Proline: an essential intermediate in arginine degradation in Saccharomyces cerevisiae.

Results of studies on proline-nonutilizing (Put-) mutants of the yeast Saccharomyces cerevisiae indicate that proline is an essential intermediate in the degradation of arginine. Put- mutants excreted proline when grown on arginine or ornithine as the sole nitrogen source. Yeast cells contained a single enzyme, delta 1-pyrroline-5-carboxylate (P5C) dehydrogenase, which is essential for the complete degradation of both proline and arginine. The sole inducer of this enzyme was found to be proline. P5C dehydrogenase converted P5C to glutamate, but only when the P5C was derived directly from proline. When the P5C was derived from ornithine, it was first converted to proline by the enzyme P5C reductase. Proline was then converted back to P5C and finally to glutamate by the Put enzymes proline oxidase and P5C dehydrogenase.     

Arginine utilization by Chlorella autotrophica and Chlorella saccharophila

Chlorella saccharophila can utilize the amino acids arginine, glutamate. ornithine and proline as sole sources of nitrogen for growth. By comparison C. autotrophica utilized only arginine and ornithine. Following osmotic shock of Chlorella autotrophica from 50 to 150% artificial seawater rapid synthesis of proline (the main osmoregulatory solute in this alga) occurred in cells grown on arginine or citrulline. However, little proline synthesis occurred in ornithine-grown cells. Distribution of radiolabelled carbon from [¹⁴C]-arginine assimilation following osmotic shock of C. autotrophica agrees with the following pathway of arginine utilization: arginine→citrulline→ornithine→glutamate semialdehyde→pyrroline-5-carboxylate→proline. These 4 steps are catalysed by arginine deiminase (EC 3.5.3.6), citrullinase (EC 3.5.1.20), ornithine transaminase (EC 2.6.1.13) and pyrroline-5-carboxylate reductase (EC 1.5.1.2), respectively. Of these 4 enzymes, only arginine deiminase and pyrroline-5-carboxylate reductase were detected in the crude extract of the 2 Chlorella species. Arginine deiminase did not require specific cations for optimal activity. The deimi-nase showed maximal activity at pH 8.0 and followed Michaelis-Menten kinetics with an apparent K_m for L-arginine of 0.085 m M for the C. autotrophica enzyme and 0.097 m M for that of C. saccharophila. The activity of arginine deiminase was not influen-ced by growing C. saccharophila on arginine. Ornithine competitively inhibited arginine deiminase with an apparent K, of 2.4 m M for the C. autotrophica enzyme, and 3.8 m M for that of C. saccharophila . Arginine utilization by Chlorella is discussed in relation to that of other organisms.     

Regulation of proline catabolism in Pseudomonas aeruginosa PAO

Mutants of Pseudomonas aeruginosa deficient in the utilization of l-proline as the only carbon and nitrogen source have been found to be defective either in proline dehydrogenase activity or in both proline dehydrogenase and ¹-pyrroline-5-carboxylate dehydrogenase activities of the bifunctional proline degradative enzyme. The latter type of mutants was unable to utilize l-ornithine, indicating that a single ¹-pyrroline-5-carboxylate dehydrogenase activity is involved in the degradation of ornithine and proline. Proline dehydrogenase and ¹-pyrroline-5-carboxylate dehydrogenase activities were strongly and coordinately induced by proline. It was excluded that ¹-pyrroline-5-carboxylate acted as an inducer of the bifunctional enzyme and it was shown that the low level induction observed during growth on ornithine was due to the intracellular formation of proline. The formation of the proline degradative enzyme was shown to be subject to catabolite repression by citrate and nitrogen control.Abbreviations EMS Ethylmethane sulfonate - NG N-methyl-N-nitro-N-nitrosoguanidine - P Minimal medium P - Pro-DH Proline dehydro-genase - P5C ¹-Pyrroline-5-carboxylate - P5C-DH ¹-Pyrroline-5-carboxylate dehydrogenase     

Phenotypic and reversion analysis of a Salmonella typhimurium constructed to have an arginine codon at the hisG46 missense codon

Of the 6 single-base mutations that would be predicted to change the missense mutation hisG46 away from a proline codon in the Salmonella/microsome mutagen selection assay for histidine-independent revertants, only 5 have been observed. We have used site-specific mutagenesis to make the unobserved mutant [CCC (proline)----CGC (arginine)] codon in the Salmonella genome. Experiments with this arginine mutant demonstrate that, like bacteria containing the hisG46 mutation, bacteria with the arginine missense mutation are histidine auxotrophs which are capable of reversion to histidine independence. However, unlike the ATP phosphoribosyltransferase coded by the hisG46 his G gene (with a proline), the arginine mutant enzyme is partially active. This is indicated by a histidine-independent phenotype when the arginine hisG gene is present in multiple copies.     

Nutritional and metabolic interrelationships of arginine, glutamic acid and proline in the chicken.

Proline satisfies by a narrow margin the criterion for dietary essentially for the chick. It is estimated that the chick may synthesize 80-90% of the total proline needed for growth. Although the metabolism of arginine, ornithine and glutamic acid is expected to give rise to proline, dietary supplements to these amino acids are relatively ineffective in reducing the proline requirement of chicks. Studies of the efficacy of dietary ornithine for growth, and tracer studies using L-(5-3H)arginine indicate that the conversion of ornithine to proline in vivo is limited, and the amount of proline synthesized from arginine is but a small fraction of that needed for growth. The limiting processes in proline synthesis from glutamic acid and ornithine are not known. In Escherichia coli, where the biosynthetic pathway from glutamate to proline has been elucidated, a glutamate kinase, NADP-dependent delta1-pyrroline-5-carboxylic acid (P5C) dehydrogenase and P5C reductase catalyze proline synthesis. P5C reductase is present in the soluble fraction of chicken liver and kidney. An NADP-dependent P5C dehydrogenase activity has also been observed in this fraction of liver. Further studies are required to assess the importance of these enzymes in proline biosynthesis and to determine the limiting process in proline formation in the chicken.     

delta]1-Pyrroline-5-Carboxylate Dehydrogenase from Cultured Cells of Potato (Purification and Properties)

[delta]1-Pyrroline-5-carboxylate (P5C) dehydrogenase (EC 1.5.1.12), the second enzyme in the proline catabolic pathway and a catalyst for the oxidation of P5C to glutamate, was purified from cultured potato (Solanum tuberosum L. var Desiree) cells. Homogeneous enzyme preparations were obtained by a three-step procedure that used anion-exchange, adsorption, and substrate elution chromatography. A 1600-fold purification was achieved, with a recovery of one-third of the initial activity. The purified enzyme was characterized with respect to structural, kinetic, and biochemical properties. It appeared to be an [alpha]-4 tetramer with subunits of an apparent molecular mass of about 60 kD and had a mildly acidic isoelectric point value. Potato P5C dehydrogenase had Michaelis constant values of 0.11 and 0.46 mM for NAD+ and P5C, respectively. Although NAD+ was the preferred electron acceptor, NADP+ also yielded an unusually high rate, and thus was found to serve as a substrate. Maximal activity was observed at pH values in the 7.3 to 8.3 range, and was progressively inhibited by chloride ions, a finding that strengthens recent suggestions that hyperosmotic stress negatively modulates in vivo proline oxidation.     

A multitracer stable isotope quantification of the effects of arginine intake on whole body arginine metabolism in neonatal piglets

We have previously shown that deficient arginine intake increased the rate of endogenous arginine synthesis from proline. In this paper, we report in vivo quantification of the effects of arginine intake on total endogenous arginine synthesis, on the rates of conversion between arginine, citrulline, ornithine, and proline, and on nitric oxide synthesis. Male piglets, with gastric catheters for diet and isotope infusion and femoral vein catheters for blood sampling, received a complete diet for 2 days and then either a generous (+Arg; 1.80 g x kg(-1) x day(-1); n = 5) or deficient (-Arg; 0.20 g.kg(-1).day(-1); n = 5) arginine diet for 5 days. On day 7, piglets received a primed, constant infusion of [guanido-(15)N(2)]arginine, [ureido-(13)C;5,5-(2)H(2)]citrulline, [U-(13)C(5)]ornithine, and [(15)N;U-(13)C(5)]proline in an integrated study of the metabolism of arginine and its precursors. Arginine synthesis (micromol x kg(-1) x h(-1)) from both proline (+Arg: 42, -Arg: 74, pooled SE: 5) and citrulline (+Arg: 67, -Arg: 120; pooled SE: 15) were higher in piglets receiving the -Arg diet (P < 0.05); and for both diets proline accounted for approximately 60% of total endogenous arginine synthesis. The conversion of proline to citrulline (+Arg: 39, -Arg: 67, pooled SE: 6) was similar to the proline-to-arginine conversion, confirming that citrulline formation limits arginine synthesis from proline in piglets. Nitric oxide synthesis (micromol x kg(-1) x h(-1)), measured by the rate conversion of [guanido-(15)N(2)]arginine to [ureido-(15)N]citrulline, was greater in piglets receiving the +Arg diet (105) than in those receiving the -Arg diet (46, pooled SE: 10; P < 0.05). This multi-isotope method successfully allowed many aspects of arginine metabolism to be quantified simultaneously in vivo.     

Suppression of Proline Requirement of proA and proAB Deletion Mutants in Salmonella typhimurium by Mutation to Arginine Requirement

Eleven variants able to grow without proline (provided arginine was absent) were obtained by spontaneous mutation from Salmonella typhimurium LT7 proA and proAB deletion mutants. Suppression resulted from mutation at argG, which specifies N(alpha)-acetylornithine delta-transaminase. In the absence of exogenous arginine, deficiency of this enzyme would cause derepression of the arginine pathway and accumulation of N-acetylglutamic gamma-semialdehyde. N-acetylglutamic gamma-semialdehyde, if deacetylated, would produce glutamic gamma-semialdehyde, the proline precursor whose synthesis from glutamate is blocked in proA and proAB mutants. All of the mutants grew only slowly (some very slowly) if not supplied with arginine. Sonic-treated preparations of eight mutants had no measurable acetylornithine delta-transaminase activity, but those of the three mutants least dependent on arginine had 0.11, 0.28, and 1.48 of wild-type activity; presumably, their enzymes have low specific activity, at least in vivo. Phage P22 cotransduced argG and strA. Genetic analysis showed that the minor degree of arginine dependence of the mutant with greater than wild-type in vitro enzyme activity was a characteristic of its argG allele, not the result of modification of the argG phenotype by mutation elsewhere.     

Water and salt stress-induced alterations in proline metabolism of Triticum durum seedlings

Many plants accumulate proline as a non-toxic and protective osmolyte under saline or dry conditions. Its accumulation is caused by both the activation of its biosynthesis and inactivation of its degradation. We report here on the alterations induced by water and salt stress in the proline metabolism and amino acid content of 5-day-old seedlings of Triticum durum cv. Simeto. Most of the amino acids showed an increase with the induction of either stress, but proline increased more markedly than did other amino acids. We also measured the activities of two enzymes, Δ¹-pyrroline-5-carboxylate (P5C) reductase (EC 1.5.1.2) and proline dehydrogenase (EC 1.5.1.2), which are involved in proline biosynthesis and catabolism, respectively. The activity of P5C reductase was enhanced during both water and salt stress, while proline dehydrogenase was inhibited only during salt stress. The results indicate that synthesis de novo is the predominant mechanism in proline accumulation in durum wheat. Use of a cDNA clone that encodes P5C-reductase from Arabidopsis thaliana , showed no differences in the gene expression between controls and stressed plants, implying that the increase in enzyme activity is unrelated to the expression of this gene.     

Purification and properties of the bifunctional proline dehydrogenase/1-pyrroline-5-carboxylate dehydrogenase from Pseudomonas aeruginosa

Proline dehydrogenase/1-pyrroline-5-carboxylate dehydrogenase (Pro/P5C dehydrogenase), a bifunctional enzyme catalyzing the two consecutive reactions of the oxidation of proline to glutamic acid, was purified from Pseudomonas aeruginosa strain PAO1. Pro/P5C dehydrogenase oxidized L-proline in an FAD-dependent reaction to L-delta 1-pyrroline-5-carboxylic acid and converted this intermediate with NAD or NADP as cosubstrates to L-glutamic acid. The purification procedure involved DEAE-cellulose chromatography, affinity chromatography on Matrex gel red A and gel filtration on Sephadex G-200. It resulted, after 40-fold purification with 11% yield, in a homogeneous preparation (greater than 98% pure). The molecular weight of the single subunit was determined as 119,000. Gel filtration of purified Pro/P5C dehydrogenase yielded a molecular weight of 242,000 while polyacrylamide gel electrophoresis under native conditions led to the appearance of two catalytically active forms of the enzyme with molecular weights of 241,000 and 470,000. Manual Edman degradation revealed proline, alanine and aspartic acid as the N-terminal amino acid sequence. Pro/P5C dehydrogenase was highly specific for the L-forms of proline and delta 1-pyrroline-5-carboxylic acid. Its apparent Km values were 45 mM for L-proline, 0.03 mM for NAD and 0.17 mM for NADP. The saturation function for delta 1-pyrroline-5-carboxylic acid was non-hyperbolic.     

Arginine synthesis is regulated by dietary arginine intake in the enterally fed neonatal piglet

Arginine is conditionally indispensable in the neonate, and its synthesis in the intestine is not sufficient to meet requirements. It is not known how neonatal endogenous arginine synthesis is regulated and the degree to which proline and glutamate are used as precursors. Primed, constant intraportal and intragastric infusions of L-[U-14C]proline and L-[3,4-3H]glutamate, and intragastric L-[guanido-14C]arginine were used to measure whole body and first-pass intestinal arginine synthesis in 10 neonatal piglets fed generous (1.80 g.kg(-1).day(-1)) or deficient (0.20 g.kg(-1).day(-1)) quantities of arginine for 5 days. Glutamate tracer was not detected in arginine, indicating a biologically insignificant conversion of <1% of arginine flux. Endogenous arginine synthesis from proline had obligatory (0.36 g.kg(-1).day(-1)) and maximal (0.68 g.kg(-1).day(-1)) levels (P < 0.05, pooled SE 0.05). Although first-pass gut metabolism is responsible for 42-63% of whole body arginine synthesis, the gut is incapable of upregulating proline to arginine conversion during arginine deficiency, compared with a more than threefold increase without first-pass gut metabolism. These data suggest that upregulation of proline-to-arginine conversion occurs via increased arterial extraction of proline by the gut or in nonintestinal tissues. This study demonstrates that dietary arginine is an important regulator of endogenous arginine synthesis in the neonatal piglet and that proline, but not glutamate, is an important precursor for arginine synthesis in the neonate.     

Inhibition of yeast Δ1-pyrroline-5-carboxylate dehydrogenase by common amino acids and the regulation of proline catabolism

A yeast glutamate auxotroph (glt_{1 ? 1}), blocked in the tricarboxylic acid cycle at aconitase, is shown to possess catabolic pathways to glutamate from proline, arginine and glutamine, and grows on any of these amino acids in a minimal medium. This mutant does not, however, grow on these amino acids in a medium containing the full complement of common amino acids minus glutamate. The mechanism of this growth failure involves partial inhibition of the catabolic routes to glutamate by more than half the common amino acids. In the case of proline catabolism, this inhibition is localized principally at the enzyme Δ¹-pyrroline-5-carboxylate: NAD(P)⁺ oxidoreductase by in vitro studies. Similar results with this enzyme prepared both from yeast and from beef kidney mitochondria suggest that the inhibition observed may be the basis of a regulatory mechanism of general significance.     

Arginine synthesis does not occur during first-pass hepatic metabolism in the neonatal piglet

We have shown that first-pass intestinal metabolism is necessary for approximately 50% of whole body arginine synthesis from its major precursor proline in neonatal piglets. Furthermore, the intestine is not the site of increased arginine synthesis observed during dietary arginine deficiency. Primed constant intravenous (iv) and intraportal (ip) infusions of L-[U-14C]proline, and iv infusion of either L-[guanido-14C]arginine or L-[4,5-3H]arginine were used to measure first-pass hepatic arginine synthesis in piglets enterally fed either deficient (0.20 g.kg(-1).day(-1)) or generous (1.80 g.kg(-1).day(-1)) quantities of arginine for 5 days. Conversion of arginine to other urea cycle intermediates and arginine recycling were also calculated for both dietary treatments. Arginine synthesis (g.kg(-1).day(-1)) from proline was greater in piglets (P < 0.05) fed the deficient arginine diet in both the presence (generous: 0.07; deficient: 0.17; pooled SE = 0.01) and absence (generous: 0.06; deficient: 0.20; pooled SE = 0.01) of first-pass hepatic metabolism. There was no net arginine synthesis from proline during first-pass hepatic metabolism regardless of arginine intake. Arginine conversion to urea, citrulline, and ornithine was significantly greater (P < 0.05) in piglets fed the generous arginine diet. Calculated arginine fluxes were significantly lower (P = 0.01) for [4,5-3H]arginine than for [guanido-14C]arginine, and the discrepancy between the values was greater in piglets fed the deficient arginine diet (35% vs. 20%). Collectively, these findings show that first-pass hepatic metabolism is not a site of net arginine synthesis and that piglets conserve dietary arginine in times of deficiency by decreasing hydrolysis and increasing recycling.     

Characterization of the gene for Δ1-pyrroline-5-carboxylate synthetase and correlation between the expression of the gene and salt tolerance in Oryza sativa L.

A cDNA for ¹-pyrroline-5-carboxylate (P5C) synthetase (cOsP5CS), an enzyme involved in the biosynthesis of proline, was isolated and characterized from a cDNA library prepared from 14-day-old seedlings of Oryza sativa cv. Akibare. The deduced amino acid sequence of the P5CS protein (OsP5CS) from O. sativa exhibited 74.2% and 75.5% homology to that of the P5CS from Arabidopsis thaliana and Vigna aconitifolia, respectively. Northern blot analysis revealed that the gene for P5CS (OsP5CS) was induced by high salt, dehydration, treatment of ABA and cold treatment, while it was not induced by heat treatment. Simultaneously, accumulation of proline was observed as a result of high salt treatment in O. sativa. Moreover, the levels of expression of OsP5CS mRNA and content of proline under salt stress condition were compared between a salt-tolerant cultivar, Dee-gee-woo-gen (DGWG) and a salt-sensitive breeding line, IR28. It was observed that the expression of the P5CS gene and the accumulation of proline in DGWG steadily increased, whereas those in IR28 increased slightly.     

Selection and characterisation of mutants lacking arginase in Neurospora crassa

Summary A Neurospora mutant (aga) lacking arginase was selected by virtue of its inability to utilize arginine as a source of ornithine, using a strain in which ornithine was needed to satisfy a proline requirement. It mapped in linkage group VII (right arm), close to wc. The most important characteristic of the mutant was its extreme sensitivity to arginine. Inclusion of 1 mM arginine in the medium lead to a 40-fold increase in the arginine pool and a 90% inhibition of growth. This inhibition was relieved by the addition of ornithine or proline. The high arginine pool was associated with only a slight repression of two biosynthetic enzymes examined and with a five-fold induction of ornthine transaminase, the second enzyme of arginine catabolism. It is expected that the aga mutant will be of value in further work on the regulation of arginine biosynthesis in Neurospora.     

Arginine,ornithine, and proline interconversion is dependent on small intestinal metabolism in neonatal pigs

We have previously shown that arginine deficiency is exacerbated by the removal of dietary proline in orally, but not parenterally, fed piglets. Therefore, we hypothesized that the net interconversions of proline, ornithine, and arginine primarily occur in the small intestine of neonatal piglets. Ten intragastrically fed piglets received either intraportal (IP) or intragastric (IG) primed, constant infusions of [guanido-(14)C]arginine and [U-(14)C]ornithine + [2,3-(3)H]proline. By infusing amino acid isotopes via the stomach compared with the portal vein, we isolated small intestinal first-pass metabolism in vivo. During IP infusion, fractional net conversions (%) from proline to ornithine (0), ornithine to arginine (11 +/- 6), and ornithine to proline (5 +/- 1) were lower (P < 0.05) than during IG infusion (39 +/- 8, 18 +/- 6, and 42 +/- 12, respectively); we speculate that these data are due to the localization of ornithine aminotransferase to the gut. The balance of these conversions indicated a large synthesis of arginine (70.0 micromol. kg(-1). h(-1)) by the gut, with a corresponding degradation of ornithine (70.8 micromol. kg(-1). h(-1)) and no change in proline balance. Gut synthesis of arginine from proline (48.1 micromol. kg(-1). h(-1)) was 50% of its requirement, whereas proline synthesis from arginine (33.0 micromol. kg(-1). h(-1)) amounted to 10% of its requirement. Overall, arginine synthesis is more dependent on the gut than proline synthesis. In situations in which gut metabolism is compromised, such as during parenteral nutrition or gastrointestinal disease, arginine and proline are individually indispensable because their biosyntheses are negligible.     

Subcellular location of delta-pyrroline-5-carboxylate reductase in root/nodule and leaf of soybean

The expression of Δ¹-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 (AJ Delauney, DPS Verma [1990] 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 NaCl). 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.     

The CbrA-CbrB two-component regulatory system controls the utilization of multiple carbon and nitrogen sources in Pseudomonas aeruginosa

A novel two-component system, CbrA-CbrB, was discovered in Pseudomonas aeruginosa; cbrA and cbrB mutants of strain PAO were found to be unable to use several amino acids (such as arginine, histidine and proline), polyamines and agmatine as sole carbon and nitrogen sources. These mutants were also unable to use, or used poorly, many other carbon sources, including mannitol, glucose, pyruvate and citrate. A 7 kb EcoRI fragment carrying the cbrA and cbrB genes was cloned and sequenced. The cbrA and cbrB genes encode a sensor/histidine kinase (Mr 108 379, 983 residues) and a cognate response regulator (Mr 52 254, 478 residues) respectively. The amino-terminal half (490 residues) of CbrA appears to be a sensor membrane domain, as predicted by 12 possible transmembrane helices, whereas the carboxy-terminal part shares homology with the histidine kinases of the NtrB family. The CbrB response regulator shows similarity to the NtrC family members. Complementation and primer extension experiments indicated that cbrA and cbrB are transcribed from separate promoters. In cbrA or cbrB mutants, as well as in the allelic argR9901 and argR9902 mutants, the aot-argR operon was not induced by arginine, indicating an essential role for this two-component system in the expression of the ArgR-dependent catabolic pathways, including the aruCFGDB operon specifying the major aerobic arginine catabolic pathway. The histidine catabolic enzyme histidase was not expressed in cbrAB mutants, even in the presence of histidine. In contrast, proline dehydrogenase, responsible for proline utilization (Pru), was expressed in a cbrB mutant at a level comparable with that of the wild-type strain. When succinate or other C4-dicarboxylates were added to proline medium at 1 mM, the cbrB mutant was restored to a Pru+ phenotype. Such a succinate-dependent Pru+ property was almost abolished by 20 mM ammonia. In conclusion, the CbrA-CbrB system controls the expression of several catabolic pathways and, perhaps together with the NtrB-NtrC system, appears to ensure the intracellular carbon: nitrogen balance in P. aeruginosa.     

Oscillation and regulation of proline content by P5CS and ProDH gene expressions in the light/dark cycles in Arabidopsis thaliana L

The fluctuation of proline content, and protein and mRNA levels of delta1-pyrroline-5-carboxylate synthetase (P5CS) and proline dehydrogenase (ProDH), both of which are involved in proline biosynthesis and degradation, in the shoots of Arabidopsis grown in light/dark cycles were demonstrated under salt-stressed and unstressed conditions. Proline content, as well as proteins and mRNAs of these enzymes, clearly oscillated in the light/dark cycles under the stressed and unstressed conditions. A reciprocal relationship between P5CS and ProDH was observed. Protein levels of P5CS and ProDH were well synchronized with their mRNA levels, although the fluctuation of protein levels was not as significant as that of their mRNA levels. Both mRNA and protein levels of the two enzymes as well as the proline content did not oscillate under the continuous light or the dark conditions. Thus, P5CS and ProDH gene expressions seemed to be involved in light irradiation. Moreover, relative water content (RWC) in the plants oscillated in the light/dark cycles. The fluctuations of proline content in shoot reversely responded to that of RWC. It is suggested that the expression of two genes responds sensitively to a subtle change of cellular water status, and accumulated proline keeps the osmotic balance between cells and the outer environment.