Proline Dehydrogenase Activity Research Articles

Structural basis of substrate channeling in the PutA flavoenzyme

Proline utilization A (PutA) is a high-hanging fruit of X-ray crystallography. PutA is a membrane-associated bifunctional flavoenzyme that catalyzes the 4-electron oxidation of proline to glutamate by the sequential activities of proline dehydrogenase and aldehyde dehydrogenase domains. PutAs are challenging crystallography targets because of their long polypeptide chain length (1000-1300 residues) and multidomain architecture. In this talk, I will present new crystal structures and SAXS analysis of two PutAs. Seven high resolution crystal structures of a 1004-residue minimalist PutA were determined using Hg SIRAS phasing, and the oligomeric state and quaternary structure were determined with SAXS [1]. The structures reveal an elaborate and dynamic tunnel system featuring a 75-Å long tunnel that links the two active sites. Also, a novel mechanism-based inactivation strategy allowed the trapping of the elusive PutA-quinone complex in the crystalline state. These structures provide insight into the mechanism of substrate channeling and how the enzyme changes conformation during the catalytic cycle. I will conclude by describing the first structure of a new type of PutA that contains an additional C-terminal domain of unknown function (CTDUF) that is not present in the smaller minimalist enzyme [2]. This larger PutA reveals an unexpectedly different structural solution to the problem of sequestering the reaction intermediate.

Kinetic and structural characterization of tunnel-perturbing mutants in Bradyrhizobium japonicum proline utilization A.

Proline utilization A from Bradyrhizobium japonicum (BjPutA) is a bifunctional flavoenzyme that catalyzes the oxidation of proline to glutamate using fused proline dehydrogenase (PRODH) and Δ1-pyrroline-5-carboxylate dehydrogenase (P5CDH) domains. Recent crystal structures and kinetic data suggest an intramolecular channel connects the two active sites, promoting substrate channeling of the intermediate Δ1-pyrroline-5-carboxylate/glutamate-γ-semialdehyde (P5C/GSA). In this work, the structure of the channel was explored by inserting large side chain residues at four positions along the channel in BjPutA. Kinetic analysis of the different mutants revealed replacement of D779 with Tyr (D779Y) or Trp (D779W) significantly decreased the overall rate of the PRODH–P5CDH channeling reaction. X-ray crystal structures of D779Y and D779W revealed that the large side chains caused a constriction in the central section of the tunnel, thus likely impeding the travel of P5C/GSA in the channel. The D779Y and D779W mutants have PRODH activity similar to that of wild-type BjPutA but exhibit significantly lower P5CDH activity, suggesting that exogenous P5C/GSA enters the channel upstream of Asp779. Replacement of nearby Asp778 with Tyr (D778Y) did not impact BjPutA channeling activity. Consistent with the kinetic results, the X-ray crystal structure of D778Y shows that the main channel pathway is not impacted; however, an off-cavity pathway is closed off from the channel. These findings provide evidence that the off-cavity pathway is not essential for substrate channeling in BjPutA.

Lead tolerance mechanism in sterilized seedlings of Potamogeton crispus L.: Subcellular distribution, polyamines and proline

The effects of increasing concentrations of lead (Pb) on malondialdehyde (MDA) content, soluble protein, Pb accumulation, nutrients, polyamines (PAs) and proline metabolism were investigated in sterilized seedlings of Potamogeton crispus L. after 5d exposure. Significant oxidative stress was not caused, indicated by a little induction of MDA content and soluble proteins. Pb accumulation increased in a concentration-dependent manner and most of Pb was stored in the cell wall. Total P, Mg, Na and Zn rose and total Fe fell; total Ca increased at 25μM Pb but then declined. The nutrients in cell wall fraction changed in the same pattern as total nutrients, whereas those in soluble and organelle fraction declined. Total putrescine (Put) decreased markedly, while total spermidine (Spd), spermine (Spm) and (Spd+Spm)/Put ratio increased progressively but then declined. The trends for free, perchloric acid soluble conjugated (PS-conjugated) and perchloric acid insoluble bound (PIS-bound) PAs were similar to those on total PAs, except that PIS-bound Spm increased significantly. Arginine decarboxylase (ADC) and ornithine decarboxylase (ODC) activities rose gradually, while diamine oxidase (DAO) initially increased but then declined. Proline content increased initially only to decline later, due to the increase of r-glutamyl kinase (GK) activity. Meanwhile, ornithine-d-aminotransferase (OAT) activity gradually reduced, while no significant change was observed in proline dehydrogenase (PDH) activity. Our results indicated that the tolerance of P. crispus to Pb stress was based on cell wall compartmentalization combined with increase of nutrients, alterations of PAs, and induction of proline.

Biochemical characterization of proline dehydrogenase in Arabidopsis mitochondria.

Proline has multiple functions in plants. Besides being a building block for protein biosynthesis proline plays a central role in the plant stress response and in further cellular processes. Here, we report an analysis on the integration of proline dehydrogenase (ProDH) into mitochondrial metabolism in Arabidopsis thaliana. An experimental system to induce ProDH activity was established using cell cultures. Induction of ProDH was measured by novel photometric activity assays and by a ProDH in gel activity assay. Effects of increased ProDH activity on other mitochondrial enzymes were systematically investigated. Activities of the protein complexes of the respiratory chain were not significantly altered. In contrast, some mitochondrial dehydrogenases had markedly changed activities. Activity of glutamate dehydrogenase substantially increased, indicating upregulation of the entire proline catabolic pathway, which was confirmed by co-expression analyses of the corresponding genes. Furthermore, activity of d-lactate dehydrogenase was increased. d-lactate was identified to be a competitive inhibitor of ProDH in plants. We suggest that induction of d-lactate dehydrogenase activity allows rapid upregulation of ProDH activity during the short-term stress response in plants.

Effect of exogenous γ-aminobutyric acid (GABA) treatment on chilling injury and antioxidant capacity in banana peel

In order to evaluate the effects of γ-aminobutyric acid (GABA) on chilling injury (CI) of banana fruit, CI index, malondialdehyde (MDA), total phenolics, proline, the activities of peroxidase (POD), phenylalanine ammonia-lyase (PAL), Δ1-pyrroline-5-carboxy-late synthetase (P5CS), proline dehydrogenase (PDH), ferric reducing antioxidant potential (FRAP) assay and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity were analyzed after pretreated 20mM GABA vacuum infiltration and stored at 7°C for 20 days. GABA treatment retarded the increase of CI index, electrolyte leakage and MDA content, while promoted the accumulation of total phenolics and proline. GABA treatment significantly enhanced the activities of P5CS, POD and PAL, while inhibited the activity of PDH. FRAP assay and DPPH radical scavenging activity of GABA-treated banana fruit were significantly higher than control fruit in the first 10 days. The results suggest that application of GABA plays an important role in alleviating banana fruit chilling injury through proline accumulation and enhancement of antioxidant defense system. GABA treatment may act as a promising approach in inhibiting chilling injury and maintaining quality of banana fruit.

Context of action of Proline Dehydrogenase (ProDH) in the Hypersensitive Response of Arabidopsis

BackgroundProline (Pro) dehydrogenase (ProDH) potentiates the oxidative burst and cell death of the plant Hypersensitive Response (HR) by mechanisms not yet elucidated. ProDH converts Pro into ∆1 pyrroline-5-carboxylate (P5C) and can act together with P5C dehydrogenase (P5CDH) to produce Glu, or with P5C reductase (P5CR) to regenerate Pro and thus stimulate the Pro/P5C cycle. To better understand the effects of ProDH in HR, we studied the enzyme at three stages of the defense response differing in their ROS and cell death levels. In addition, we tested if ProDH requires P5CDH to potentiate HR.ResultsControl and infected leaves of wild type and p5cdh plants were used to monitor ProDH activity, in vivo Pro catabolism, amino acid content, and gene expression. Wild type plants activated ProDH at all HR stages. They did not consume Pro during maximal ROS accumulation, and maintained almost basal P5C levels at all conditions. p5cdh mutants activated ProDH as wild type plants. They achieved maximum oxidative burst and cell death levels producing normal HR lesions, but evidenced premature defense activation.ConclusionProDH activation has different effects on HR. Before the oxidative burst it leads to Pro consumption involving the action of P5CDH. During the oxidative burst, ProDH becomes functionally uncoupled to P5CDH and apparently works with P5CR. The absence of P5CDH does not reduce ROS, cell death, or pathogen resistance, indicating this enzyme is not accompanying ProDH in the potentiation of these defense responses. In contrast, p5cdh infected plants displayed increased ROS burst and earlier initiation of HR cell death. In turn, our results suggest that ProDH may sustain HR by participating in the Pro/P5C cycle, whose action on HR must be formally evaluated in a future.

Effects of exogenous ornithine on resistance of <i>Potamogeton crispus</i> L. to cadmium stress

本文以组织培养技术培养的菹草无菌苗为实验材料,研究了2 mmol/L外源鸟氨酸(L-Orn)对40μmol/L镉(Cd)胁迫下菹草体内Cd含量、光合色素水平、超氧阴离子(O₂^·-)产生速率、过氧化氢(H₂O₂)、丙二醛(MDA)与可溶性蛋白含量以及脯氨酸和多胺代谢的影响.结果显示:(1)在单一Cd胁迫下,菹草体内的Cd大量积累,达到335.00μg/g,并诱导了明显的氧化胁迫,具体表现为O₂^·-产生速率以及H₂O₂和MDA含量显著升高,可溶性蛋白以及光合色素含量明显下降.外源L-Orn的添加很大程度上抑制了菹草体内Cd的积累,Cd含量降至232.50μg/g,并降低了活性氧(ROS)水平,减轻了膜脂过氧化程度,减缓了光合色素和可溶性蛋白的分解.(2)在单一Cd胁迫下,伴随着吡咯啉-5-羧酸合成酶(P5CS)活性的增加和脯氨酸脱氢酶(ProDH)活性的下降,菹草体内的脯氨酸含量轻微增加,仅为对照组的1.91倍,而外源L-Orn显著增强了P5CS和δ-鸟氨酸转氨酶(OAT)的活性,使脯氨酸含量大幅度升高,为对照组的8.36倍.(3)单一Cd胁迫分别通过激活精氨酸脱羧酶(ADC)、鸟氨酸脱羧酶(ODC)和多胺氧化酶(PAO)的活性,使腐胺(Put)的含量增加,亚精胺(Spd)和精胺(Spm)含量减少.而施用外源L-Orn后,ADC和ODC的活性进一步增强,Put的含量大幅增加,并促进Put向Spd和Spm的转化,同时PAO的活性下降,使Spd和Spm含量增加,从而使总多胺含量增加.由此可见,外施L-Orn可通过抑制Cd的进入和参与调节脯氨酸以及多胺代谢,抑制ROS的积累、膜脂过氧化、光合色素以及蛋白质的分解,一定程度上增强菹草对Cd胁迫的抗性.;Sterile of Potamogeton crispus L. cultured by tissue culture technology, were used as experimental materials. In this study, the effects of 2 mmol/L exogenous ornithine (L-Orn) application on Cd accumulation, superoxide anion (O₂^·-) generation rate, photosynthetic pigments, hydrogen peroxide (H₂O₂), malondialdehyde (MDA) and soluble protein contents, as well as the metabolisms of proline and polyamines under 40 μmol/L Cd stress were investigated. Results indicated that (1) Cd addition alone promoted a substantial accumulation of Cd to 335.00 μg/g and apparently induced oxidative stress, specific performance as follows:obviously raised O₂^·- generation rate, H₂O₂ and MDA contents, as well as decreased photosynthetic pigments and soluble protein contents. Meanwhile, exogenous application of L-Orn markedly inhibited the accumulation of Cd to 232.50 μg/g, reduced ROS level, alleviated membrane lipid peroxidation and retarded the degradation of photosynthetic pigments and soluble protein. (2) Single Cd treatment slightly increased proline content to 1.91-fold than control by activating pyrroline-5-carboxylate (P5CS) activity and restraining proline dehydrogenase (ProDH) activity, whereas exogenous addition of L-Orn further activated P5CS and ornithine-δ-aminotransterase (OAT) activities, thus significantly increasing proline accumulation to 8.36-fold than control. (3)Cd exposure alone enhanced arginine decarboxylase (ADC), ornithine decarboxylase (ODC) and polyamine oxidase (PAO) activities, as a result, increasing putrescine (Put) content and reducing spermidine (Spd) and spermine (Spm) contents. However, adjunction of exogenous L-Orn sharply added Put content by strengthening ADC and ODC activities compared with single Cd treatment, and encouraged Put to transform into Spd and Spm, moreover reduced PAO activity, hence raised Spd and Spm contents, finally increased total polyamines content. The results demonstrated that the supplement of exogenous L-Orn restrained ROS accumulation, membrane lipid peroxidation and the degradation of photosynthetic pigments and soluble protein thought inhibiting the accumulation of Cd and involving in regulation of proline and polyamines metabolisms, consequently to a certain extent enhanced Cd stress tolerance of Potamogeton crispus L..

Effect of hydrogen peroxide on growth and activity of some enzymes involved in proline metabolism of sweet corn seedlings under copper stress

CuCl2 treatments led to a significant growth reduction, while pre-treatment with H2O2 increased plant growth compared to the control in sweet corn seedlings. The increment of the proline content in shoots followed in the order of H2O2 (6h)+CuCl2>H2O2>CuCl2>Control. During only H2O2 treatments the activities of Δ1-pyrroline-5-carboxylatesynthetase (P5CS) and glutamate dehydrogenase (GDH) of the glutamate path increased immediately, while Arginase and ornithine aminotransferase (OAT) of the ornithine path activities raised after 8h treatments. Under CuCl2 stress Arginase and OAT activities rose quickly, P5CS and GDH activities also increased after 8h treatments. The simultaneous increase of the proline content during CuCl2 stress might be related with the sequential activation of the ornithine and glutamate pathways and a reduction in ProDH. Pre-treatment with H2O2 induced an almost immediate and rapid increase of activities of Arginase and OAT, P5CS and GDH. The treatments also decreased the activity of proline dehydrogenase, a key enzyme of proline degradation. At the same time, pre-treatment with H2O2 induced an additional increase of the enzymes expression of ornithine pathway and glutamate pathway, while an additional decline was also observed in the expression of ProDH in sweet corn seedling under CuCl2 stress. While pre-treated with H2O2 under CuCl2 stress inducing proline content was a combined result of activation of the ornithine and glutamate pathways and a reduction in ProDH. It was, therefore, concluded that H2O2 ameliorated the stress generated by CuCl2 through the enhancing proline content. Overall, the adverse effects of CuCl2 stress could be alleviated by exogenous application of H2O2.

Involvement of compatible solutes in chill hardening-induced chilling tolerance in Jatropha curcas seedlings

Low temperature is a major environmental factor that affects metabolism, growth, development, distribution and production of chilling-sensitive plant, and J. curcas L. is a sustainable energy plant with great potential for biodiesel production due to the fact that its seed contains high oil content, which has attracted much attention worldwide. Our previous work found that the chill hardening improved the chilling tolerance of J. curcas seedlings (Ao et al. in Acta Physiologiae Plantarum 35:153–160, 2013), but its mechanism still remains elusive. In present work, the mechanism of chill hardening-induced chilling tolerance was further investigated in J. curcas seedlings. The results showed that chill hardening at 12 °C for 2 days markedly lowered osmotic and water potentials, which, in turn, maintained relative higher pressure potential in leaves of J. curcas seedlings compared with the control seedlings without chill hardening. In addition, chill hardening gradually increased compatible solutes proline, betaine and total soluble sugar contents compared with the control. When the control and hardened seedlings were subjected to chilling stress at 1 °C for 1–7 days, the chill-hardened seedlings significantly accumulated higher proline, betaine and total soluble sugar contents, which decreased osmotic and water potentials, and maintained higher pressure potential. To further understand the pathways of accumulation of compatible solutes, measurement of activities of ∆1-pyrroline-5-carboxylate synthetase (P5CS), glutamate dehydrogenase (GDH), ornithine aminotransferase (OAT), arginase, proline dehydrogenase (ProDH) and betaine dehydrogenase (BADH) showed that the chill hardening at 12 °C for 2 days obviously increased the activities of P5CS, GDH, OAT, arginase and BADH, as well as lowered ProDH activity both in leaves and stems of J. curcas seedlings to some extent as compared with the control. When the control and hardened seedlings were exposed to chilling stress at 1 °C for 1–7 days, the chill-hardened seedlings generally maintained significantly higher activities of P5CS, GDH, OAT, arginase and BADH. All above-mentioned results illustrated that the chill hardening could induce an accumulation of compatible solutes in leaves of J. curcas seedlings and compatible solutes play important roles in chill hardening-induced chilling tolerance.

Effect of Nitric Oxide on Antioxidative Response and Proline Metabolism in Banana during Cold Storage

The effect of exogenous nitric oxide (NO) on chilling injury to banana fruit was investigated. Banana fruit was treated with NO donor sodium nitroprusside of 0.05 mM at 20 °C for 10 min and then stored at 7 °C for up to 20 days. Banana fruit treated with NO sustained a lower chilling injury index and higher firmness and kept lower electrolyte leakage and malondialdehyde content than the control. Further investigation showed that NO treatment enhanced activities of guaiacol peroxidase, ascorbate peroxidase, and glutathione reductase compared to the control. It also maintained higher ascorbic acid, reduced glutathione content, and total antioxidant capacity but reduced hydrogen peroxide and superoxide anion to lower levels compared to control fruit during storage. NO treatment significantly enhanced the accumulation of total phenolics and proline, which resulted from the increased activities of phenylalanine ammonia-lyase and Δ¹-pyrroline-5-carboxylate synthetase and decreased proline dehydrogenase activity. We proposed that the enhanced chilling tolerance induced by NO treatment may result from the reduction of oxidative stress and proline accumulation.

Proline and its metabolism enzymes in cucumber cell cultures during acclimation to salinity

Proline is an important osmolyte appearing as the result of salt stress response of plants. In the present study, we measured the proline concentration, activities of pyrroline-5-carboxylate synthetase (P5CS), pyrroline-5-carboxylate reductase (P5CR), and proline dehydrogenase (PDH) key regulatory enzymes in the biosynthesis and degradation of proline in the acclimated (AC20) and the non-acclimated (NAC) cucumber cell suspension cultures subjected to moderate (150 mM NaCl; AC20-150, NAC-150, respectively) and severe (200 mM NaCl; AC20-200, NAC-200, respectively) salt stress. The data showed that salt stress brought about a linear increase in proline content in both types of cultures. However, in the acclimated culture proline accumulation was observed earlier, in third hour after stress. Only in the acclimated culture moderate and severe stresses up-regulated P5CS activity throughout the experiment, whereas the activity of P5CR grew in response to both NaCl concentrations only in 24th and 48th hour. The severe salt stress resulted in decrease in P5CR in NAC-200 cultures. In response to salt stress, both types of cell suspension cultures reacted with decline in PDH activity below the spectrophotometrically detected level. Cell cultures vigor correlated with salt concentration and time of exposure to the stress factor. Both NaCl concentrations caused linear decline in vigor of the non-acclimated culture up to 80-90 % at the end of the experiment, whereas in the acclimated culture significant decrease by about 30-40 % was reached in 24th hour after stress. The presented data suggest that acclimation to salt stress up-regulated proline synthesis enzyme activity and caused intensive accumulations of proline by inhibiting its oxidation.

ROS generation and proline metabolism in calli of halophyte Nitraria tangutorum Bobr. to sodium nitroprusside treatment

Nitric oxide (NO) is a stress factor or a signal molecule involved in various plant physiological and developmental processes. In the present study, the generation of reactive oxygen species and the metabolism of proline due to different sodium nitroprusside (SNP, an NO donor) concentrations were investigated in callus from halophyte Nitraria tangutorum Bobr. Treatment with SNP led to significant increases of hydrogen peroxide (H2O2) content and cell viability but notable reductions in hydrogen radical level and lipid peroxidation degree, and superoxide onion (O2 (-)) content also enhanced in 100 μM SNP-treated calli. Using a chemical inhibitor for plasma membrane (PM) NADPH oxidase diphenylene iodonium (DPI), we found low O2 (-) generation in untreated and 25 μM SNP-treated calli, whereas in those treated with 100 μM SNP O2 (-) level exhibited a very little alteration, comparable to the absence of DPI. These suggest a high activity of PM NADPH oxidase in untreated calli. H2O2 scavenging enzymes (catalase, peroxidase [POD] and ascorbate peroxidase) and H2O2 forming enzymes (superoxide dismutase [SOD], cell wall-POD and diamine oxidase [DAO]) stimulated significantly in calli treated with different SNP concentrations while glutathione reductase activity decreased. In addition, a reduction in proline content was observed in SNP-treated calli. Moreover, different SNP concentrations stimulated proline dehydrogenase (PDH) and ornithine δ-aminotransferase but inhibited r-glutamyl kinase (GK). In conclusion, our results suggest that the increasing H2O2 generation was associated with the stimulation of SOD, cell wall-POD and DAO, and that the reduction of proline content might be the consequence of increased PDH activity and decreased GK activity in N. tangutorum Bobr. calli under SNP treatment.

Pre-storage application of oxalic acid alleviates chilling injury in mango fruit by modulating proline metabolism and energy status under chilling stress

Effects of oxalic acid on chilling injury, proline metabolism and energy status in mango fruit were investigated after mango fruit (Mangifera indica L. cv. Zill) were dipped in 5mM oxalic acid solution for 10min at 25°C and then stored at low temperature (10±0.5°C) for 49days thereafter transferred to 25°C for 4days. Pre-storage application of oxalic acid apparently inhibited the development of chilling injury, notably elevated proline accumulation actually associated with increase in Δ1-pyrroline-5-carboxylate synthetase (P5CS) activity and decrease in proline dehydrogenase (PDH) activity in the peel and the flesh, without activation of ornithine-δ-aminotransferase (OAT) activity, and maintained high ATP level and energy charge in the flesh during storage. It was suggested that these effects of oxalic acid might collectively contribute to improving chilling tolerance, thereby alleviating chilling injury and maintaining quality of mango fruit in long term cold storage.

Role of Arbuscular Mycorrhizal Symbiosis in Proline Biosynthesis and Metabolism of Cicer arietinum L. (Chickpea) Genotypes Under Salt Stress

Salinity causes osmotic stress and negatively impacts plant growth and productivity. Proline is one of the most important osmoprotectants synthesized under stressed conditions. Accumulation of free proline occurs due to enhanced biosynthesis and repressed degradation, and both processes are controlled by feedback regulatory mechanisms. Arbuscular mycorrhizal (AM) fungi are considered to be bioameliorators of salinity stress due to their wide-ranging presence in contaminated soils and their role in modulation of biochemical processes. Chickpea is considered sensitive to salinity. However, reports on AM-induced osmoprotection through regulation of proline biosynthesis in chickpea genotypes are scant. The present study investigated the influence of AM symbiosis on proline metabolism in two chickpea (Cicer arietinum L.) genotypes (PBG-5 and CSG-9505) under salt stress and correlated the same with sodium (Na+) ion uptake. Salinity reduced plant biomass (roots and shoots), with roots being more negatively affected than shoots. Mycorrhizal colonization with Glomus mosseae was much stronger in PBG-5 and was correlated with reduced Na+ ion uptake and higher growth when compared with CSG-9505 under stressed and unstressed conditions. Mycorrhizal symbiosis with chickpea roots boosted proline biosynthesis by significantly increasing pyrroline-5-carboxylate synthetase (P-5-CS) and glutamate dehydrogenase (GDH) activities with a concomitant decline in proline dehydrogenase (ProDH) activity under salt stress. The enhancement of the activity of these enzymes was higher in PBG-5 than in CSG-9505 and could be directly correlated with the percent mycorrhizal colonization and Na+ uptake. The study indicated a strong role of AM symbiosis in enhancing stress tolerance in chickpea by significantly modulating proline metabolism and Na+ uptake.

Mechanism of Cu- and Cd-Induced Proline Hyperaccumulation in Triticum aestivum (Wheat)

Proline hyperaccumulation, a frequently reported phenomenon in the plant kingdom, is considered an adaptive strategy of plants to cope with a variety of stresses. The present study demonstrated the mechanism of proline hyperaccumulation in Triticum aestivum exposed to excess Cu and Cd. The intracellular level of proline was increased significantly in the plants treated with test metals. Enhanced proline accumulation due to Cu and Cd was negatively correlated with the growth of the treated plants, indicating that plants divert their resources toward induction of survival strategies rather than improving growth and development. The activities of pyrroline-5-carboxylate synthetase (P5CS) and pyrroline-5-carboxylate reductase (P5CR) were increased immediately after metal treatment and remained higher through the end of the experiment, whereas ornithine amino transferase (OAT) activity of the treated plants was lower than P5CS and P5CR enzymes. The activity of proline dehydrogenase (PDH) was decreased sharply in the early phase of metal exposure (up to 12 h) but remained unchanged thereafter until the end of the experiment. Expression analysis of the transcripts of P5CS, OAT, and PDH also supports the above findings and indicates the tissue-specific expression of these genes. Based on these results, increased proline synthesis through the glutamate pathway involving the P5CS and P5CR enzymes is a major contributor to proline hyperaccumulation followed by decreased catabolism of proline.

Hydrogen sulfide donor sodium hydrosulfide-improved heat tolerance in maize and involvement of proline

Hydrogen sulfide (H2S) has long been considered as a phytotoxin, but nowadays as a cell signal molecule involved in growth, development, and the acquisition of stress tolerance in higher plants. In the present study, hydrogen sulfide donor, sodium hydrosulfide (NaHS), pretreatment markedly improved germination percentage of seeds and survival percentage of seedlings of maize under heat stress, and alleviated an increase in electrolyte leakage of roots, a decrease in tissue vitality and an accumulation of malondialdehyde (MDA) in coleoptiles of maize seedlings. In addition, pretreatment of NaHS could improve the activity of Δ1-pyrroline-5-carboxylate synthetase (P5CS) and lower proline dehydrogenase (ProDH) activity, which in turn induced accumulation of endogenous proline in maize seedlings. Also, application of proline could enhance endogenous proline content, followed by mitigated accumulation of MDA and increased survival percentage of maize seedlings under heat stress. These results suggest that sodium hydrosulfide pretreatment could improve heat tolerance of maize and the acquisition of this heat tolerance may be involved in proline.

Expression, Purification and Characterization of the Proline Dehydrogenase Domain of PutA from Pseudomonas putida POS-F84

The objective of the present work was to express a truncated form of Pseudomonas putida PutA that shows proline dehydrogenase (ProDH) activity. The putA gene encoding ProDH enzyme was cloned into pET23a vector and expressed in Escherichia coli strain BL-21 (DE3) plysS. The recombinant P. putida enzyme was biochemically characterized and its three dimensional structure was also predicted. ProDH encoding sequence showed an open reading frame of 1,035-bp encoding a 345 amino acid residues polypeptide chain. Purified His-tagged enzyme gave a single band with a molecular mass of 40kDa on SDS-PAGE. The molecular mass of the isolated enzyme was found to be about 40kDa by gel filtration. This suggested that the enzyme of interest consists of one subunit. The K m and V max values of recombinant P. putida ProDH were estimated to be 31mM and 132μmol/min, respectively. The optimum pH and temperature for the catalytic activity of the enzyme was about pH 8.5 and 30°C. The modeling analysis of the three dimensional structure elucidated that Ser-165, Lys-195 and Ala-252 were key residues for the ProDH activity. This study provides data on the cloning, sequencing and recombinant expression of PutA ProDH domain from P. putida POS-F84.

Dissecting the stress metabolic alterations in in vitro Cyrtanthus regenerants

Metabolic plasticity in plants allows for continuous adjustments of defence strategies in suboptimal environments. Proline and other metabolites figure prominently in most stress-mediated responses. This study examined the expression of salinity and osmotic adjustments in the enzymatic activity and accumulation of solutes and metabolites in response to imposed water and salt stress in Cyrtanthus contractus N.E. Br. and Cyrtanthus guthrieae L. Bolus regenerants. Invitro-derived plantlets were cultured on solid Murashige and Skoog (MS) media with three different polyethylene glycol (PEG)-induced osmotic levels and four NaCl stress-induced levels at 25°C. The levels of proline and phenolic compounds measured at intervals of three, four and five weeks from initial plantlet culture increased in a stress-dependent pattern. The levels of these metabolites also showed a significant increase with an increase in the duration of plantlets under stress conditions. The highest proline concentration (9.98μmolg(-1) FW) was recorded in C.contractus at 300μM NaCl after five weeks. A corresponding high level of total phenolic compounds (147mg GAEg(-1) DW) was also recorded in the same treatment for the same species. The activity of proline dehydrogenase (PDH) (EC 1.5.99.8) was shown to decrease with an increase in proline levels from week three to week five in almost all the stress conditions. The high levels, particularly of phenolic compounds obtained under osmotic and salinity stress conditions in this study present a promising potential of manipulating culture and/or growing conditions for improved secondary compound production and hence medicinal benefits.

Differential performance of two forage species, Medicago truncatula and Sulla carnosa, under water-deficit stress and recovery

The response patterns during water deficit stress and subsequent recovery of two forage species, Medicago truncatula and Sulla carnosa, were studied. After germination and pre-treatment, seedlings were individually cultivated for two months under two irrigation modes: 100% and 33% of field capacity. Measured parameters were plant growth, water relations, leaf osmotic potential, lipid peroxidation, and leaf inorganic (Na+ and K+) and organic (proline and soluble sugars) solute contents, as well as delta-1-pyrroline-5-carboxylate synthase (P5CS) and proline dehydrogenase (PDH) activities. Our results showed that under control conditions, and in contrast to roots, no significant differences were observed in shoot biomass production between the two species. However, when subjected to water-deficit stress, M. truncatula appeared to be more tolerant than S. carnosa (reduction by 50 and 70%, respectively). In the two studied species, water-deficit stress led to an increase in root/shoot ratio and leaf proline and soluble sugar contents, and a decrease in leaf osmotic potential. Enzymatic assay revealed that in the two species, P5CS activity was stimulated whereas that of PDH was inhibited under stress conditions. Despite greater accumulation of proline, sugar, and potassium in leaves of S. carnosa, M. truncatula was more tolerant to water deficit. This was essentially due to its capacity to control tissue hydration and water-use efficiency, in addition to its greater ability to protect membrane integrity. Following stress relief, M. truncatula and S. carnosa showed partial re-establishment of growth capacity.

Zinc-induced oxidative damage, antioxidant enzyme response and proline metabolism in roots and leaves of wheat plants

The purpose of this study was to evaluate antioxidative responses and proline metabolism in roots and leaves of wheat seedlings after treatment with different zinc (Zn) concentrations (0, 0.5, 1 and 3mM) for 6 days. A notable reduction in Zn content was observed in 0.5mM Zn-treated leaves, but a significant elevation in response to 1 and 3mM Zn treatment. Significant increases in Zn levels were observed in roots exposed to all applied Zn concentration. The highest Zn concentration resulted in significant reduction in the amount of total chlorophyll (chl) and chl a, while chl b content decreased under all applied Zn concentrations. In wheat leaves, Zn excess caused an insignificant enhancement of hydrogen peroxide (H2O2) content as well as unaltered malondialdehyde (MDA) level. Unchanged superoxide dismutase (SOD) activity, increased peroxidase (POD), catalase (CAT), glutathione reductase (GR) and ascorbate peroxidase (APX) activities were also observed in the leaves of Zn-treated seedlings. By contrast, higher H2O2 and MDA contents in Zn-treated roots were correlated with the stimulation of SOD and the inhibition of POD and GR. There were significant enhancements of soluble sugar and proline in both leaves and roots of wheat seedlings under Zn stress, but the increased rate of proline was higher in the roots than in the leaves. Differently, soluble protein content due to Zn treatment was lower in the leaves but higher in the roots, as compared with untreated seedlings. Additionally, ornithine δ-aminotransferase in both leaves and roots was stimulated by Zn stress, but different Zn concentrations exhibited inhibitory effect on glutamate kinase activity in wheat seedlings. In contrast, all applied Zn concentration resulted in an elevation of proline dehydrogenase activity in the leaves while the highest Zn concentration inhibited this parameter in the roots.