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

Abstract

Tobacco (Nkotiana tabacum L. var Wisconsin 38) cells that are adapted to 428 millimolar NaCI accumulate proline mainly due to increased synthesis from glutamate. These cells were used to evaluate the possible role of A'-pyrroline-5-carboxylate reductase in the regulation of proline biosynthesis. No increase in the specific activity of A1-pyrroline-5-carboxylate reductase in crude extracts throughout the growth cycle was observed in NaCIadapted cells compared to unadapted cells. The enzyme from both cell types was purified extensively. On the basis of affinity for the substrates NADPH, NADH, and A1-pyrroline-5-carboxylate, pH profiles, chromatographic behavior during purification, and electrophoretic mobility of the native enzyme, the activities of the enzyme from the two sources were similar. These data suggest that the NaCI-dependent regulation of proline synthesis in tobacco cells does not involve induction of pyrroline-5-carboxylate isozymes or changes in its kinetic properties.Proline accumulation is proposed to be part of the process of osmotic adjustment that contributes to the cellular adaptation of many plant species to drought, salinity, and other stresses (9,16,26,27). Tracer studies and characterization of P5C2 reductase provide insights into the regulation of proline accumulation under osmotic stress. The metabolic causes of proline accumulation have been proposed to involve: (a) stimulation of synthesis from glutamate; (b) lowered rate of proline oxidation; and (c) slowed incorporation of proline into protein (9). Accelerated flux from glutamate to P5C (2, 3), and/or to proline (22) appears to be associated with water stress. Computer simulation analysis of ['5N]H4' incorporation into glutamine, glutamate, and proline revealed that the rate of proline synthesis from glutamate is increased in NaCl-

Overexpression of [delta]-Pyrroline-5-Carboxylate Synthetase Increases Proline Production and Confers Osmotolerance in Transgenic Plants

“…salt has been shown to increase the leve1 of P5CR, but this step does not seem to be rate limiting for Pro synthesis (LaRosa et al, 1991). We independently reached the Same conclusion by overexpressing soybean P5CR in transgenic tobacco (Szoke et al, 1992).…”

“…4, lane 8). That the reaction did not result in accumulation of P5C, instead of Pro, can be explained by the observation that P5C reductase is in excess and is not a rate-limiting factor in Pro biosynthesis in plants (LaRosa et al, 1991;Szoke et al, 1992). The partially purified P5CS from E.coli expressing Vigna P5CS cDNA catalyzed the production of P5C (Fig.…”

“…It was also found that the accumulation of C1-, K f , and Pro was concurrent with an increase in the "binding strength" of water in wheat leaves (Rascio et al, 1994). The primary function of the accumulation of these solutes may be the regulation of intracellular water activity (LaRosa et al, 1991). Both Pro and Gly betaine under water-stressed conditions may be able to induce the formation of strong H-bonded water around the protein, preserving the native state of the cell biopolymers (Rascio et al, 1994).…”

Molecular Cloning and Evidence for Osmoregulation of the Δ1-Pyrroline-5-Carboxylate Reductase (proC) Gene in Pea (Pisum sativum L.)

“…This situation is analogous to that in bacteria, in which proline synthesis is known to be regulated at the level of 'y-glutamyl kinase via proline feedback inhibition (9). In plants, however, only the last enzyme in the proline pathway, P5CR, has been characterized (7,19,21). Evidence for the first two enzymes of this pathway, -y-glutamyl kinase and y-glutamyl phosphate reductase, is not conclusive (21), and their role in the regulation of proline biosynthesis remains to be demonstrated.…”

“…The significance of this apparent osmoregulation of P5CR activity in these plants is unknown, especially because the activity of this enzyme is not modulated in response to osmotic adjustment in other plants (21). There are reports of kinetically distinguishable P5CR isozymes (18) and both cytosolic (18) and chloroplastic (26) forms of P5CR in plants.…”