The Mechanism of Abscisic Acid-induced Proline Accumulation in Barley Leaves

Abstract

When leaf blades of fully expanded second leaves of barley (cv. Prior) were excised and incubated with the cut end in a 20 milligram per liter solution of abscisic acid, they accumulated proline at the rate of about I micromole per hour per gram fresh weight after a 3-to 4-hour lag. This accumulation occurred reproducibly only if leaves were pretreated by placing the cut end in a solution consisting of 50 milhmlar sucrose and I millimolar glutamate. Treated leaves were taken from plants which had been in the light for 24 hours. Abscisic acid caused a stimulation of proline synthesis from glutamic acid. Proline oxidation rates were similar in leaves incubated in abscisic acid and in water even though the proline level in abscisic acid-treated leaves was 2.5 times the level in the water-treated controls. The incorporation of proline into protein was not affected by abscisic acid. These results are interpreted to indicate that the metabolc cause of abscisic acid-induced proline accumulation is a stimulation of proUne synthesis from glutamic acid. Inhibition of the utilzation of proUne by oxidation and protein synthesis does not contribute to proline accumulation the way it does in drought-stressed leaves. Soluble proline accumulates in plant tissues under several types of stresses including drought (4, 13, 16-19, and references cited therein), salinity (8), and low temperature (7). Treatment with ABA also causes proline to accumulate (2). The metabolic mechanism by which proline accumulates during drought stress has been studied in barley leaves which accumulate proline when subjected to any of these stresses and the results have been summarized (17). In excised leaves, proline accumulates mainly because its synthesis from glutamic acid is stimulated (4). Inhibition of proline oxidation also contributes to the accumulation, as does impaired utilization for protein synthesis (18, 19). ABA also accumulates in leaves under drought stress (20) and, in barley, ABA induces proline to accumulate in turgid leaves (2). The interaction between ABA treatment and PEG-induced water stress in barley has been studied and the results indicate that ABA-treated stressed plants accumulate more proline than those stressed without ABA (12). The time course for changes in ABA and proline levels in barley leaves during water stress and recovery from stress permit one to postulate that ABA could be the cause of proline accumulation under stress conditions (1). However, exogenous ABA does not cause proline to accumulate in turgid leaves of sunflower and tobacco, species which readily accumulate proline during drought stress (1). The experiments reported here were conducted to determine the metabolic basis for ABA-induced proline accumulation in barley leaves. The main effect of ABA on proline metabolism is stimulated synthesis from glutamate. MATERIALS AND METHODS Barley (Hordeum vulgare, cv. "Prior") seedlings were grown in soil in a growth chamber at 21 C and 2,500 ft-c. After emergence, plants were watered daily with modified Hoagland solution (9). Fully expanded second leaves were excised at the base of the leaf blade from 2-week-old plants which had been in the light for 24 h prior to excision. After excision, leaves were pretreated by placing the cut ends in a solution that was 50 mm sucrose and I mm glutamate. This pretreatment was found to be necessary for reproducible ABA-induced proline accumulation even though leaves were taken from plants which had been in the light for 24 h immediately preceding excision. Radioactive compounds,in 5 t1 of H20 were added through the cut end of the leaf. Details of the amount added and specific radioactivity are given in the figure legends. Leaves were incubated in vials with the cut end immersed in I ml of H20 (control) or I ml of 20 mg/l ABA. ABA was the (RS) cis-trans isomer obtained from Sigma Chemical Co. It was dissolved in H20 by continued stirring in darkness. Each measurement was an average of three replicate leaves. When "C-labeled precursors were fed, leaves were incubated for various times, killed by immersion in 95% v/v ethanol, and then thoroughly extracted with 80%o v/v ethanol. Extracts were dried, washed once with chloroform, and then taken up in water. After two-dimensional TLC of soluble compounds, spots were located by autoradiography and radioactivity was determined by liquid scintillation spectroscopy (18). When [5-3HJproline was added, leaves were rapidly frozen at the end of the incubation and crushed into I ml of ice-cold water. After 0.1-ml aliquots were taken for 3H20 recovery (11, 18), 10 ml of 95% v/v ethanol was added and the sample was extracted as described above. Tritiated water was recovered from 0.1 ml of the incubation solution. Proline was determined by a modification (13) of the Chinard method (6). RESULTS The time course for soluble proline accumulation in ABA-treated leaves is shown in Figure IA. Proline levels commenced increasing at 2 h and, after 4 h, increased at a rate of about 1 ,umol/g fresh weight-h. This rate is similar to the rate of accumulation of soluble proline in barley leaves wilted to 75% of their original fresh weight. Control leaves did not accumulate proline. Effects of ABA on the Incorporation of Precursors into Proline. The incorporation of radioactivity into proline from ['4CJgluta-mate is shown in Figure IB. There was a 20-fold stimulation of incorporation by ABA. This enhanced conversion represents stimulated synthesis because the specific radioactivity of the proline in the ABA-treated leaves was higher than in the control. At the 6-h time, the specific radioactivity of the ABA-treated leaves was