Abscisic Acid Metabolism in Water-stressed Bean Leaves

Abstract

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...

Abscisic Acid Metabolism by a Cell-free Preparation from Echinocystis lobata Liquid Endoserum

“…The turnover of ABA to DPA appears to be a continuous process since DPA is present in both immature and mature seeds and its concentration far exceeds ABA in the mature seed. DPA also occurs endogenously in Echinocystis macrocarpa endosperm (2), and P. vulgaris leaves (5) and seeds (24), suggesting that metabolism plays a role in regulating the total level of ABA in these tissues as well as in E. lobata. We believe that the demonstration of a cell-free system is the first step in understanding the specific way in which ABA is metabolized.…”

“…The accumulation of DPA in bean seeds (24), as well as the continuous formation of PA and DPA in water-stressed leaves (5), indicates that this pathway has an active role in regulating total ABA concentration within the plant. The decreased activity of PA in bioassay systems for growth inhibition (9,22), abscission promotion (1,4), and stomatal closure (7) suggests that the DPA pathway has a role in regulating the hormonal activity of ABA.…”

“…The partially purified ABA and DPA were methylated (18) and further purified by TLC in hexane-ethyl acetate (1:1), and in 2-propanol-hexane (1:4) for ABA and DPA methyl esters, respectively. The ABA and DPA were quantified by gas chromatography as previously described (5).…”

Plant Cold Acclimation: The Role of Abscisic Acid

“…As ABA enters into the cell, the enzymes for its catabolism are induced and (+)ABA is oxidized to ()) phaseic acid (PA) or conjugated irreversibly with glucose to form a glucose ester (Oritani and Kiyota 2003). In wilted bean leaves the half-time of turnover of ABA to PA was about 3 h (Harrison and Walton 1975). Neither PA nor the ABA glucose ester was effective in inducing freezing tolerance in plant cells (Churchill and others 1994;Robertson and others 1994).…”

The effects of root cooling and excision treatments on the growth of primary leaves of Phaseolus vulgar is L.

“…The accumulation of ABA at the rate of 1-4 pmol mg d.wth ' as a result of induced leaf synthesis is plausible. Pierce & Raschke (1981) estimated the maximum rate of synthesis in terminal trifoliate leaflets of droughted bean plants to be 15 pmol mg~' d.wth ' and from the data of Harrison & Walton (1975) for two-week-old water-stressed primary leaves a rate of ABA synthesis of 3-8 pmol mg"' d.wt h ' can be calculated. Young expanding leaves of Xanthium accumulate ABA at an average rate of 5-5 pmol mg ' d .…”