracted. Unwilted control leaves were wrapped in foil after an identical prelabeling period and also maintained at 25 C until extracted. Each sample consisted of a pair of leaves (4-5 g fresh weight). Extraction and Purification of Radioactive Compounds. Leaves were frozen in dry ice and then macerated with a VirTis homogenizer in 20 ml of ice-cold 80% methanol/g of leaf fresh weight. The macerate was stirred overnight in the cold, the methanol was removed by filtration, and the residue was extracted for 4 additional hr with 10 ml methanol g fresh weight. The combined methanolic extracts were reduced to a small aqueous volume in vacuo at 25 C, and the insoluble material was removed by centrifugation. The supernatant was adjusted to pH 3.5 with HCl and extract...