Proline Reduction Research Articles

Halotolerant and plant growth-promoting endophytic fungus Aspergillus terreus CR7 alleviates salt stress and exhibits genoprotective effect in Vigna radiata.

Soil salinity is one of the major environmental stresses that results in reduction of cultivable land and decreased productivity. In the present study, halotolerant and plant growth-promoting endophytic fungi were isolated from Catharanthus roseus, and their effect in mitigating salt stress in Vigna radiata was evaluated. An isolate CR7, identified to be Aspergillus terreus, showing plant growth promotion activities, viz. IAA production (23.43 ± 0.79 μg/ml), phosphate solubilization (133.63 ± 6.40 μg/ml), ACC deaminase activity (86.36 ± 2.70 μmol α-ketobutyrate/h/mg protein) etc. and ability to grow at 15% NaCl was selected for further in vivo studies. Colonization of CR7 was carried out in V. radiata which was subjected to different concentrations of salt (150, 200, and 250 mM NaCl). Under salt stress, A. terreus CR7 inoculated plants showed substantially improved root and shoot length, biomass, chlorophyll content, relative water content, phenolics, protein content, and DPPH scavenging activity. Endogenous IAA level was enhanced by 5.28-fold in treated plants at maximum salt stress. Inoculation of A. terreus CR7 affected oxidative stress parameters, exhibiting an increase in catalase and superoxide dismutase and reduction in proline, electrolyte leakage, and malondialdehyde content. Fluorescent microscopic analysis of roots revealed improved cell viability and decreased levels of glutathione and hydrogen peroxide under salt stress in treated plants. The isolate A. terreus CR7 also protected against DNA damage induced by salt stress which was evaluated using comet assay. A decrease in DNA tail length, tail moment, and olive tail moment to the extent of 19.87%, 19.76%, and 24.81%, respectively, was observed in A. terreus CR7-colonized plants under salt stress. It can be concluded that A. terreus CR7 can be exploited for alleviating the impact of salt stress in crop plants.

Silver nanoparticles enhance the mitigation of osmotic stress in Chenopodium quinoa microshoots grown under in vitro osmo-stressing conditions.

Osmotic stress is one of the main destructive abiotic factors that hinder plant growth and development. In this research, the role of silver nanoparticles (Ag NPs) in mitigating the negative impact of osmotic stress on in vitro grown Chenopodium quinoa (Quinoa 6 Line; Q6) was investigated to determine whether Ag NPs were able to reduce the negative effects on the in vitro grown cultures of the Q6 line. The explants were subcultured onto a special osmostressing media containing sucrose, sorbitol, or mannitol at different levels (0.1, 0.2, 0.3, and 0.4 mol/L) to mimic the osmotic stressing environment for four weeks. Then, stress physiological responses of in vitro grown Q6 under the induced osmotic stress were investigated to determine the highest stress level that the microshoots could tolerate. Next, Ag NPs; 25, 50, and 75 mg/L were added to the medium that contained the highest stress level of the induced osmotic stress to determine if their addition improved the physiological performance of the Q6 microshoots under the most severe osmotic agent levels. The results revealed that 0.4 mol/L sucrose, 0.3 mol/L sorbitol, and 0.3 mol/L mannitol were the highest stress levels that the microshoots could tolerate. The addition of 75 mg/L Ag NPs to the previous highest stress levels resulted in a significant increase in the following: stem length (SL), leaves number (LN), fresh weight (FW), dry weight (DW), total chlorophyll, protein, calcium (Ca), and phosphorus (P) contents, while it caused a reduction in proline, sodium (Na) ions, and potassium (K) ions. These results indicate that the negative consequences of osmotic stress on Q6 quinoa microshoots could be mitigated by adding specific concentrations of Ag NPs to the culture medium.

Isolation of Yeast Strains with Higher Proline Uptake and Their Applications to Beer Fermentation.

Although proline is the most or second most abundant amino acid in wort and grape must, it is not fully consumed by the yeast Saccharomyces cerevisiae during alcoholic fermentation, unlike other amino acids. Our previous studies showed that arginine, the third most abundant amino acid in wort, inhibits the utilization of proline in most strains of S. cerevisiae. Furthermore, we found that some non-Saccharomyces yeasts utilized proline in a specific artificial medium with arginine and proline as the only nitrogen source, but these yeasts were not suitable for beer fermentation due to their low alcohol productivity. For yeasts to be useful for brewing, they need to utilize proline and produce alcohol during fermentation. In this study, 11 S. cerevisiae strains and 10 non-Saccharomyces yeast strains in the Phaff Yeast Culture Collection were identified that utilize proline effectively. Notably, two of these S. cerevisiae strains, UCDFST 40-144 and 68-44, utilize proline and produce sufficient alcohol in the beer fermentation model used. These strains have the potential to create distinctive beer products that are specifically alcoholic but with a reduction in proline in the finished beer.

Exogenous Allantoin Confers Rapeseed (Brassica campestris) Tolerance to Simulated Drought by Improving Antioxidant Metabolism and Physiology.

Allantoin is an emerging plant metabolite, but its role in conferring drought-induced oxidative stress is still elusive. Therefore, an experiment was devised to explore the role of allantoin (0.5 and 1.0 mM; foliar spray) in rapeseed (Brassica campestris cv. BARI Sarisha-17) under drought. Seedlings at fifteen days of age were subjected to drought, maintaining soil moisture levels at 50% and 25% field capacities, while well-irrigated plants served as the control group. Drought-stressed plants exhibited increased levels of lipid peroxidation and hydrogen peroxide, electrolyte leakage, and impaired glyoxalase systems. Thus, the growth, biomass, and yield attributes of rapeseed were significantly impaired under drought. However, the allantoin-supplemented plants showed a notable increase in their contents of ascorbate and glutathione and decreased dehydroascorbate and glutathione disulfide contents under drought. Moreover, the activity of antioxidant enzymes such as ascorbate peroxidase, dehydroascorbate reductase, glutathione reductase, glutathione peroxidase, and catalase were accelerated with the allantoin spray and the glyoxalase system was also enhanced under drought. Moreover, the improvement in water balance with reduction in proline and potassium ion contents was also observed when allantoin was applied to the plants. Overall, the beneficial effects of allantoin supplementation resulted in the improved plant growth, biomass, and yield of rapeseed under drought conditions. These findings suggest that allantoin acts as an efficient metabolite in mitigating the oxidative stress caused by reactive oxygen species by enhancing antioxidant defense mechanisms and the glyoxalase system.

Influence of Thermotolerant Rhizobacteria Bacillus spp. on Biochemical Attributes and Antioxidant Status of Mustard Under High Temperature Stress

Due to global warming, increasing incidences of higher-than-normal temperatures have been observed, which adversely affect seed germination, crop growth, and productivity. Several reports are available on the effect of inoculation with rhizobacteria on plant growth and biochemical attributes; however, information on their influence on seed germination and plant stress levels is lacking. In the present study, under heat stress, we studied the effect of three thermotolerant rhizobacterial strains on mustard seed germination, seedling vigor, and plant growth. Effect of inoculation with the rhizobacterial strains on the plant stress levels, biochemical attributes and antioxidant activity was also determined. Under heat stress, inoculation with the rhizobacterial strains improved seed germination and seedling fresh weight and plumule length; while only Bacillus licheniformis SSA 61 inoculated plants showed better radicle length. There was a concomitant decrease in the plant ethylene levels in the inoculated treatments. Inoculated plants showed higher shoot fresh weight, however, Bacillus sp. MRD-17 inoculated plants only improved root growth. There was significant increase in most of the plant biochemical parameters and activities of antioxidant enzymes superoxide dismutase, catalase, and ascorbate peroxidase. Significant reduction in proline and total sugar content was noted in the inoculated treatments; while increase in the amino acid and phenolics content was observed. A further increase in the antioxidant enzyme activity was recorded in most of the inoculated treatments compared with no stress. Thus, our study indicated that thermotolerant rhizobacterial strains reduced plant stress levels; enhanced seed germination, seedling vigor, plant biomass, and thermotolerance of mustard.

Clostridioides difficile minimal nutrient requirements for flagellar motility.

As many gastro-intestinal pathogens, the majority of Clostridioides difficile strains express flagella together with a complete chemotaxis system. The resulting swimming motility is likely contributing to the colonization success of this important pathogen. In contrast to the well investigated general energy metabolism of C. difficile, little is known about the metabolic requirements for maintaining the ion motive force across the membrane, which in turn powers the flagellar motor. We studied here systematically the effect of various amino acids and carbohydrates on the swimming velocity of C. difficile using video microscopy in conjunction with a software based quantification of the swimming speed. Removal of individual amino acids from the medium identified proline and cysteine as the most important amino acids that power swimming motility. Glycine, which is as proline one of the few amino acids that are reduced in Stickland reactions, was not critical for swimming motility. This suggests that the ion motive force that powers the flagellar motor, is critically depending on proline reduction. A maximal and stable swimming motility was achieved with only four compounds, including the amino acids proline, cysteine and isoleucine together with a single, but interchangeable carbohydrate source such as glucose, succinate, mannose, ribose, pyruvate, trehalose, or ethanolamine. We expect that the identified "minimal motility medium" will be useful in future investigations on the flagellar motility and chemotactic behavior in C. difficile, particularly for the unambiguous identification of chemoattractants.

Use of a Biostimulant to Mitigate the Effects of Excess Salinity in Soil and Irrigation Water in Tomato Plants.

Global warming is linked to progressive soil salinisation, which reduces crop yields, especially in irrigated farmland on arid and semiarid regions. Therefore, it is necessary to apply sustainable and effective solutions that contribute to enhanced crop salt tolerance. In the present study, we tested the effects of a commercial biostimulant (BALOX®) containing glycine betaine (GB) and polyphenols on the activation of salinity defense mechanisms in tomato. The evaluation of different biometric parameters and the quantification of biochemical markers related to particular stress responses (osmolytes, cations, anions, oxidative stress indicators, and antioxidant enzymes and compounds) was carried out at two phenological stages (vegetative growth and the beginning of reproductive development) and under different salinity conditions (saline and non-saline soil, and irrigation water), using two formulations (different GB concentrations) and two doses of the biostimulant. Once the experiments were completed, the statistical analysis revealed that both formulations and doses of the biostimulant produced very similar effects. The application of BALOX® improved plant growth and photosynthesis and assisted osmotic adjustment in root and leaf cells. The biostimulant effects are mediated by the control of ion transport, reducing the uptake of toxic Na+ and Cl- ions and favoring the accumulation of beneficial K+ and Ca2+ cations, and a significant increase in leaf sugar and GB contents. BALOX® significantly reduced salt-induced oxidative stress and its harmful effects, as evidenced by a decrease in the concentration of oxidative stress biomarkers, such as malondialdehyde and oxygen peroxide, which was accompanied by the reduction of proline and antioxidant compound contents and the specific activity of antioxidant enzymes with respect to the non-treated plants.

Proline Reduction and Increasing Growth of Vanilla Plants Induced by BNR Fungus with Dose of Goat Manure and Husk Charcoal

Goat manure and husk charcoal are agricultural wastes that have not been used optimally, contain soil microorganisms that are effective in converting nutrients into more perfect forms, play a role in improving soil physical properties and increase the ability of soil to bind nutrients. The purpose of this study is to determine the effect of goat manure and husk charcoal to proline reduction and increase growth of vanilla plant induced by Binucleate Rhizoctonia (BNR). The study was designed with a factorial completely randomized block design. The first factor is the dose of manure (0,5,10,15) g/plant, the second factor is the dose of husk charcoal (0,5, 10, 15) g/plant. Data were analyzed by analysis of variance ANOVA with Duncan's Multiple Distance Test (DMRT) with a significant difference of 5%. The effect of doses of goat manure and husk charcoal induced by BNR can reduce proline contents and increase growth of vanilla plant. The benefit of this research is to provide treatment information to reduce proline levels and increase plant growth with the application of goat fertilizer and husk charcoal.

Changes in the growth, nutritional quality and gut microbiota of the adult Chinese mitten crab, Eriocheir sinensis , following overwinter rearing

Every autumn, large numbers of Chinese mitten crabs, Eriocheir sinensis, are sold, creating a backlog in availability reducing their sales. Crabs require a period of controlled rearing prior to their sale, which alleviates this fall backlog. This means that it has become increasingly important to establish an effective method for the overwinter rearing of E. sinensis. This study was designed to evaluate changes in the growth, nutritional quality and the composition of the gut microbiota of adult E. sinensis following overwinter rearing. The results revealed that the hepatosomatic index (HIS) significantly decreased while the gonadosomatic index (GSI) increased after overwinter rearing (p < 0.05) for 80 days. Percentages of total saturated fatty acid (ΣSFA) and monounsaturated fatty acid (ΣMUFA) in the three edible tissues (muscle, gonad and hepatopancreas) decreased after 80 days of overwintering. In addition, the amino acid content in all the three edible tissues diminished additionally, with the reduction in proline being the most significant (p < 0.05). Analyses demonstrated that overwintering affected the overall microbial community composition, and elevated the abundance of Candidatus Bacilloplasma; however, these changes were not significant (p > 0.05). Therefore, our results provide a key reference standard for the overwinter rearing of E. sinensis.

Effect of the Pesticide Endosulfan and Two Different Biostimulants on the Stress Responses of Phaseolus leptostachyus Plants Grown in a Saline Soil

Soil salinity and the indiscriminate use of agrochemicals has significantly reduced the productivity of the ‘Chinampas’ agroecosystem in Mexico City. Crop improvement under these stressful conditions may be achieved by soil bioremediation. In this study, we checked the effects of the organochlorine pesticide endosulfan and bioremediation with Penicillium crustosum or a citric waste on the growth of Phaseolus leptostachyus plants in saline soil from the Chinampas area. Biochemical markers associated with specific stress responses were also determined after one month of growth in the different substrates. Plant growth was stimulated by bioremediation of the soil. Both biostimulants reduced the degree of stress affecting the plants, as shown by the increase in photosynthetic pigments and the reduction of proline, malondialdehyde (MDA), and H2O2 contents, and the activation of antioxidant systems. However, the biostimulants appeared to mitigate oxidative stress through different mechanisms. Endosulfan contamination inhibited seed germination—which was reverted to control values in the presence of the biostimulants—and further decreased plant growth. No clear patterns of variation of biochemical stress markers were observed combining endosulfan and the biostimulants. In any case, bioremediation with P. crustosum and/or citric waste is recommended to improve the germination and growth of P. leptostachyus plants.

Unique genomic traits for cold adaptation in Naganishia vishniacii, a polyextremophile yeast isolated from Antarctica.

Cold environments impose challenges to organisms. Polyextremophile microorganisms can survive in these conditions thanks to an array of counteracting mechanisms. Naganishia vishniacii, a yeast species hitherto only isolated from McMurdo Dry Valleys, Antarctica, is an example of a polyextremophile. Here we present the first draft genomic sequence of N. vishniacii. Using comparative genomics, we unraveled unique characteristics of cold associated adaptations. 336 putative genes (total: 6183) encoding solute transfers and chaperones, among others, were absent in sister species. Among genes shared by N. vishniacii and its closest related species we found orthologs encompassing possible evidence of positive selection (dN/dS>1). Genes associated with photoprotection were found in agreement with high solar irradiation exposure. Also genes coding for desaturases and genomic features associated with cold tolerance (i.e. trehalose synthesis and lipid metabolism) were explored. Finally, biases in amino acid usage (namely an enrichment of glutamine and a trend in proline reduction) were observed, possibly conferring increased protein flexibility. To the best of our knowledge, such a combination of mechanisms for cold tolerance has not been previously reported in fungi, making N. vishniacii a unique model for the study of the genetic basis and evolution of cold adaptation strategies.

Plant growth-promoting Bacillus sp. strain SDA-4 confers Cd tolerance by physio-biochemical improvements, better nutrient acquisition and diminished Cd uptake in Spinacia oleracea L.

Cadmium (Cd) is highly toxic metal for plant metabolic processes even in low concentration due to its longer half-life and non-biodegradable nature. The current study was designed to assess the bioremediation potential of a Cd-tolerant phytobeneficial bacterial strain Bacillus sp. SDA-4, isolated, characterized and identified from Chakera wastewater reservoir, Faisalabad, Pakistan, together with spinach (as a test plant) under different Cd regimes. Spinach plants were grown with and without Bacillus sp. SDA-4 inoculation in pots filled with 0, 5 or 10mgkg-1 CdCl2-spiked soil. Without Bacillus sp. SDA-4 inoculation, spinach plants exhibited reduction in biomass accumulation, antioxidative enzymes and nutrient retention. However, plants inoculated with Bacillus sp. SDA-4 revealed significantly augmented growth, biomass accumulation and efficiency of antioxidative machinery with concomitant reduction in proline and MDA contents under Cd stress. Furthermore, application of Bacillus sp. SDA-4 assisted the Cd-stressed plants to sustain optimal levels of essential nutrients (N, P, K, Ca and Mg). It was inferred that thecharacterized Cd-tolerant PGPR strain, Bacillus sp. SDA-4 has a potential to reduce Cd uptake and lipid peroxidation which in turn maintained the optimum balance of nutrients and augmented the growth of Cd-stressed spinach. Analysis of bioconcentration factor (BCF) and translocation factor (TF) revealed that Bacillus sp. SDA-4 inoculation with spinach sequestered Cd in rhizospheric zone. Research outcomes are important for understanding morpho-physio-biochemical attributes of spinach-Bacillus sp. SDA-4 synergy which might provide efficient strategies to decrease Cd retention in edible plants and/or bioremediation of Cd polluted soil colloids.

Melatonin Counteracts Drought Induced Oxidative Damage and Stimulates Growth, Productivity and Fruit Quality Properties of Tomato Plants.

Melatonin “N-Acetyl-5-methoxytryptamine” (MT) has recently been considered as a new plant growth regulator with multiple physiological functions. Although many previous studies have confirmed that exogenous applied-MT can alleviate the deleterious effects of drought stress in many plant species, most of these studies were exclusive on seeds, seedlings, and young plants for a short period of their life cycles. Therefore, the knowledge of using MT as a potential promising agricultural foliar application to improve crop productivity and quality is still insufficient under adverse open field conditions. In this study, we investigated the effect of MT as a foliar application at 0, 20, and 40 ppm on tomato plants that were grown in the open field under the long term of optimal and deficit irrigation conditions. The results indicated that exogenous MT significantly enhanced plant growth, chlorophyll and activities of antioxidant enzymes, including ascorbate peroxidase (APX), catalase (CAT), and peroxidase (POX). This improvement was associated with a marked reduction in proline and soluble sugars. In addition, applied-MT worked as a protective agent against oxidative damage by reducing the cellular content of toxic substances such as H2O2 and malondialdehyde (MDA). Similarly, MT-treated plants showed greater total fruit yield with improving its quality attributes like total soluble solids (TSS), ascorbic acid, and lycopene. Generally, the highest significant fruit yield either under well-watered (13.7%) or water deficit (37.4%) conditions was achieved by the treatment of 20 ppm MT. These results indicate that exogenous MT played an essential role in enhancing tomato tolerance to deficit irrigation and could be recommended as a promising agricultural treatment under such conditions.

Above-Belowground Herbivore Interactions in Mixed Plant Communities Are Influenced by Altered Precipitation Patterns.

Root- and shoot-feeding herbivores have the capacity to influence one another by modifying the chemistry of the shared host plant. This can alter rates of nutrient mineralization and uptake by neighboring plants and influence plant–plant competition, particularly in mixtures combining grasses and legumes. Root herbivory-induced exudation of nitrogen (N) from legume roots, for example, may increase N acquisition by co-occurring grasses, with knock-on effects on grassland community composition. Little is known about how climate change may affect these interactions, but an important and timely question is how will grass–legume mixtures respond in a future with an increasing reliance on legume N mineralization in terrestrial ecosystems. Using a model grass–legume mixture, this study investigated how simultaneous attack on lucerne (Medicago sativa) by belowground weevils (Sitona discoideus) and aboveground aphids (Acyrthosiphon pisum) affected a neighboring grass (Phalaris aquatica) when subjected to drought, ambient, and elevated precipitation. Feeding on rhizobial nodules by weevil larvae enhanced soil water retention under ambient and elevated precipitation, but only when aphids were absent. While drought decreased nodulation and root N content in lucerne, grass root and shoot chemistry were unaffected by changes in precipitation. However, plant communities containing weevils but not aphids showed increased grass height and N concentrations, most likely associated with the transfer of N from weevil-attacked lucerne plants containing more nodules and higher root N concentrations compared with insect-free plants. Drought decreased aphid abundance by 54% but increased total and some specific amino acid concentrations (glycine, lysine, methionine, tyrosine, cysteine, histidine, arginine, aspartate, and glutamate), suggesting that aphid declines were being driven by other facets of drought (e.g., reduced phloem hydraulics). The presence of weevil larvae belowground decreased aphid numbers by 30%, likely associated with a significant reduction in proline in weevil-treated lucerne plants. This study demonstrates how predicted changes to precipitation patterns and indirect interactions between herbivores can alter the outcome of competition between N-fixing legumes and non-N-fixing grasses, with important implications for plant community structure and productivity.

Targeted modification of storage protein content resulting in improved amino acid composition of barley grain.

C-hordein in barley and ω-gliadins in wheat are members of the prolamins protein families. Prolamins are the major component of cereal storage proteins and composed of non-essential amino acids (AA) such as proline and glutamine therefore have low nutritional value. Using double stranded RNAi silencing technology directed towards C-hordein we obtained transgenic barley lines with up to 94.7% reduction in the levels of C-hordein protein relative to the parental line. The composition of the prolamin fraction of the barley parental line cv. Golden Promise was resolved using SDS-PAGE electrophoresis, the protein band were excised and the proteins identified by quadrupole-time-of-flight mass spectrometry. Subsequent SDS-PAGE separation and analysis of the prolamin fraction of the transgenic lines revealed a reduction in the amounts of C-hordeins and increases in the content of other hordein family members. Analysis of the AA composition of the transgenic lines showed that the level of essential amino acids increased with a concomitant reduction in proline and glutamine. Both the barley C-hordein and wheat ω-gliadin genes proved successful for RNAi-gene mediated suppression of barley C-hordein level. All transgenic lines that exhibited a reduction for C-hordein showed off-target effects: the lines exhibited increased level of B/γ-hordein while D-hordein level was reduced. Furthermore, the multicopy insertions correlated negatively with silencing.

Variation in protein intake induces variation in spider silk expression.

BackgroundIt is energetically expensive to synthesize certain amino acids. The proteins (spidroins) of spider major ampullate (MA) silk, MaSp1 and MaSp2, differ in amino acid composition. Glutamine and proline are prevalent in MaSp2 and are expensive to synthesize. Since most orb web spiders express high proline silk they might preferentially attain the amino acids needed for silk from food and shift toward expressing more MaSp1 in their MA silk when starved.Methodology/Principal FindingsWe fed three spiders; Argiope aetherea, Cyrtophora moluccensis and Leucauge blanda, high protein, low protein or no protein solutions. A. aetherea and L. blanda MA silks are high in proline, while C. moluccesnsis MA silks are low in proline. After 10 days of feeding we determined the amino acid compositions and mechanical properties of each species' MA silk and compared them between species and treatments with pre-treatment samples, accounting for ancestry. We found that the proline and glutamine of A. aetherea and L. blanda silks were affected by protein intake; significantly decreasing under the low and no protein intake treatments. Glutmaine composition in C. moluccensis silk was likewise affected by protein intake. However, the composition of proline in their MA silk was not significantly affected by protein intake.ConclusionsOur results suggest that protein limitation induces a shift toward different silk proteins with lower glutamine and/or proline content. Contradictions to the MaSp model lie in the findings that C. moluccensis MA silks did not experience a significant reduction in proline and A. aetherea did not experience a significant reduction in serine on low/no protein. The mechanical properties of the silks could not be explained by a MaSp1 expressional shift. Factors other than MaSp expression, such as the expression of spidroin-like orthologues, may impact on silk amino acid composition and spinning and glandular processes may impact mechanics.

Rapid Reaction Kinetics of Proline Dehydrogenase in the Multifunctional Proline Utilization A Protein

The multifunctional proline utilization A (PutA) flavoenzyme from Escherichia coli catalyzes the oxidation of proline to glutamate in two reaction steps using separate proline dehydrogenase (PRODH) and Δ(1)-pyrroline-5-carboxylate (P5C) dehydrogenase domains. Here, the kinetic mechanism of PRODH in PutA is studied by stopped-flow kinetics to determine microscopic rate constants for the proline:ubiquinone oxidoreductase mechanism. Stopped-flow data for proline reduction of the flavin cofactor (reductive half-reaction) and oxidation of reduced flavin by CoQ(1) (oxidative half-reaction) were best-fit by a double exponential from which maximum observable rate constants and apparent equilibrium dissociation constants were determined. Flavin semiquinone was not observed in the reductive or oxidative reactions. Microscopic rate constants for steps in the reductive and oxidative half-reactions were obtained by globally fitting the stopped-flow data to a simulated mechanism that includes a chemical step followed by an isomerization event. A microscopic rate constant of 27.5 s(-1) was determined for proline reduction of the flavin cofactor followed by an isomerization step of 2.2 s(-1). The isomerization step is proposed to report on a previously identified flavin-dependent conformational change [Zhang, W. et al. (2007) Biochemistry 46, 483-491] that is important for PutA functional switching but is not kinetically relevant to the in vitro mechanism. Using CoQ(1), a soluble analogue of ubiquinone, a rate constant of 5.4 s(-1) was obtained for the oxidation of flavin, thus indicating that this oxidative step is rate-limiting for k(cat) during catalytic turnover. Steady-state kinetic constants calculated from the microscopic rate constants agree with the experimental k(cat) and k(cat)/K(m) parameters.

Stimulation of the proline cycle and anthraquinone accumulation in Rubia tinctorum cell suspension cultures in the presence of glutamate and two proline analogs

Anthraquinone biosynthesis in Rubia tinctorum L. involves different metabolic routes. Chorismic acid, the end-product of the shikimate pathway, becomes the branch point between primary and secondary metabolism. It has been proposed that the proline cycle could be coupled with the pentose phosphate pathway (PPP), since the NADP+ generated by proline reduction from glutamate could act as a cofactor of the first enzymes of the PPP. This pathway generates erythrose-4-phosphate, the substrate of the shikimate pathway. The aim of the present work was to study the effect of the addition of glutamate and two proline analogs, azetidine-2-carboxylic acid and thiazolidine-4-carboxylic acid (T4C), on the PPP, the proline cycle, and anthraquinone production in R. tinctorum cell suspension cultures. The addition of 5 mM of glutamate enhanced both anthraquinone (up to 30%) and total phenolic content (12%), which correlated well with proline accumulation. Only the addition of 200 μM of T4C resulted in an increase in anthraquinone production, which was accompanied by a rise in the proline content. Neither the addition of glutamate nor proline analogs resulted in the induction of PPP, so this route was not a limiting factor as a carbon donor to the shikimate pathway.

Renal imino acid and glycine transport system ontogeny and involvement in developmental iminoglycinuria

Renal maturation occurs post-natally in many species and reabsorption capacity at birth can vary substantially from the mature kidney. However, little is known regarding the maturation of amino acid transport mechanisms, despite the well-known physiological state of developmental iminoglycinuria. Commonly seen during early infancy, developmental iminoglycinuria is a transient version of the persistent inherited form of the disorder, referred to as iminoglycinuria, and manifests as a urinary hyperexcretion of proline, hydroxyproline and glycine. The transporters involved in developmental iminoglycinuria and their involvement in the improvement of renal reabsorption capacity remain unknown. qPCR (quantitative real-time PCR) and Western blot analysis in developing mouse kidney revealed that the expression of Slc6a18, Slc6a19, Slc6a20a and Slc36a2 was lower at birth (approx. 3.4-, 5.0-, 2.4- and 3.0-fold less than adult kidney by qPCR respectively) and increased during development. Furthermore, immunofluorescence confocal microscopy demonstrated the absence of apical expression of Slc6a18, Slc6a19, Slc6a20a and the auxiliary protein collectrin in kidneys of mice at birth. This correlated with the detection of iminoglycinuria during the first week of life. Iminoglycinuria subsided (proline reduction preceded glycine) in the second week of life, which correlated with an increase in the expression of Slc6a19 and Slc6a20a. Mice achieved an adult imino acid and glycine excretion profile by the fourth week, at which time the expression level of all transporters was comparable with adult mice. In conclusion, these results demonstrate the delayed expression and maturation of Slc6a18, Slc6a19, Slc6a20a and Slc36a2 in neonatal mice and thus the molecular mechanism of developmental iminoglycinuria.

Modulation of intracellular proline levels affects flowering time and inflorescence architecture in Arabidopsis

We reported previously that the plant oncogene rolD anticipates and stimulates flowering in Nicotiana tabacum, and encodes ornithine cyclodeaminase, an enzyme catalysing the conversion of ornithine to proline. To investigate on the possible role of proline in flowering, we altered the expression of AtP5CS1, encoding the rate-limiting enzyme of proline biosynthesis in plants. Accordingly we characterized a mutant line containing a T-DNA insertion into AtP5CS1 and introduced in Arabidopsis thaliana AtP5CS1 under the control of the CaMV35S promoter. As expected homozygous p5cs1 mutants behaved as late flowering. In addition p5cs1 mutants exhibited a shorter size and contained lower levels of proline, compared to wild type. 35S-P5CS1 plants, manifested, early in development, overexpression of P5CS1 and accumulation of proline, leading to early flowering, both under long- and short-day conditions. Later in development, down-regulation of P5CS1 occurred in 35S-P5CS1 leaves, leading to proline reduction, and, in turn, impaired bolting and stunted growth. Salt-stress restored expression of P5CS1 and proline accumulation in P5CS1-transformed plants, as well as rescuing growth. Our data suggest that proline plays a key role in flower transition, bolting and coflorescence formation.