Proline Metabolic Enzymes Research Articles

Proline metabolism in cancer.

Cancer cells often change their metabolism to support uncontrolled proliferation. Proline is the only proteogenic secondary amino acid that is abundant in the body. Recent studies have shown that proline metabolism plays an important role in metabolic reprogramming and affects the occurrence and development of cancer. Proline metabolism is related to ATP production, protein and nucleotide synthesis, and redox homeostasis in tumor cells. Proline can be synthesized by aldehyde dehydrogenase family 18 member A1 (ALDH18A1) and delta1-pyrroline-5-carboxylate reductase (PYCR), up-regulating ALDH18A1 and PYCR can promote the proliferation and invasion of cancer cells. As the main storage of proline, collagen can influence cancer cells proliferation, invasion, and metastasis. Its synthesis depends on the hydroxylation of proline catalyzed by prolyl 4-hydroxylases (P4Hs), which will affect the plasticity and metastasis of cancer cells. The degradation of proline occurs in the mitochondria and involves an oxidation step catalyzed by proline dehydrogenase/proline oxidase (PRODH/POX). Proline catabolism has a dual role in cancer, linking apoptosis with the survival and metastasis of cancer cells. In addition, it has been demonstrated that the regulation of proline metabolic enzymes at the genetic and post-translational levels is related to cancer. This article reviews the role of proline metabolic enzymes in cancer proliferation, apoptosis, metastasis, and development. Research on proline metabolism may provide a new strategy for cancer treatment.

5-aminolevulinic acid improves salt tolerance mediated by regulation of tetrapyrrole and proline metabolism in Brassica napus L. seedlings under NaCl stress

5-aminolevulinic acid (ALA), a key biosynthetic precursor of tetrapyrroles, is vital for plant growth and adaptation to stress environments. Although exogenous ALA could enhance photosynthesis and biomass accumulation in plants under stress conditions, the underlying physiological and molecular mechanisms governed by ALA in promoting salt tolerance in Brassica napus L. are not yet clearly understood. In the present study, exogenous ALA with the concentration of 30 mg L−1 was applied to the leaves of B. napus seedlings subjected to 200 mM NaCl. The results showed that NaCl stress decreased the photosynthesis, biomass accumulation, and levels of chlorophyll and heme with the reduction of the concentrations of intermediates including ALA, protoporphyrin IX (Proto IX), Mg-Proto IX, and Pchlide in the tetrapyrrole (chlorophyll and heme) biosynthetic pathway. The transcript levels of genes encoding ALA-associated enzymes and genes encoding Mg-chelatase in the chlorophyll biosynthetic branch were down-regulated, while the expression levels of genes encoding Fe-chelatase in the heme branch were not significantly altered by NaCl stress. Foliar application with ALA enhanced the aboveground biomass, net photosynthetic rate, activities of antioxidant enzymes, accumulation of chlorophyll and heme, and concentrations of intermediates related to chlorophyll and heme biosynthesis in B. napus under 200 mM NaCl. The expression of most genes mentioned above remained constant in ALA-treated plants in comparison with non-ALA-treated plants under NaCl stress. Additionally, exogenous ALA synchronously induced the proline concentration and up-regulated the expression of genes P5CS and ProDH encoding proline metabolic enzymes in the NaCl treatment. These findings suggested that ALA improved salt tolerance through promoting the accumulation of chlorophyll and heme resulting from the increase of intermediate levels in the tetrapyrrole biosynthetic pathway, along with enhancing the proline accumulation in B. napus.

Role of Proline in Pathogen and Host Interactions.

Proline metabolism has complex roles in a variety of biological processes, including cell signaling, stress protection, and energy production. Proline also contributes to the pathogenesis of various disease-causing organisms. Understanding the mechanisms of how pathogens utilize proline is important for developing new strategies against infectious diseases. Recent Advances: The ability of pathogens to acquire amino acids is critical during infection. Besides protein biosynthesis, some amino acids, such as proline, serve as a carbon, nitrogen, or energy source in bacterial and protozoa pathogens. The role of proline during infection depends on the physiology of the host/pathogen interactions. Some pathogens rely on proline as a critical respiratory substrate, whereas others exploit proline for stress protection. Disruption of proline metabolism and uptake has been shown to significantly attenuate virulence of certain pathogens, whereas in other pathogens the importance of proline during infection is not known. Inhibiting proline metabolism and transport may be a useful therapeutic strategy against some pathogens. Developing specific inhibitors to avoid off-target effects in the host, however, will be challenging. Also, potential treatments that target proline metabolism should consider the impact on intracellular levels of Δ1-pyrroline-5-carboxylate, a metabolite intermediate that can have opposing effects on pathogenesis. Further characterization of how proline metabolism is regulated during infection would provide new insights into the role of proline in pathogenesis. Biochemical and structural characterization of proline metabolic enzymes from different pathogens could lead to new tools for exploring proline metabolism during infection and possibly new therapeutic compounds.

Structural Biology of Proline Catabolic Enzymes.

Proline catabolism refers to the 4-electron oxidation of proline to glutamate catalyzed by the enzymes proline dehydrogenase (PRODH) and l-glutamate γ-semialdehyde dehydrogenase (GSALDH, or ALDH4A1). These enzymes and the intermediate metabolites of the pathway have been implicated in tumor growth and suppression, metastasis, hyperprolinemia metabolic disorders, schizophrenia susceptibility, life span extension, and pathogen virulence and survival. In some bacteria, PRODH and GSALDH are combined into a bifunctional enzyme known as proline utilization A (PutA). PutAs are not only virulence factors in some pathogenic bacteria but also fascinating systems for studying the coordination of metabolic enzymes via substrate channeling. Recent Advances: The past decade has seen an explosion of structural data for proline catabolic enzymes. This review surveys these structures, emphasizing protein folds, substrate recognition, oligomerization, kinetic mechanisms, and substrate channeling in PutA. Major unsolved structural targets include eukaryotic PRODH, the complex between monofunctional PRODH and monofunctional GSALDH, and the largest of all PutAs, trifunctional PutA. The structural basis of PutA-membrane association is poorly understood. Fundamental aspects of substrate channeling in PutA remain unknown, such as the identity of the channeled intermediate, how the tunnel system is activated, and the roles of ancillary tunnels. New approaches are needed to study the molecular and in vivo mechanisms of substrate channeling. With the discovery of the proline cycle driving tumor growth and metastasis, the development of inhibitors of proline metabolic enzymes has emerged as an exciting new direction. Structural biology will be important in these endeavors.

Epibrassinolide reverses the stress generated by combination of excess aluminum and salt in two wheat cultivars through altered proline metabolism and antioxidants

This study was conducted to unravel the EBL mediated changes on the enzymes of proline metabolism under combined stress of aluminum and salt through varied mode of application. The results of the study revealed that application of EBL through different mode significantly increased the activities of P5CS and δ-OAT, proline content, photosynthetic efficiency and various enzymes of antioxidant system whereas, these parameters were further accelerated in the plants exposed to combined stress of aluminum and salt followed by EBL treatment. These findings indicate that exogenous application of EBL modified the activity of enzymes of proline metabolism and also scavenges the excess reactive oxygen species through improved activities of antioxidant enzymes which could have provided the ability to confer tolerance in wheat plants under combination of aluminum and salt stress.

Alleviation of waterlogged stress in peach seedlings inoculated with Funneliformis mosseae: Changes in chlorophyll and proline metabolism

Peach plants are highly susceptible to soil waterlogging, which can strongly inhibit plant growth and fruit production, and even cause tree death. A pot study was conducted to investigate the effects of an arbuscular mycorrhizal fungus (AMF), Funneliformis mosseae, on chlorophyll concentration, proline concentration, and activity of proline metabolic enzymes (Δ1-pyrroline-5-carboxylate synthase, P5CS; orn-δ-aminotransferase, δ-OAT; and proline dehydrogenase, ProDH) in leaves of peach [Prunes persica (L.) Batsch] seedlings under soil waterlogged and non-waterlogged conditions. After 12 days of waterlogged treatment, root mycorrhizal colonization and entry points were significantly decreased. Inoculation with F. mosseae significantly increased chlorophyll a, b, and a+b concentrations under waterlogged and non-waterlogged conditions, as compared with non-AMF inoculation. AMF inoculation led to more proline accumulation in leaves, accompanied an increase of P5CS activity and a decrease of δ-OAT and ProDH activity under waterlogged and non-waterlogged conditions. It was concluded that F. mosseae conferred a positive contribution to waterlogged tolerance of the peach plant through promotion of proline and chlorophyll biosysthesis.

播种期对晚季香稻香气2-乙酰-1-吡咯啉含量和产量的影响

PDF HTML阅读 XML下载 导出引用 引用提醒 播种期对晚季香稻香气2-乙酰-1-吡咯啉含量和产量的影响 DOI: 10.5846/stxb201305211129 作者: 作者单位: 华南农业大学农学院,华南农业大学农学院,华南农业大学农学院,华南农业大学农学院,华南农业大学农学院,华南农业大学农学院 作者简介: 通讯作者: 中图分类号: 基金项目: 国家自然科学基金项目（31271646）；广东省自然科学基金项目（8151064201000017）；广东省农业攻关项目（2011AO20202001）；广东省农业标准化项目（4100F10003） Effects of sowing date on 2-acetyl-1-pyrroline content and yield of late season aromatic rice Author: Affiliation: College of Agriculture,South China Agricultural University,College of Agriculture,South China Agricultural University,College of Agriculture,South China Agricultural University,College of Agriculture,South China Agricultural University,College of Agriculture,South China Agricultural University, Fund Project: 摘要 | 图/表 | 访问统计 | 参考文献 | 相似文献 | 引证文献 | 资源附件 | 文章评论 摘要:为了确定华南双季稻区晚季香稻的适宜播种期，在大田条件下以桂香占和农香18为材料，研究了播种期对香稻香气2-乙酰-1-吡咯啉（2-AP）、脯氨酸代谢酶活性和产量的影响。结果表明：（1）7月20日播种的香稻的2-AP含量最高，2-AP含量较高的播期其游离脯氨酸含量、籽粒脯氨酸氧化酶活性以及脯氨酸脱氢酶活性维持在较高水平。（2）7月15日播种的桂香占和7月20日播种的农香18的产量最高，产量较高的播期具有较高的收获指数、干物质积累量和群体生长率。（3）试验条件下晚季香稻的适宜播种期为7月15日-7月20日。 Abstract:Aromatic rice, a famous rice type with high nutritional value and a distinct aroma, has been planted in South and Southeast Asian countries since ancient times. Recently, it has gained in popularity and gained a significant market share in the global rice trade resulting in higher prices and increased production in many countries. However, in China, a minimal area has been planted with aromatic rice because of its low resistance to disease, low yield and other disadvantages of the aromatic rice cultivars. As the standard of living has improved, China has begun to focus on the production of quality food, so researchers have started to pay more attention to aromatic rice and have bred some aromatic rice cultivars. However, finding ways to increase the concentration of the aroma of aromatic rice has proved challenging based on the low yield and poor quality of existing cultivars. 2-Acetyl-1-pyrroline (2-AP) is a buttered-popcorn-like aromatic compound found in both basmati and jasmine aromatic rice, and is considered a primary aromatic component of aromatic rice; this highly volatile and lipophilic compound occurs in the stems, leaves and grains of aromatic rice. L-proline is widely considered to be the nitrogen source for 2-AP and Δ-pyrroline-5-carboxylic acid (P5C) and has been proposed as one of the main precursors required for 2-AP synthesis. P5C is the common intermediate in the metabolic pathways of proline, ornithine and glutamic acid synthesis. Among these three pathways, the proline pathway is the major pathway used for the synthesis of 2-AP, in which proline to P5C is mediated by proline dehydrogenase, which is then used to synthetize 2-AP. Genetic factors play a major role in determining the aroma content of aromatic rice, and environmental factors and cultivation practices have a substantial effect on the quality of aromatic rice. The aroma of aromatic rice can be increased using low-density cultivation techniques, shallow irrigation, low levels of nitrogen, and moderate use of trace element fertilizers containing elements such as zinc, iron and lanthanum. However, unsuitable post-harvest conditions and some plant growth regulators can reduce the 2-AP content in aromatic rice considerably. Using better packaging materials, such as vacuum packing in nylon pouches, can retard volatilization of the aromatic compounds in aromatic rice. Temperature during grain filling, ripening and storage also plays an important role in the development of the aroma of aromatic rice; lower temperature, especially at night, has positive effects on aroma formation during the grain filling and ripening stages compared with higher temperature. Additionally, 2-AP content declines over time and is significantly affected by the temperature during storage. The sowing date affects proline and 2-AP content in aromatic rice as a result of variations in light and temperature conditions. Hence, a field experiment was carried out using Guixiangzhan and Nongxiang18 cultivars to determine an appropriate sowing date for late season aromatic rice in South China's double-cropping region and to study the effects of different sowing dates on aroma (2-AP), activities of proline metabolic enzymes and yield. The results show (1) Aromatic rice sown on 20 July achieved the highest 2-AP content. Treatments having higher 2-AP content also had a higher free proline content, higher proline oxidase activities in grain and higher levels of proline dehydrogenase. (2) Guixiangzhan sown on 15 July and Nongxiang18 sown on 20 July achieved the highest grain yield, and both had a high harvest index, high levels of dry matter accumulation and rapid crop growth rates. (3) The appropriate late season sowing date for aromatic rice was between 15 July and 20 July. 参考文献 相似文献 引证文献

Effect of spermine treatment on the functioning of Thellungiella salsuginea antioxidant system

The effect of spermine (Spm) treatment on the content of polyamines (PAs) and activities of antioxidant enzymes in the roots and leaves of Thellungiella salsuginea (Pall.) O.E. Schulz plants grown under optimal conditions were studied. The genes encoding three forms of ascorbate peroxidase (APX; APX1, APX2, and APX4) and genes of key enzymes of proline metabolism (Pro, P5CS1, 1P5CD) were identified, and their expression intensity was measured. Six-day-old plants were treated with Spm (1 and 2 mM) and with the inhibitor of polyamine oxidase (PAO) activity, N,N-(2-hydroxyethyl)hydrazine (HEH, 1 and 2 mM) separately or in combination by adding these compounds to nutrient medium. Roots and leaves responded differently to Spm treatment. In the leaves, the content of PAs reduced due to a decreased in the spermidine (Spd) content, whereas in the roots the total pool of PAs increased due to putrescine (Put) and Spd accumulation. Treatment with Spm activated PAO in the roots but not in the leaves; HEH removed this increase, but the intercellular Spm concentration was not substantially changed. It was suggested that treatment with Spm suppressed the biosynthesis of intracellular Spm and, on the other hand, stimulated the reverse conversion of Spm into Spd and further into Put due to the activation of one of the PAO isoforms. Plant treatment with Spm was not accompanied by a noticeable activation enzymes degrading hydrogen peroxide in the roots: APX, (except of peroxidase II), and catalase. However, the activity of Cu/Zn-SOD doubled and the activity of Mn-SOD reduced. In the leaves, slight activation of peroxidases I and III, the inhibition of Cu/Zn- and Mn-SOD, differential changes in the time-coursed of gene expression of three APX isoforms, and activated gene expression of key enzymes of Pro metabolism were observed. At the same time, the level of MDA did not increase either in the leaves or in the roots. This indicates that treatment of Th. salsuginea plants with Spm under optimal growing conditions did not enhance ROS generation and did not manifest prooxidant properties.

Proline Mechanisms of Stress Survival

The imino acid proline is utilized by different organisms to offset cellular imbalances caused by environmental stress. The wide use in nature of proline as a stress adaptor molecule indicates that proline has a fundamental biological role in stress response. Understanding the mechanisms by which proline enhances abiotic/biotic stress response will facilitate agricultural crop research and improve human health. It is now recognized that proline metabolism propels cellular signaling processes that promote cellular apoptosis or survival. Studies have shown that proline metabolism influences signaling pathways by increasing reactive oxygen species (ROS) formation in the mitochondria via the electron transport chain. Enhanced ROS production due to proline metabolism has been implicated in the hypersensitive response in plants, lifespan extension in worms, and apoptosis, tumor suppression, and cell survival in animals. The ability of proline to influence disparate cellular outcomes may be governed by ROS levels generated in the mitochondria. Defining the threshold at which proline metabolic enzyme expression switches from inducing survival pathways to cellular apoptosis would provide molecular insights into cellular redox regulation by proline. Are ROS the only mediators of proline metabolic signaling or are other factors involved? New evidence suggests that proline biosynthesis enzymes interact with redox proteins such as thioredoxin. An important future pursuit will be to identify other interacting partners of proline metabolic enzymes to uncover novel regulatory and signaling networks of cellular stress response.

The relationship of proline content and metabolism on the productivity of maize plants

The free proline content in maize ear-leaves, silk and pollen were analyzed in field grown plants which had matured to the pollination stage. Using maize hybrids PR34F02, PR35P12 and PR36B08 field trials were set up at two locations in eastern Croatia in two different years. Two enzymes of proline metabolism were analyzed in the same leaf samples and specific activities of synthetase (P5CS) and proline dehydrogenase (PDH). Plant productivity was evaluated at harvest by the estimation of total and fully developed grain number per ear and per plant, the mean single grain mass, and the mass of grain per plant. The year in which the plants were grown had a very significant effect on the free proline content in the leaf and pollen, as well as on the enzyme activities assayed. The differences between the plants from the two localities were very significant in all tested parameters of plant grain productivity. There was a significant genotype effect on proline content and P5CS total activity in leaf and on all the productivity parameters. Some of the correlations established suggest that the rate of proline synthesis and degradation in maize ear-leaf at pollination might contribute to the final grain production of the maize plant. Multiple regression analyses was used to further analyze the relationship between proline and grain productivity, but it is clear that future work should include other environmental conditions, plant species and organs such as roots.

In planta function of compatible solute transporters of the AtProT family

The three proline transporters of Arabidopsis thaliana (AtProTs) transport the compatible solutes proline and glycine betaine and the stress-induced compound γ-aminobutyric acid when expressed in heterologous systems. The aim of the present study was to show transport and physiological relevance of these three AtProTs in planta. Using single, double, and triple knockout mutants and AtProT-overexpressing lines, proline content, growth on proline, transport of radiolabelled betaine, and expression of AtProT genes and enzymes of proline metabolism were analysed. AtProT2 was shown to facilitate uptake of L- and D-proline as well as [14C]glycine betaine in planta, indicating a role in the import of compatible solutes into the root. Toxic concentrations of L- and D-proline resulted in a drastic growth retardation of AtProT-overexpressing plants, demonstrating the need for a precise regulation of proline uptake and/or distribution. Furthermore evidence is provided that AtProT genes are highly expressed in tissues with elevated proline content—that is, pollen and leaf epidermis.

Proline metabolism in response to salt stress in common reed [Phragmites australis (Cav.) Trin. ex Steud

Abstract We studied effects of salt stress on proline accumulation and the activities of proline metabolic enzymes in reed, a salt-tolerant, cosmopolitan, aquatic grass. A concentration-dependent inhibition of plant growth occurred in the presence of NaCl, which was accompanied by high amounts of proline accumulation. Reed tolerance of salt stress reaches a limit at approximately 200 mm NaCl. Free proline content and pyrroline-5-carboxylate synthetase (P5CS) activity both increased with salt stress treatment, while ornithine aminotransferase (OAT) activity appeared to be unaffected, suggesting that the glutamate pathway, rather than the ornithine pathway, plays a vital role in proline accumulation during osmotic regulation. The activity of the proline degrading enzyme, proline dehydrogenase (PDH), decreased under salt stress, indicating that proline catabolism may also be responsible for proline accumulation in response to salt stress. Furthermore, high salinity ≥250 mm NaCl, which stops growth of reed, decreases proline accumulation and P5CS, OAT, and PDH activities. This suggests that changes in enzyme activities of proline metabolism coupled with increased proline accumulation are involved in osmotic regulation of reed plants only when they are grown at salinities ≤200 mm NaCl.

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, Delta(1)-pyrroline-5-carboxylate (P5C) reductase (P5CR), proline oxidase, P5C dehydrogenase, P5C synthase and ornithine-delta-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.

Proline modulates the intracellular redox environment and protects mammalian cells against oxidative stress

The potential of proline to suppress reactive oxygen species (ROS) and apoptosis in mammalian cells was tested by manipulating intracellular proline levels exogenously and endogenously by overexpression of proline metabolic enzymes. Proline was observed to protect cells against H 2O 2, tert-butyl hydroperoxide, and a carcinogenic oxidative stress inducer but was not effective against superoxide generators such as menadione. Oxidative stress protection by proline requires the secondary amine of the pyrrolidine ring and involves preservation of the glutathione redox environment. Overexpression of proline dehydrogenase (PRODH), a mitochondrial flavoenzyme that oxidizes proline, resulted in 6-fold lower intracellular proline content and decreased cell survival relative to control cells. Cells overexpressing PRODH were rescued by pipecolate, an analog that mimics the antioxidant properties of proline, and by tetrahydro-2-furoic acid, a specific inhibitor of PRODH. In contrast, overexpression of the proline biosynthetic enzymes Δ 1-pyrroline-5-carboxylate (P5C) synthetase (P5CS) and P5C reductase (P5CR) resulted in 2-fold higher proline content, significantly lower ROS levels, and increased cell survival relative to control cells. In different mammalian cell lines exposed to physiological H 2O 2 levels, increased endogenous P5CS and P5CR expression was observed, indicating that upregulation of proline biosynthesis is an oxidative stress response.

Functioning of defense systems in halophytes and glycophytes under progressing salinity

Six-week-old Plantago major L. and Thellungiella halophila Mey. plants were subjected to progressing salinity by a daily increase in the NaCl concentration by 100 mM until the final concentration of 400 mM. A dynamics of stress-dependent accumulation of Na+ and Cl− ions, proline, and free polyamines and also activities of antioxidant enzymes, superoxide oxidase (SOD) and free, ion-bound, and covalently bound guaiacol-dependent peroxidases was studied. We also examined the intensity of gene expression encoding enzymes of proline metabolism and polyamine biosynthesis. It was shown that the high salt-resistance of the halophyte T. halophila was determined by plant capability of ion accumulation and stress-dependent proline accumulation. An important role in the maintenance of this plant homeostasis under salinity plays a high constitutive levels of activities of three types of peroxidases tested and also of proline manifesting a polyfunctional protective action. In contrast, P. major plants characterized by a lower tolerance to salt excess did not display a high constitutive level of proline or the activity of guaiacol-dependent peroxidases; they also were not capable of stress-induced accumulation of compatible osmolytes and did not accumulate the salt. However, this glycophyte contained relatively much spermidine and active SOD, which provided for a decrease in the damaging effects of reactive oxygen species under salt shock. In both plant species, it was established that salinity changed the intracellular content of polyamines, which was not dependent on the activity of gene transcription encoding the enzymes of their biosynthesis. The results obtained support a hypothesis that halophytes and glycophytes have some common mechanisms of tolerance to salinity, but the control of these mechanisms differs substantially.

Investigation of Transcriptional Regulation of the put Regulon from Escherichia coli

All organisms can use proline as energy source via proline oxidation. In bacteria the proline utilization genes are organized in the put regulon, composed of putA and putP genes. In some gram-negative bacteria PutA also functions both as a proline metabolic enzymes and a transcriptional repressor of put genes by binding to the put control DNA through its N-terminal ribbon-helix-helix DNA binding domain. Here we identified the PutA binding sites on the put control DNA in E. coli, which all share a gGTTGCAc sequence, by systematic evaluation of the different regions of the put control DNA. Cell-based reporter gene assays indicated three binding sites that are critical for PutA autorepression and two binding sites that are important for regulation of the putP gene. To investigate the mechanism by which put gene expression is activated by proline, we performed the reporter gene assays in the presence of proline and the non-reducing analog L-Tetrahydro-2-furoic acid (THFA). Proline was observed to induce putA gene expression, while THFA had no effect. Membrane binding deficient PutA mutants showed no response to proline in reporter gene assays. Therefore, both flavin reduction and membrane binding are both required to activate put genes by proline. We also investigated global factors, such as the cAMP receptor protein (CRP) and the nitrogen assimilation control protein (NAC), that are presumed to be involved in regulating the put genes. CRP regulates put genes by binding directly to the put control DNA, while NAC appears to be mainly involved in regulating the putP gene.

Proline accumulation and real time PCR expression analysis of genes encoding enzymes of proline metabolism in relation to drought tolerance in Andean potato

Six potato varieties belonging to four dif- ferent sub-species were submitted to drought stress during tuberization under controlled field conditions, resulting in contrasting responses of the genotypes to water stress with yield losses varying between 27 and 75%. In all clones free proline accumulated under drought, however, proline levels increased earlier in drought-susceptible varieties than in more tolerant ones. The expression of two key genes in proline metabolism, D 1 -pyrroline-5-carboxylate synthase and proline dehydrogenase, was monitored in the leaves of the experimental plants by r2eal time PCR 23 and 42 days after drought onset. Expression of both en- zymes did not correlate with the proline levels found in leaf tissue indicating that mechanisms other than transcription participate in the regulation of proline accumulation in potato leaves.

Physiological and biochemical aspects of flight metabolism in cocoon-enclosed adults of the fruit beetle, Pachnoda sinuata

We studied several aspects of flight metabolism in cocoon-enclosed adults of the fruit beetle Pachnoda to investigate their flight capability. The majority of adults which were forcefully removed from their pupal cocoon flew off within 5 min of exposure to bright sunlight. Most of the beetles which did not fly voluntarily were, however, capable of flight. Compared with 2–4 week old adults of the same species, cocoon-enclosed adults have higher reserves of glycogen in flight muscles and fat body, whereas the level of total carbohydrates in the haemolymph and the concentration of proline in haemolymph, flight muscles and fat body were similar.Enzymes involved in carbohydrate breakdown (MDH, GAPDH) were more active in flight muscles and fat body of cocoon-enclosed adults compared with adults, while enzymes of proline metabolism in the flight muscles (AlaT, NAD-ME) and fat body (AlaT, NADP-ME) had similar activities in cocoon-enclosed adults and adults. An enzyme of the β-oxidation of fatty acids (HOAD) had similar activities in flight muscles and fat body of cocoon-enclosed adults and adults.Mitochondria isolated from flight muscles of adults removed prematurely from their cocoon favour the oxidation of proline and pyruvate. Pyruvate, however, is oxidized at higher rates than by mitochondria isolated from flight muscles of adults.During a short lift-generating flight, cocoon-enclosed adults proved that their flight muscles are capable of strong flight performance. During these flights, cocoon-enclosed adults consume proline and carbohydrates at a similar rate to that of adults.The endogenous AKH peptide, Mem-CC, has hyperprolinaemic and hypertrehalosaemic activity in cocoon-enclosed adults. The hypertrehalosaemic effect, however, is stronger in cocoon-enclosed adults than in adults.The content of Mem-CC in corpora cardiaca of larvae (3rd instar), cocoon-enclosed adults and 1 day-old adults is similar at 5–6 pmol per pair of corpora cardiaca, whereas it is higher in 10 day-old adults and 20 day-old adults (37 and 15 pmol per pair corpora cardiaca, respectively).From these results we conclude that cocoon-enclosed adults comply with all the prerequisites for flight performance before they leave their pupal cocoon. Furthermore, cocoon-enclosed adults have a more pronounced carbohydrate-based metabolism before they leave their cocoon compared with adults, which suggests that carbohydrate breakdown is mainly involved in such activities as leaving the cocoon and burrowing activity thereafter.

Proline and Proline Metabolising Enzymes in in-vitro Selected NaCl-tolerant Brassica juncea L. under Salt Stress

The effect of salt stress was studied on proline accumulation and the activities of proline metabolic pathway enzymes in seedling and leaf tissue of two genetically stable lines (SR2P1-2 and SR3P6-2) of in vitro selected NaCl-tolerant plants and parent cultivar Prakash of Brassica juncea L. Salt stress caused differential enhancement in proline level in both seedlings and leaf tissue of plants at different developmental stages. The magnitude of increase in proline content was higher in SR3P6-2 line in seedlings (34 fold at 140 meq-1 NaCl) as well as leaves (16 fold at 40 d after sowing at 100 meq-1 NaCl) compared to the parent cv. Prakash (29 fold in seedlings and five fold in leaves) and SR2P1-2 (21 fold in seedlings and five fold in leaves) at similar stress levels. Salt stress also resulted in changes in the activities of enzymes of proline metabolism. The activities of proline biosynthetic enzymes, pyrroline-5-carboxylate reductase and ornithine aminotransferase, increased under salt stress both in the seedlings and leaves. The range of increase in the activities of the two enzymes was relatively higher in SR3P6-2 (3·3-3·9 fold) compared to the SR2P1-2 (1·8-2·8 fold) and parent cv. Prakash (1·5-2·8 fold). The activity of proline degrading enzyme, proline oxidase, decreased under salt stress in both the tissues of all the lines; the reduction in activity was relatively greater in SR3P6-2 compared to SR2P1-2 or cv. Prakash. The trend of changes in the enzyme activities was in tune with the increase in proline level, the magnitude of change did not match the extent of increase in proline level.

Activities of the Pentose Phosphate Pathway and Enzymes of Proline Metabolism in Legume Root Nodules

Based on localization and high activities of pyrroline-5-carboxylate reductase and proline dehydrogenase activities in soybean nodules, we previously suggested two major roles for pyrroline-5-carboxylate reductase in addition to the production of the considerable quantity of proline needed for biosynthesis; namely, transfer of energy to the location of biological N(2) fixation, and production of NADP(+) to drive the pentose phosphate pathway. The latter produces ribose-5-phosphate which can be used in de novo purine synthesis required for synthesis of ureides, the major form in which biologically fixed N(2) is transported from soybean root nodules to the plant shoot. In this paper, we report rapid induction (in soybean nodules) and exceptionally high activities (in nodules of eight species of N(2)-fixing plants) of pentose phosphate pathway and pyrroline-5-carboxylate reductase. There was a marked increase in proline dehydrogenase activity during soybean (Glycine max) ontogeny. The magnitude of proline dehydrogenase activity in bacteroids of soybean nodules was sufficiently high during most of the time course to supply a significant fraction of the energy requirement for N(2) fixation. Proline dehydrogenase activity in bacteroids from nodules of other species was also high. These observations support the above hypothesis. However, comparison of pentose phosphate pathway and pyrroline-5-carboxylate reductase activities of ureide versus amide-exporting nodules offers no support. The hypothesis predicts that pyrroline-5-carboxylate and pentose phosphate pathway activities should be higher in ureide-exporting nodules than in amide-exporting nodules. This predicted distinction was not observed in the results of in vitro assays of these activities.