Determination of P5C reductase activity in the proline mutant

The biosynthesis of proline in all organisms so far studied proceeds from either glutamic acid or ornithine via glutamic-g-semialdehyde which is in equilibrium with D'-pyrroline-5-carboxylic acid (P5C) (H. J. Vogel and B. D. Davis, J. Am. Chem. Soc., 74:109, 1952). P5C is metabolized to proline by P5C reductase, while proline degradation is accomplished by proline dehydrogenase (Mazelis M. and L. Fowden, J. Exp. Bot. 22:137, 1971) and proline oxidase (Boggess, Koeppe and Stewart, Plant Physiol. 63:22-25, 1978).

In our laboratory we isolated a proline requiring mutant, pro (Gavazzi, Racchi and Tonelli, Theoret. Appl. Genet. 46:339, 1975); that is seemingly the result of a genetic block between P5C and proline (Racchi, Gavazzi, Monti and Manitto, Plant Science Letters in press). Accordingly we analyzed the enzyme involved in this biosynthetic step.

The enzyme was extracted from embryos of homozygous pro-1, pro-2 and pro-3 seeds (Racchi, Gavazzi this M.N.L.) previously soaked in water for 16 hours and from callus of homozygous pro-3 mutant grown on Murashige and Skoog medium supplemented with proline (2mM). The enzymatic assay was carried out following the procedure of Noguchi, Koiwai and Tamaki (Agr. Biol. Chem. 30: 452-456, 1966). An enzyme unit is defined as the enzyme amount which produces a decrease in optical density of 0.001 per minute at 340 nm. Soluble proteins were estimated by the method of J. Sedmak (Anal. Bioch. 79:544-552, 1977). ?-pyrroline-5-carboxylate was prepared by the method of H. J. Strecker (J. Biol. Chem. 235:2045, 1960).

The results (Table 1) indicate that P5C reductase is present in both normal and mutant embryos; the mutant rates of pro-2 and pro-3 are higher than the normal. The enzymatic activity of pro-3/pro-3 and nonmutant callus parallels that found in the embryo tissues (Table 2). Presence of enzymatic activity in mutant tissues is in contrast with the hypothesis of a genetic block between P5C and proline. In view of these results the P5C reductase of normal and mutant tissues should be further characterized in terms of Km, pH optimum and so on. Such studies are under way.

Table 1.

Table 2.

Chiara Tonelli and Alcide Bertani

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