The Effect of Wilting on Proline Metabolism in Excised Bean Leaves in the Dark

Abstract

The effects of wilting on the fate of proline and on the rates of nonprotein proline formation and utilization have been determined in excised bean leaves. Wilting did not alter the fate of exogenously added 14C-L-proline (2 mM) in either non-starved leaves (from plants previously in the light) or starved leaves (from plants previously in the dark). The fate of proline in nonstarved leaves was protein synthesis and in starved leaves was protein synthesis and oxidation to other compounds. Wilting caused an increase in non-protein proline formation, possibly including release by proteolysis and synthesis from precursors in both starved and nonstarved leaves. Wilting caused a decrease in proline utilization in nonstarved leaves by decreasing protein synthesis. In starved leaves, wilting caused an increase in the rate of proline utilization but this is due to the higher content of proline in wilted leaves compared to the turgid leaves which causes more proline utilization by oxidation. Thus, the primary effects of wilting which lead to the accumulation of proline were to decrease protein synthesis and to increase proline formation. The source of the proline is not known but the increased formation due to wilting is not affected by the carbohydrate content of the leaf. The role of carbohydrates is to prevent the loss of accumulating proline by oxidation. It has been shown (1, 6, 9, 11) that nonprotein proline accumulates in wilted leaves except when the leaves have a low carbohydrate content (9). The amount of nonprotein proline in a tissue is determined by the relative rates of formation and utilization. Proline formation occurs primarily by proteolysis and by de novo synthesis. Proline is utilized mainly in protein synthesis and by oxidation. Since the increase in proline during wilting exceeds the proline released from protein (11) de novo synthesis must account for the increase in nonprotein proline. Also since proline oxidation is inhibited (5, 8) when carbohydrates are present, protein synthesis is responsible for proline utilization when proline accumulates. Thus proline accumulation could be due to an increase in de novo proline synthesis or a decrease in protein synthesis or both. This paper demonstrates that in water stress there is an increase in proline synthesis and a decrease in protein synthesis. Further, the in-' Supported by Iowa State University Research Foundation. creased proline synthesis does not require the presence of high levels of carbohydrates. MATERIALS AND METHODS Most of the methods used in these experiments have been described (7, 8). Fully expanded primary leaves of bean (Phaseolus vulgaris L. var. Tendergreen) were used. Starved leaves were from plants which had been in the dark for 48 hr. Nonstarved leaves were from plants which had been in the light (2500 ft-c) for 16 hr or more. Wilting, sampling, and incubation at a constant water content have been described (7). Addition of metabolites by vacuum infiltration, collection of "4CO2, extraction, fractionation, chromatography of amino acids, and determination of radioactivity have been described (4, 8, 10). Proline was determined by the method of Chinard (2). The rate of proline utilization (,moles/hr-g fresh weight) was calculated from the rate of loss of 14C from nonprotein proline (cpm/hr g fresh weight) divided by the specific radioactivity of the nonprotein proline (counts/min-umole). The rate of change in nonprotein proline content (/imoles/ hr g fresh weight) was calculated from the slope of the time course of changes in nonprotein proline content. The rate of proline formation (t,moles/hr g fresh weight) was calculated from the following formula: rate of change in nonprotein proline = rate of formation minus rate of utilization. RESULTS The fate of exogenously added 2 mm proline in turgid (A) and wilted (B) nonstarved leaves is shown in Figure 1. As shown previously (8), proline was incorporated into protein in nonstarved leaves and its oxidation to other amino acids, organic acids, and CO, was minimal due to the presence of carbohydrates in the leaves. Wilting did not affect the fate of exogenously added proline qualitatively but did quantitatively. The organic acid fraction is omitted from Figure 1 because it contained 2% or less of the total 14C recovered and the other amino acids are omitted from Figure lB because they contained 1 % or less of the total 14C recovered. Figure 2 shows the fate of exogenously added 2 mm`4Cmm`4C-proline for turgid (A) and wilted (B) starved leaves. In starved leaves, the oxidation of proline to other amino acids, organic acids, and CO2 represented a major fate of metabolized pro-line in addition to the incorporation into protein. Wilting did not alter the fate of added "4C-proline in these leaves but decreased the rate (see below). The effect of wilting on the nonprotein proline content during incubation after adding 2 mm proline to nonstarved leaves is shown in Figure 3A. In the turgid leaves, there was a gradual decrease in proline throughout the incubation. In the wilted 508