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

Abstract

The expression of A'-pyrroline-5-carboxylate reductase (P5CR) gene was found to be higher in soybean root nodules than in leaves and roots, and its expression in roots appeared to be osmoregulated Mol Gen Genet 221: 299-305).P5CR was purified to homogeneity as a monomeric protein of 29 kilodaltons by overexpression of a soybean P5CR cDNA clone in Escherichia coli. The pH optimum of the purified P5CR was altered by increasing the salt concentration, and maximum enzyme activity was attainable at a lower pH under high salt (0.2-1 molar NaCI). Kinetic studies of the purified enzyme suggested that nicotinamide adenine dinucleotide phosphate' inhibited P5CR activity, whereas nicotinamide adenine dinucleotide+ did not. Subcellular fractionation and antibodies raised against purified soybean P5CR were used to investigate location of the enzyme in different parts of soybean as well as in leaves of transgenic tobacco plants synthesizing soybean P5CR. P5CR activity was present in cytoplasm of soybean roots and nodules as well as in leaves, but in leaves, about 15% of the activity was detected in the plastid fraction. The location of P5CR was further confirmed by western blot assay of the proteins from cytosol and plastid fractions of different parts of the plant. Expression of soybean nodule cytosolic P5CR in transgenic tobacco under the control of cauliflower mosaic virus 35S promoter led to the accumulation of this protein exclusively in the cytoplasm, suggesting that the chloroplastic activity may be due to the presence of a plastid form of the enzyme. The different locations of P5CR in root and leaf suggested that proline may be synthesized in different subcellular compartments in root and leaf. Proline concentration was not significantly increased in transgenic plants exhibiting high level P5CR activity, indicating that reduction of P5C is not a rate-limiting step in proline production.Proline accumulation is one of the adaptations of plants to salinity and water deficit (4,22,34). The proline biosynthetic pathway in plants and its regulation in response to osmotic stress is, however, not well understood. A precise determination of the proline pathway and its compartmentalization in different tissues is essential not only for understanding the regulation of proline production, but also for establishing the role of this amino acid in conferring tolerance to salt and drought stresses. The proline pathway in plants has been proposed to be analogous to that in bacteria (7, 21), although proline can also be made from omithine (1).

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

“…We independently reached the Same conclusion by overexpressing soybean P5CR in transgenic tobacco (Szoke et al, 1992). We also demonstrated that the synthesis of Pro from Orn in plants proceeds via the 8-transamination of Orn to P5C and subsequent reduction to pro, and we have isolated a c~~~ clone encoding OAT.…”

“…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.…”

“…Corresponding to the level of expression of P5CS, Pro contents were increased in transgenic lines 22, 88, 136, and 162. Because overproduction of P5CR does not increase Pro content in transgenic plants (Szoke et al, 1992), a direct correlation between P5CS expression level and Pro accumulation in the transgenic plants clearly suggests that P5CS, catalyzing the first two steps in the pathway, is rate limiting in Pro biosynthesis.…”

“…The products (2 /nL of the mixture) were resolved by TLC on a silica gel (Analtech, Inc., Newark, DE). P5C was prepared as described earlier (Szoke et al, 1992). Glu, Gin, Pro, and P5C (1 /j,g of each) were used as reference markers.…”

Comparative Analysis of the Regulation of Expression and Structures of Two Evolutionarily Divergent Genes for Δ1-Pyrroline-5-Carboxylate Synthetase from Tomato

“…The observation that salinity or dehydration stress stimulated the accumulation of the transcripts for the Pro-biosynthetic genes has been interpreted to mean that the transcriptional control of these genes is important for the regulation of Pro synthesis by osmotic stress. However, 50-fold overproduction of P5C reductase in transgenic tobacco plants did not lead to increased Pro accumulation (Szoke et al, 1992), indicating that the much smaller induction of P5C reductase in NaCl-stressed plants is not likely to be of significance for the Pro accumulation.…”