Pyrroline-5-Carboxylate Reductase in Chlorella autotrophica and Chlorella saccharophila in Relation to Osmoregulation

Abstract

Pyrroline-5-carboxylate (P5C) reductase (EC 1.5.1.2), which catalyzes the reduction of P5C to proline, was partially purified from two Chlorella species; Chlorella autotrophica, a euryhaline marine alga that responds to increases in salinity by accumulating proline and ions, and Chlorella saccharophila, which does not accumulate proline for osmoregulation. From the elution profile of this enzyme from an anion exchange column in Tris-HCI buffer (pH 7.6), containing sorbitol and glycine betaine, it was shown that P5C reductase from C. autotrophica was a neutral protein whereas the enzyme from C. saccharophila was negatively charged. The kinetic mechanisms of the reductase was characteristic of a ping-pong mechanism with double competitive substrate inhibition. Both enzymes showed high specificity for NADH as cofactor. The affinities of the reductases for their substrates did not change when the cells were grown at different salinities. In both algae, the apparent Km values of the reductase for P5C and NADH were 0.17 and 0.10 millimolar, respectively. A fourfold increase in maximal velocity of the reductase was observed when C. autotrophica was transferred from 50 to 150% artificial sea water. Even though the reductase was inhibited by NaCI, KCI, and proline, it still showed appreciable activity in the presence of these compounds at molar concentrations. A possible role for the regulation of proline synthesis at the step catalyzed by P5C reductase is discussed in relation to the specificity of P5C reductase for NADH and its responses to salt treatments.A-Pyrroline-5-carboxylate reductase (L-Proline:NAD(P)-5-oxidoreductase, EC 1.5.1.2) catalyzes the final step in the biosynthetic pathway leading from glutamic acid to proline. The enzyme has been partially purified and characterized for many higher plants (7,9,13,17,21,23). None ofthese studies suggest a major role for P5C2 reductase in the regulation of proline biosynthesis. However, the activity of P5C reductase has been shown to increase 3.5-fold when seedlings of Pennisetum typhoides were grown in the presence of 17 mm NaCl with a concomitant increase in proline concentration (7). More recently, Treichel (23) (15). In these cells, glucose-6-phosphate dehydrogenase, which catalyzes the rate-limiting step of the pentose phosphate pathway, is dependent on the availability of NADP+ and is inhibited by NADPH. It is believed that P5C reductase by reducing P5C to proline with NADPH as cofactor serves to furnish NADP+ to glucose-6-phosphate dehydrogenase for the synthesis of purine nucleotides. Recently, it has been shown that in soybean nodules P5C reductase can indeed play an important role for the support of purine biosynthesis in addition to producing proline for incorporation into protein (8). In both erythrocytes and soybean nodules the P5C reductases are characterized by a higher affinity for NADPH than NADH, and a sensitivity to inhibition by NADP+ but not by proline. Finally, as pointed out by Bellinger and Larher (2), in glycophytes and in some halophyt...

Elevated Accumulation of Proline in NaCl-Adapted Tobacco Cells Is Not Due to Altered Δ1-Pyrroline-5-Carboxylate Reductase

“…3C, Table IV). P5C reductase from cultured cells had a smooth, broad response to changes in pH, which compares favorably to the response observed for the enzyme from tobacco leaves and other plant sources (12,14,17,25). In contrast to other reports (8,19), no major shoulders or dual pH optima were apparent.…”

“…Treichel (28) proline biosynthesis in cultured cells of Mesembryanthemum nodiflorum because ofthe observation that NaCl-adapted cells had increased P5C reductase activity. In Chlorella autotrophica, which accumulates proline in response to salt treatment, a possible role for the regulation of proline synthesis at the step catalyzed by P5C reductase was suggested by the fourfold increase in maximal velocity of this enzyme (12). A nearly sixfold increase in relative abundance of soybean P5C reductase mRNA was observed in soybean seedling roots subjected to short-term salinization (6).…”

“…Also, because P5C reductase may be a branch point between the pathway from glutamate and the pathway to and from arginine, P5C reductase seems a logical control point. A fourfold increase in the maximal velocity of P5C reductase concomitant with proline accumulation in the halophytic alga Chlorella autotrophica and the halophytic higher plant Mesembryanthemum nodiflorum L. corresponding to osmotic adjustment to salinization has led to suggestions that P5C reductase plays some role in the regulation of the proline biosynthetic pathway (12,28). Tobacco cells adapted to 428 mm NaCl are analogous to some halophytes in the sense that the cells accumulate proline probably via an increased conversion of glutamate (1,21).…”

Subcellular Location of Δ1-Pyrroline-5-Carboxylate Reductase in Root/Nodule and Leaf of Soybean

“…Analysis of P5CR activity in soybean leaves further revealed that the bulk of this activity is present in the cytosolic fraction (Table IV), with about 15% of P5CR activity detected in the chloroplast fraction. The activity of a known chloroplast marker enzyme, TPDH, was found exclusively in the chloroplast preparation (Table IV (2,18,32) as proline accumulates (10,19). By isolating genes encoding enzymes involved in biosynthesis, we have demonstrated that both routes are operational in higher plants (14).…”

Photosynthetic and Growth Responses of Olive to Proline and Salicylic Acid under Salinity Condition

“…In addition, root vegetative characteristics such as root dry weight and root volume were increased by Pro application in both saline and non-saline conditions. Increased activity of P5CR and Pro accumulation has been reported in Chlorella (Laliberté and Hellebust, 1989) and in NaCl-adapted cells of Mesembryanthemum (Treichel, 1986). Accumulation of Pro in the leaf helps plant to cope with abiotic stress conditions.…”