Interaction of an Antinucleating Chemical and an Ice Nucleation Active Bacterium: a Case Study with an n-Octylbenzyldimethyl-ammonium Salt and Erwinia ananas

Natto is a traditional Japanese food made from soybeans fermented by strains of Bacillus subtilis natto. It gives off a strong ammonia smell during secondary fermentation, and the biochemical basis for this ammonia production was investigated in this study. When natto was fermented by strain r22, ammonia production was shown to involve degradation of soybean proteins releasing amino acids, and only the glutamate contained in the natto obviously decreased, while the other amino acids increased during secondary fermentation. Strain r22 has two active glutamate dehydrogenase genes, rocG and gudB, and inactivating both genes reduced ammonia production by half, indicating that deamination of glutamate was one of the major ammonia-releasing reactions. In addition, urease encoded by ureABC was found to degrade urea during secondary fermentation. A triple mutant lacking rocG, gudB, and ureC exhibited minimal ammonia production, suggesting that the degradation of urea might be a further ammonia-releasing reaction.     

Metabolism of Proline, Glutamate, and Ornithine in Proline Mutant Root Tips of Zea mays (L.)

In excised pro_1-1 mutant and corresponding normal type roots of Zea mays L. the uptake and interconversion of [¹⁴C]proline, [¹⁴C]glutamic acid, [¹⁴C]glutamine, and [¹⁴C]ornithine and their utilization for protein synthesis was measured with the intention of finding an explanation for the proline requirement of the mutant. Uptake of these four amino acids, with the exception of proline, was the same in mutant and normal roots, but utilization differed. Higher than normal utilization rates for proline and glutamic acid were noted in mutant roots leading to increased CO₂ production, free amino acid interconversion, and protein synthesis. Proline was synthesized from either glutamic acid (or glutamine) or ornithine in both mutant and normal roots; it did not accumulate but rather was used for protein synthesis. Ornithine was not a good precursor for proline in either system, but was preferentially converted to arginine and glutamine, particularly in mutant roots. The pro_1-1 mutant was thus not deficient in its ability to make proline. Based on these findings, and on the fact that ornithine, arginine, glutamic acid and aspartic acid are elevated as free amino acids in mutant roots, it is suggested that in the pro_1-1 mutant proline catabolism prevails over proline synthesis.     

Selection-Driven Accumulation of Suppressor Mutants in Bacillus subtilis: The Apparent High Mutation Frequency of the Cryptic gudB Gene and the Rapid Clonal Expansion of gudB+ Suppressors Are Due to Growth under Selection

Soil bacteria like Bacillus subtilis can cope with many growth conditions by adjusting gene expression and metabolic pathways. Alternatively, bacteria can spontaneously accumulate beneficial mutations or shape their genomes in response to stress. Recently, it has been observed that a B. subtilis mutant lacking the catabolically active glutamate dehydrogenase (GDH), RocG, mutates the cryptic gudB^CR gene at a high frequency. The suppressor mutants express the active GDH GudB, which can fully replace the function of RocG. Interestingly, the cryptic gudB^CR allele is stably inherited as long as the bacteria synthesize the functional GDH RocG. Competition experiments revealed that the presence of the cryptic gudB^CR allele provides the bacteria with a selective growth advantage when glutamate is scarce. Moreover, the lack of exogenous glutamate is the driving force for the selection of mutants that have inactivated the active gudB gene. In contrast, two functional GDHs are beneficial for the cells when glutamate was available. Thus, the amount of GDH activity strongly affects fitness of the bacteria depending on the availability of exogenous glutamate. At a first glance the high mutation frequency of the cryptic gudB^CR allele might be attributed to stress-induced adaptive mutagenesis. However, other loci on the chromosome that could be potentially mutated during growth under the selective pressure that is exerted on a GDH-deficient mutant remained unaffected. Moreover, we show that a GDH-proficient B. subtilis strain has a strong selective growth advantage in a glutamate-dependent manner. Thus, the emergence and rapid clonal expansion of the active gudB allele can be in fact explained by spontaneous mutation and growth under selection without an increase of the mutation rate. Moreover, this study shows that the selective pressure that is exerted on a maladapted bacterium strongly affects the apparent mutation frequency of mutational hot spots.     

Mycobacterium tuberculosis Is a Natural Ornithine Aminotransferase (rocD) Mutant and Depends on Rv2323c for Growth on Arginine

Mycobacterium tuberculosis (Mtb) possesses a genetic repertoire for metabolic pathways, which are specific and fit to its intracellular life style. Under in vitro conditions, Mtb is known to use arginine as a nitrogen source, but the metabolic pathways for arginine utilization have not been identified. Here we show that, in the presence of arginine, Mtb upregulates a gene cluster which includes an ornithine aminotransferase (rocD) and Rv2323c, a gene of unknown function. Isotopologue analysis by using ¹³C- or ¹⁵N-arginine revealed that in Mtb arginine is not only used as nitrogen source but also as carbon source for the formation of amino acids, in particular of proline. Surprisingly, rocD, which is widespread in other bacteria and is part of the classical arginase pathway turned out to be naturally deleted in Mtb, but not in non-tuberculous mycobacteria. Mtb lacking Rv2323c showed a growth defect on arginine, did not produce proline from arginine, and incorporated less nitrogen derived from arginine in its core nitrogen metabolism. We conclude that the highly induced pathway for arginine utilization in Mtb differs from that of other bacteria including non-tuberculous mycobacteria, probably reflecting a specific metabolic feature of intracellular Mtb.     

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.     

Catabolic pathway of arginine in Anabaena involves a novel bifunctional enzyme that produces proline from arginine

Arginine participates widely in metabolic processes. The heterocyst‐forming cyanobacterium Anabaena catabolizes arginine to produce proline and glutamate, with concomitant release of ammonium, as major products. Analysis of mutant Anabaena strains showed that this catabolic pathway is the product of two genes, agrE (alr4995) and putA (alr0540). The predicted PutA protein is a conventional, bifunctional proline oxidase that produces glutamate from proline. In contrast, AgrE is a hitherto unrecognized enzyme that contains both an N‐terminal α/β propeller domain and a unique C‐terminal domain of previously unidentified function. In vitro analysis of the proteins expressed in Escherichia coli or Anabaena showed arginine dihydrolase activity of the N‐terminal domain and ornithine cyclodeaminase activity of the C‐terminal domain, overall producing proline from arginine. In the diazotrophic filaments of Anabaena, β‐aspartyl‐arginine dipeptide is transferred from the heterocysts to the vegetative cells, where it is cleaved producing aspartate and arginine. Both agrE and putA were found to be expressed at higher levels in vegetative cells than in heterocysts, implying that arginine is catabolized by the AgrE‐PutA pathway mainly in the vegetative cells. Expression in Anabaena of a homolog of the C‐terminal domain of AgrE obtained from Methanococcus maripaludis enabled us to identify an archaeal ornithine cyclodeaminase.     

Metabolism of arginine in lactating rat mammary gland.

Significant activities of the four enzymes needed to convert arginine into proline and glutamate (arginase, ornithine aminotransferase, pyrroline-5-carboxylate reductase and pyrroline-5-carboxylate dehydrogenase) develop co-ordinately in lactating rat mammary glands in proportion to the increased production of milk. No enzymes were detected to carry out the reactions of proline oxidation or reduction of glutamate to pyrroline-5-carboxylate. Minces of the gland converted ornithine into proline and into glutamate plus glutamine. These conversions increased during the cycle of lactation in proportion to the increased milk production and to the content of the necessary enzymes. The minced gland did not convert labelled ornithine into citrulline, confirming the absence from the gland of a functioning urea cycle, and did not convert labelled proline or glutamate into ornithine. A metabolic flow of labelled arginine to proline and glutamate in mammary gland was confirmed in intact animals with experiments during which the specific radioactivity of proline in plasma remained below that of the proline being formed from labelled arginine within the gland. It was concluded that arginase in this tissue had a metabolic role in the biosynthesis of extra proline and glutamate needed for synthesis of milk proteins.     

Metabolism of absorbed aspartate,asparagine, and arginine by rat small intestine in vivo

l-[U-¹⁴C]aspartate, l-[U-¹⁴C]asparagine, and l-[U-¹⁴C]arginine were administered luminally into isolated segments of rat jejunum in situ, and the radioactive products appearing in venous blood from the segment were identified and quantified, in a continuation of similar studies with l-glutamate and l-glutamine (Windmueller H.G. and Spaeth, A. E. (1975) Arch. Biochem. Biophys. 171, 662–672). Aspartate, administered alone (6 mm) or with 18 other amino acids plus glucose, was absorbed more rapidly than glutamate, but, as with glutamate, less than 1% was recovered intact in intestinal venous blood. More than 50% of aspartate carbon was recovered in CO₂, 24% in organic acids, mostly lactate, 12% in other amino acids (alanine, glutamate, proline, ornithine, and citrulline), and 10% in glucose, apparently the first demonstration of gluconeogenesis by intestine in vivo. In contrast to aspartate and glutamine, nearly all asparagine was absorbed intact, less than 1% being catabolized. About 4% of the absorbed dose was incorporated into the acid-insoluble fraction of intestine, as was the case with all the amino acids studied. In conventional or germ-free rats, only 60% of arginine was absorbed intact, while 33% was hydrolyzed to ornithine and urea. The urea and 38% of the ornithine were released into the blood; the remaining ornithine was metabolized further by intestine to citrulline, proline, glutamate, organic acids, and CO₂. Catabolism of several amino acids from the lumen plus glutamine from arterial blood may provide an important energy source in small intestine.     

Metabolic engineering of Corynebacterium glutamicum for increasing the production of l-ornithine by increasing NADPH availability

The experiments presented here were based on the conclusions of our previous proteomic analysis. Increasing the availability of glutamate by overexpression of the genes encoding enzymes in the l-ornithine biosynthesis pathway upstream of glutamate and disruption of speE, which encodes spermidine synthase, improved l-ornithine production by Corynebacterium glutamicum. Production of l-ornithine requires 2 moles of NADPH per mole of l-ornithine. Thus, the effect of NADPH availability on l-ornithine production was also investigated. Expression of Clostridium acetobutylicum gapC, which encodes NADP-dependent glyceraldehyde-3-phosphate dehydrogenase, and Bacillus subtilis rocG, which encodes NAD-dependent glutamate dehydrogenase, led to an increase of l-ornithine concentration caused by greater availability of NADPH. Quantitative real-time PCR analysis demonstrates that the increased levels of NADPH resulted from the expression of the gapC or rocG gene rather than that of genes (gnd, icd, and ppnK) involved in NADPH biosynthesis. The resulting strain, C. glutamicum ΔAPRE::rocG, produced 14.84 g l^?1 of l-ornithine. This strategy of overexpression of gapC and rocG will be useful for improving production of target compounds using NADPH as reducing equivalent within their synthetic pathways.     

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.     

Transgenic manipulation of a single polyamine in poplar cells affects the accumulation of all amino acids

The polyamine metabolic pathway is intricately connected to metabolism of several amino acids. While ornithine and arginine are direct precursors of putrescine, they themselves are synthesized from glutamate in multiple steps involving several enzymes. Additionally, glutamate is an amino group donor for several other amino acids and acts as a substrate for biosynthesis of proline and γ-aminobutyric acid, metabolites that play important roles in plant development and stress response. Suspension cultures of poplar (Populus nigra × maximowiczii), transformed with a constitutively expressing mouse ornithine decarboxylase gene, were used to study the effect of up-regulation of putrescine biosynthesis (and concomitantly its enhanced catabolism) on cellular contents of various protein and non-protein amino acids. It was observed that up-regulation of putrescine metabolism affected the steady state concentrations of most amino acids in the cells. While there was a decrease in the cellular contents of glutamine, glutamate, ornithine, arginine, histidine, serine, glycine, cysteine, phenylalanine, tryptophan, aspartate, lysine, leucine and methionine, an increase was seen in the contents of alanine, threonine, valine, isoleucine and γ-aminobutyric acid. An overall increase in percent cellular nitrogen and carbon content was also observed in high putrescine metabolizing cells compared to control cells. It is concluded that genetic manipulation of putrescine biosynthesis affecting ornithine consumption caused a major change in the entire ornithine biosynthetic pathway and had pleiotropic effects on other amino acids and total cellular carbon and nitrogen, as well. We suggest that ornithine plays a key role in regulating this pathway.     

Exogenous ornithine is an effective precursor and the δ-ornithine amino transferase pathway contributes to proline accumulation under high N recycling in salt-stressed cashew leaves

The role of the δ-ornithine amino transferase (OAT) pathway in proline synthesis is still controversial and was assessed in leaves of cashew plants subjected to salinity. The activities of enzymes and the concentrations of metabolites involved in proline synthesis were examined in parallel with the capacity of exogenous ornithine and glutamate to induce proline accumulation. Proline accumulation was best correlated with OAT activity, which increased 4-fold and was paralleled by NADH oxidation coupled to the activities of OAT and Δ¹-pyrroline-5-carboxylate reductase (P5CR), demonstrating the potential of proline synthesis via OAT/P5C. Overall, the activities of GS, GOGAT and aminating GDH remained practically unchanged under salinity. The activity of P5CR did not respond to NaCl whereas Δ¹-pyrroline-5-carboxylate dehydrogenase was sharply repressed by salinity. We suggest that if the export of P5C from the mitochondria to the cytosol is possible, its subsequent conversion to proline by P5CR may be important. In a time-course experiment, proline accumulation was associated with disturbances in amino acid metabolism as indicated by large increases in the concentrations of ammonia, free amino acids, glutamine, arginine and ornithine. Conversely, glutamate concentrations increased moderately and only within the first 24 h. Exogenous feeding of ornithine as a precursor was very effective in inducing proline accumulation in intact plants and leaf discs, in which proline concentrations were several times higher than glutamate-fed or salt-treated plants. Our data suggest that proline accumulation might be a consequence of salt-induced increase in N recycling, resulting in increased levels of ornithine and other metabolites involved with proline synthesis and OAT activity. Under these metabolic circumstances the OAT pathway might contribute significantly to proline accumulation in salt-stressed cashew leaves.     

Isolation and characterization of Pseudomonas putida mutants affected in arginine, ornithine and citrulline catabolism: function of the arginine oxidase and arginine succinyltransferase pathways.

Pseudomonas putida mutants impaired in the utilization of arginine are affected in either the arginine succinyltransferase pathway, the arginine oxidase route, or both. However, mutants affected in one of the pathways still grow on arginine as sole carbon source. Analysis of the products excreted by both wild-type and mutant strains suggests that arginine is mainly channelled by the oxidase route. Proline non-utilizing mutants are also affected in ornithine utilization, confirming the role of proline as an intermediate in ornithine catabolism. Mutants affected in ornithine cyclodeaminase activity still grow on proline and become unable to use ornithine. Both proline non-utilizing mutants and ornithine-cyclodeaminase-minus mutants are unable to use citrulline. These results, together with induction of ornithine cyclodeaminase when wild-type P. putida is grown on citrulline, indicate that utilization of citrulline as a carbon source proceeds via proline with ornithine as an intermediate. Thus in P. putida, the aerobic catabolism of arginine on the one hand and citrulline and ornithine on the other proceed by quite different metabolic segments.     

Arginine catabolism in the cyanobacterium Synechocystis sp. Strain PCC 6803 involves the urea cycle and arginase pathway

Cells of the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 supplemented with micromolar concentrations of L-[(14)C]arginine took up, concentrated, and catabolized this amino acid. Metabolism of L-[(14)C]arginine generated a set of labeled amino acids that included argininosuccinate, citrulline, glutamate, glutamine, ornithine, and proline. Production of [(14)C]ornithine preceded that of [(14)C]citrulline, and the patterns of labeled amino acids were similar in cells incubated with L-[(14)C]ornithine, suggesting that the reaction of arginase, rendering ornithine and urea, is the main initial step in arginine catabolism. Ornithine followed two metabolic pathways: (i) conversion into citrulline, catalyzed by ornithine carbamoyltransferase, and then, with incorporation of aspartate, conversion into argininosuccinate, in a sort of urea cycle, and (ii) a sort of arginase pathway rendering glutamate (and glutamine) via Delta(1)pyrroline-5-carboxylate and proline. Consistently with the proposed metabolic scheme (i) an argF (ornithine carbamoyltransferase) insertional mutant was impaired in the production of [(14)C]citrulline from [(14)C]arginine; (ii) a proC (Delta(1)pyrroline-5-carboxylate reductase) insertional mutant was impaired in the production of [(14)C]proline, [(14)C]glutamate, and [(14)C]glutamine from [(14)C]arginine or [(14)C]ornithine; and (iii) a putA (proline oxidase) insertional mutant did not produce [(14)C]glutamate from L-[(14)C]arginine, L-[(14)C]ornithine, or L-[(14)C]proline. Mutation of two open reading frames (sll0228 and sll1077) putatively encoding proteins homologous to arginase indicated, however, that none of these proteins was responsible for the arginase activity detected in this cyanobacterium, and mutation of argD (N-acetylornithine aminotransferase) suggested that this transaminase is not important in the production of Delta(1)pyrroline-5-carboxylate from ornithine. The metabolic pathways proposed to explain [(14)C]arginine catabolism also provide a rationale for understanding how nitrogen is made available to the cell after mobilization of cyanophycin [multi-L-arginyl-poly(L-aspartic acid)], a reserve material unique to cyanobacteria.     

Functional genomics and SNP analysis of human genes encoding proline metabolic enzymes

Proline metabolism in mammals involves two other amino acids, glutamate and ornithine, and five enzymatic activities, Δ¹-pyrroline-5-carboxylate (P5C) reductase (P5CR), proline oxidase, P5C dehydrogenase, P5C synthase and ornithine-δ-aminotransferase (OAT). With the exception of OAT, which catalyzes a reversible reaction, the other four enzymes are unidirectional, suggesting that proline metabolism is purpose-driven, tightly regulated, and compartmentalized. In addition, this tri-amino-acid system also links with three other pivotal metabolic systems, namely the TCA cycle, urea cycle, and pentose phosphate pathway. Abnormalities in proline metabolism are relevant in several diseases: six monogenic inborn errors involving metabolism and/or transport of proline and its immediate metabolites have been described. Recent advances in the Human Genome Project, in silico database mining techniques, and research in dissecting the molecular basis of proline metabolism prompted us to utilize functional genomic approaches to analyze human genes which encode proline metabolic enzymes in the context of gene structure, regulation of gene expression, mRNA variants, protein isoforms, and single nucleotide polymorphisms.     

Arginine and nitrogen mobilization in cyanobacteria

Cyanobacteria have evolved mechanisms to adapt to environmental stress and nutrient availability, including accumulation of storage compounds in inclusions and granules. As arginine is a key building block of cyanophycin, a dynamic nitrogen reservoir in many cyanobacteria, arginine metabolism plays a key role in cyanobacterial nitrogen storage and remobilization. Recently, an arginine dihydrolase AgrE/ArgZ was identified as a major arginine‐degrading enzyme in nondiazotrophic Synechocystis, which catalyzes the conversion of arginine into ornithine and ammonia. The N‐terminal domain of AgrE/ArgZ is responsible for arginine dihydrolase activity. Burnat et al. (2019) identified the arginine catabolic pathway in diazotrophic Anabaena, which starts with the reaction catalyzed by AgrE/ArgZ. Moreover, this study identified the C‐terminal domain of AgrE/ArgZ as an ornithine cyclodeaminase that catalyze the conversion of ornithine to proline. The results demonstrated that arginine is catabolized to generate glutamate by the concerted action of AgrE/ArgZ and bifunctional proline oxidase PutA in the vegetative cells of Anabaena. These findings expand our knowledge on nitrogen mobilization and redistribution in Anabaena under nitrogen‐fixation conditions. AgrE/ArgZ is widely present in many diazotrophic cyanobacteria and may be important for their contribution to marine nitrogen fixation. AgrE/ArgZ may have potential applications in metabolic engineering and biotechnology.     

Molecular Characterization and Regulation of an Operon Encoding a System for Transport of Arginine and Ornithine and the ArgR Regulatory Protein in Pseudomonas aeruginosa

The complete nucleotide sequence for the aot operon of Pseudomonas aeruginosa PAO1 was determined. This operon contains six open reading frames. The derived sequences for four of these, aotJ, aotQ, aotM, and aotP, show high similarity to those of components of the periplasmic binding protein-dependent ABC (ATP binding cassette) transporters of enteric bacteria. Transport studies with deletion derivatives established that these four genes function in arginine-inducible uptake of arginine and ornithine but not lysine. The aotO gene, which encodes a polypeptide with no significant similarity to any known proteins, is not essential for arginine and ornithine uptake. The sixth and terminal gene in the operon encodes ArgR, which has been recently shown to function in regulation of arginine metabolism. Studies with an aotJ::lacZ translational fusion showed that expression of the aot operon is strongly induced by arginine and that this effect is mediated by ArgR. S1 nuclease and primer extension experiments showed the presence of two promoters, P1 and P2. The downstream promoter, P2, is induced by arginine and appears to be subject to carbon catabolite repression. The upstream promoter, P1, is induced by glutamate. Footprinting experiments established the presence of a 44-bp ArgR binding site that overlaps the −35 region for P2, as was shown to be the case for the arginine-inducible aru promoter, and the −10 region for P1, as was shown to be the case for arginine-repressible operons in P. aeruginosa. Sequence alignment confirms the architecture and the consensus sequence of the ArgR binding sites, as was previously reported.     

Osmotically Induced Proline Accumulation in <Emphasis Type="Italic">Lotus Corniculatus</Emphasis> Leaves is Affected by Light and Nitrogen Source

Proline accumulation in osmotically stressed leaves of Lotus corniculatus was stimulated by increasing light intensity (photon fluence density, PFD). Treatment with propanil limited proline accumulation in response to light and osmotic stress, indicating a dependence of proline synthesis on photosynthetic NADPH. Drought stress induced proline accumulation in L. corniculatus both in nitrate-fed plant (NFP) and ammonium-fed plants (AFP), although higher proline concentration was observed in AFP than in NFP after 24 h of drought stress. Changes in proline accumulation induced by drought stress in plants grown under different nitrogen regimes could not be explained by changes of either total protein or amino acids, consistent with specifically altered regulation of proline synthesis. Under control conditions, alanine, aspartate and glutamate were the predominant amino acids in NFP; conversely, in AFP, arginine and ornithine were the predominant amino acids. Only the NFP regime showed changes in the concentrations of specific amino acids under drought stress a decrease in alanine, aspartate and glutamate and increased gama-aminobutyric acid. In AFP and especially NFP, proline accumulation under osmotic stress was associated with increased ornithine amino transferase activity. An increase of both activity and protein of ferredoxin-dependent glutamate synthase was observed in osmotic-stressed NFP; inversely both decreased in drought-stressed AFP. PFD and nitrogen source are therefore shown to be regulators of proline accumulation in L. corniculatus osmotically stressed plants.     

Production of secondary metabolites from cell and organ cultures: strategies and approaches for biomass improvement and metabolite accumulation

Pyrroline-5-carboxylate reductase (P5CR) lies at the converging point of the glutamate and ornithine pathways and is the last and critical enzyme in proline biosynthesis. In the present study, a P5CR gene, named IbP5CR, was isolated from salt-tolerant sweetpotato line ND98. Expression of IbP5CR was up-regulated in sweetpotato under salt stress. The IbP5CR-overexpressing sweetpotato (cv. Kokei No. 14) plants exhibited significantly higher salt tolerance compared with the wild-type. Proline content and superoxide dismutase and photosynthetic activities were significantly increased, whereas malonaldehyde content was significantly decreased in the transgenic plants. H₂O₂ was also found to be significantly less accumulated in the transgenic plants than in the wild-type. Overexpression of IbP5CR up-regulated pyrroline-5-carboxylate synthase gene and down-regulated proline dehydrogenase and P5C dehydrogenase genes under salt stress. The systemic up-regulation of reactive oxygen species (ROS) scavenging genes was found in the transgenic plants under salt stress. These findings suggest that overexpression of IbP5CR increases proline accumulation, which enhances salt tolerance of the transgenic sweetpotato plants by regulating osmotic balance, protecting membrane integrity and photosynthesis and activating ROS scavenging system. This study indicates that IbP5CR gene has the potential to be used for improving salt tolerance of plants.