Transfer of reducing equivalents into mitochondria by the interconversions of proline and Δ1-pyrroline-5-carboxylate

Direct evidence is presented for a proline cycle using a cell-free experimental system which sequentially transfers ³H from [1-³H]glucose to NADP⁺ to Δ¹-pyrroline-5-carboxylate and yields [³H]proline. The formation of [³H]proline depends on the presence of NADP, Δ¹-pyrroline-5-carboxylate, and the enzymes glucose-6-phosphate dehydrogenase and Δ¹-pyrroline-5-carboxylate reductase. The production of [³H]proline from unlabeled proline in the presence of mitochondria provides direct evidence for one complete turn of a proline cycle which transfers reducing equivalents produced by glucose oxidation in the pentose pathway into mitochondria. In this cycle, proline is oxidized to Δ¹-pyrroline-5-carboxylate by mitochondrial proline oxidase. Δ¹-pyrroline-5-carboxylate is released from mitochondria and is recycled back to proline by Δ¹-pyrroline-5-carboxylate reductase with concomitant oxidation of NADPH. At the maximal rate observed, 60% of Δ¹-pyrroline-5-carboxylate produced is recycled back to proline. This cycle provides a mechanism for transferring reducing equivalents from NADPH into mitochondria and is linked to glucose oxidation in the pentose pathway by NADPH turnover.     

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

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

Evidence for Hysteretic Substrate Channeling in the Proline Dehydrogenase and Δ1-Pyrroline-5-carboxylate Dehydrogenase Coupled Reaction of Proline Utilization A (PutA)

PutA (proline utilization A) is a large bifunctional flavoenzyme with proline dehydrogenase (PRODH) and Δ¹-pyrroline-5-carboxylate dehydrogenase (P5CDH) domains that catalyze the oxidation of l-proline to l-glutamate in two successive reactions. In the PRODH active site, proline undergoes a two-electron oxidation to Δ¹-pyrroline-5-carboxlylate, and the FAD cofactor is reduced. In the P5CDH active site, l-glutamate-γ-semialdehyde (the hydrolyzed form of Δ¹-pyrroline-5-carboxylate) undergoes a two-electron oxidation in which a hydride is transferred to NAD⁺-producing NADH and glutamate. Here we report the first kinetic model for the overall PRODH-P5CDH reaction of a PutA enzyme. Global analysis of steady-state and transient kinetic data for the PRODH, P5CDH, and coupled PRODH-P5CDH reactions was used to test various models describing the conversion of proline to glutamate by Escherichia coli PutA. The coupled PRODH-P5CDH activity of PutA is best described by a mechanism in which the intermediate is not released into the bulk medium, i.e., substrate channeling. Unexpectedly, single-turnover kinetic experiments of the coupled PRODH-P5CDH reaction revealed that the rate of NADH formation is 20-fold slower than the steady-state turnover number for the overall reaction, implying that catalytic cycling speeds up throughput. We show that the limiting rate constant observed for NADH formation in the first turnover increases by almost 40-fold after multiple turnovers, achieving half of the steady-state value after 15 turnovers. These results suggest that EcPutA achieves an activated channeling state during the approach to steady state and is thus a new example of a hysteretic enzyme. Potential underlying causes of activation of channeling are discussed.     

Proline Accumulation in Transgenic Tobacco as a Result of Expression of Arabidopsis Δ1-Pyrroline-5-carboxylate synthetase (P5CS) During Osmotic Stress

The entire coding sequence of the bi-functional enzyme, Δ¹-Pyrroline-5-carboxylate synthetase (P5CS) from Arabidopsis thaliana was reverse-transcribed, amplified and expressed under the control of CaMV 35S promoter in transgenic tobacco plants. Several lines were established and tested for the expression of P5CS. Drought and salinity were applied as osmotic stresses and proline content of the transformed plants was compared with that of non-transformed controls. Results indicate that transgenic lines express higher levels of proline and show enhanced resistance to the applied osmotic stress as compared to the non-transgenic plants.     

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

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

Codisterol and other Δ5-sterols in the seeds of Cucurbita maxima

A substantial amount (ca 18%) of the sterol found in the seeds of Cucurbita maxima had a Δ-bond and consisted of seven components. They were identified as 25(27)-dehydroporiferasterol, clerosterol, isofucosterol, stigmasterol, sitosterol, campesterol and codisterol. The C-24 configuration of each of the sterols was unequivocally established by a ¹H NMR spectral comparison with authentic standards. This is the first time codisterol has been found in a higher plant and also the first time the structures and configurations of the Δ⁵-sterols from a Cucurbitaceae species have been clearly characterized.     

A novel Δ-pyrroline-5-carboxylate synthetase gene of Medicago truncatula plays a predominant role in stress-induced proline accumulation during symbiotic nitrogen fixation

Proline accumulates in environmentally stressed plant cells including those of legume roots and nodules, but how its level is regulated is poorly understood. Δ¹-Pyrroline-5-carboxylate synthetase (P5CS), the committed-step enzyme of proline biosynthesis, is encoded by two duplicated genes in many plants. Here, we isolated MtP5CS3, a third gene, from Medicago truncatula, whose predicted polypeptide sequence is highly similar to those of previously isolated MtP5CS1 and MtP5CS2 except an extra amino-terminal segment. MtP5CS3 was strongly expressed under salinity and drought in shoots and nodulating roots, while MtP5CS1 was constitutive and MtP5CS2 induced by abscisic acid. Under salinity, MtP5CS3 promoter was more active than those of MtP5CS1 and MtP5CS2, as shown by GUS fusions. Translationally fused MtP5CS1-GFP was localized in the cytoplasm, whereas significant proportions of MtP5CS2-GFP and MtP5CS3-GFP were co-localized with rubisco small subunit protein-fused RFP in transformed hairy root cells. Under salinity, RNA silencing of MtP5CS1 or MtP5CS2 strongly induced MtP5CS3 expression, while that of MtP5CS3 decreased free proline content and nodule number. Consistently, Mtp5cs3, a loss-of-function mutant, accumulated much less proline, formed fewer nodules, and fixed nitrogen significantly less efficiently than the wild type under salinity. Thus, MtP5CS3 plays a critical role in regulating stress-induced proline accumulation during symbiotic nitrogen fixation.     

Effect of ploidy levels on the activities of Δ1-pyrroline-5-carboxylate synthetase,superoxide dismutase and peroxidase in Cenchrus species grown under water stress

The effects of ploidy levels on the activities of Δ¹-pyrroline-5-carboxylate synthetase (P5CS; EC not assigned), superoxide dismutase (SOD; EC 1.15.1.1) and guaiacol peroxidase (POX; EC 1.11.1.7), as well as malondialdehyde (MDA) and proline contents were studied in two months old plants of Cenchrus species. The Cenchrus species represent three ploidy levels: diploid, tetraploid, hexaploid and two life spans: annual and perennial. Plants were subjected to water stress for 2, 4, 6 and 8 d by withholding water under glasshouse conditions. Although the levels of proline increased with the magnitude of water stress, the P5CS activity did not show a corresponding increase in all species. Peroxidase and superoxide dismutase activities showed an increase or steady state in the early phase of drought and then declined with the further increase in the magnitude of water stress, indicating differing behaviors of species towards drought tolerance. Under drought, diploid Cenchrus species had a higher POX activity, MDA accumulation and lower proline content than tetraploid species. Lower POX and higher P5CS activities and proline contents, however, were observed in hexaploid and tetraploid species. Taken together, our findings suggest that diploid species have a less efficient antioxidant system to scavenge reactive oxygen species than tetra and hexaploid Cenchrus. This may result in a corresponding variability in growth and persistence under natural grasslands. The study also paves the way for investigations on the molecular events associated with drought in Cenchrus species differing in ploidy and life span.     

The importance of Δ1-pyrroline in the aetiology of cerebrocortical necrosis

Bacterial thiaminase I associated with cerebrocortical necrosis of cattle and sheep is shown to utilise Δ¹-pyrroline and related compounds as cosubstrates. The product resulting from the reaction of thiamine and Δ¹-pyrroline is 1-(4-amino-2-methylpyrimidin-5-ylmethyl)-1-pyrrolinium chloride. This compound has been identified in the brains of calves suffering from cerebrocortical necrosis. The implications of these findings in the aetiology of other thiamine-responsive diseases of the central nervous system are briefly discussed.     

Effects of Δ9-tetrahydrocannabinol on neurotransmitter accumulation and release mechanisms in rat forebrain synaptosomes

The effects of (?)?Δ⁹-THC were studied on the release and accumulation of ³H5HT and ³HNE in a rat forebrain synaptosomal preparation. These studies were designed to evaluate the possible sites of action of Δ⁹-THC on these two processes. Δ⁹-THC inhibited the accumulation of ³H-leucine, ³HNE, and ³H5HT, as well as facilitated the release of the latter two amines (to a lesser degree), but had no effect on the release of ³H-leucine. Eighteen-hour pre-treatment with reserpine diminished the ability of Δ⁹-THC to induce release of ³H5HT, but had no effect on the $\text{in vitro}$ inhibition of synaptosomal uptake of this amine. Concentrations of Δ⁹-THC which blocked the uptake of ³H5HT also reduced the conversion of ³H5HT to ³H-5-hydroxy-3-indoleacetic acid. However, Δ⁹-THC, at concentrations which facilitated release of ³H5HT from preloaded synaptosomes, increased the amount of ³H5HIAA found in the medium. Taken together, these data suggest that Δ⁹-THC facilitates release from the synaptic vesicle and retards accumulation at the neuronal membrane.     

Investigations on the Δ23-, Δ24(28)- and Δ25-Sterols of zea mays

The sterols of Zea mays shoots were isolated and characterized by TLC, HPLC, GC/MS and ¹H NMR techniques. In all, 22 4-demethyl sterols were identified and they included trace amounts of the Δ²³-, Δ²⁴- and Δ²⁵-sterols, 24-methylcholesta-5,E-23-dien-3β-ol, 24-methylcholesta-5,Z-23-dien-3β-ol, 24-methylcholesta-5,25-dien-3β-ol, 24-ethylcholesta-5,25-dien-3β-ol and 24-ethylcholesta-5,24-dien-3β-ol. In the 4,4-dimethyl sterol fraction, cycloartenol and 24-methylenecycloartanol were the major sterol components but small amounts of the Δ²³-compound, cyclosadol, and the Δ²⁵-compound, cyclolaudenol, were recognized. These various Δ²³- and Δ²⁵-sterols may have some importance in alternative biosynthetic routes to the major sterols, particularly the 24β-methylcholest-5-en-3β-ol component of the C₂₈-sterols. Radioactivity from both [2-¹⁴C]MVA and [methyl-¹⁴C]methionine was incorporated by Z. mays shoots into the sterol mixture. Although 24-methylene and 24-ethylidene sterols were relatively highly labelled, the various Δ²³- and Δ²⁵-sterols contained much lower levels of radioactivity, which is possibly indicative of their participation in alternative sterol biosynthetic routes. (24R)-24-Ethylcholest-5-en-3β-ol (sitosterol) had a significantly higher specific activity than the 24-methylcholest-5-en-3β-ol indicating that the former is synthesized at a faster rate.     

Isolation and sequence analysis of the cDNA encoding Δ1-pyrroline-5-carboxylate reductase from Zalerion arboricola

A cDNA encoding Δ¹-pyrroline-5-carboxylate reductase (P5CR) was isolated from the pneumocandin(Pmo)-producing fungus, Zalerion arboricola (Za), by complementation of a P5CR-deficient mutant (pro3) of Saccharomyces cerevisiae (Sc). The cloned cDNA was placed under control of the Sc galactokinase (GAL1) promoter and restored P5CR activity to the pro3 mutant. Sequence analysis revealed that the Za P5CR-encoding cDNA encodes an approx. 35 kDa protein with substantial amino acid (aa) identity to P5CR from another filamentous fungus, Neurospora crassa (Nc). Za P5CR exhibits a moderate degree of aa identity to P5CR from plants, bacteria, human and Sc. This is the first gene to be isolated from Za.     

Stress-induced Δ1-pyrroline-5-carboxylate synthetase (P5CS) gene confers tolerance to salt stress in transgenic sugarcane

High salinity interferes in sugarcane growth and development, affecting not only crop yield but also reducing sucrose concentration in culms. Sugarcane plants submitted to salt stress can accumulate compatible solutes, such as proline, which may counteract the effects of salt accumulation in the vacuole and scavenge reactive oxygen species. The objective of this study was to evaluate the response to salt stress of sugarcane plants transformed with the Vigna aconitifolia P5CS gene, which encodes ?1-pyrroline-5-carboxylate synthetase, under the control of a stress-induced promoter AIPC (ABA-inducible promoter complex). For this, 4-month-old clonally multiplied sugarcane plants from two transformation events were irrigated every 2 days with 1/10 Hoagland’s solution supplemented with 100, 150 and 200 NaCl, progressively, during 28 days. Transgenic lines showed increased transgene expression in 3.75-fold when compared with the control plants after 9 days of irrigation with saline water, which can explain the higher proline concentration found in these plants. At the end of the experiment (day 28), the transgenic lines accumulated up to 25 % higher amounts of proline when compared with non-transformed control plants. Stress response in transgenic plants was also accompanied by a reduction of malondialdehyde (MDA) derived from cellular lipid peroxidation in leaves, lower Na⁺ accumulation in leaves and maintenance of photochemical efficiency of PSII. Thus, proline contributed to the protection of the photosynthetic apparatus and the prevention of oxidative damage in transgenic sugarcane under salt stress.     

Co-expression of the borage Δ6 desaturase and the Arabidopsis Δ15 desaturase results in high accumulation of stearidonic acid in the seeds of transgenic soybean

Two relatively rare fatty acids, γ-linolenic acid (GLA) and stearidonic acid (STA), have attracted much interest due to their nutraceutical and pharmaceutical potential. STA, in particular, has been considered a valuable alternative source for omega-3 fatty acids due to its enhanced conversion efficiency in animals to eicosapentaenoic acid when compared with the more widely consumed omega-3 fatty acid, α-linolenic acid (ALA), present in most vegetable oils. Exploiting the wealth of information currently available on in planta oil biosynthesis and coupling this information with the tool of genetic engineering it is now feasible to deliberately perturb fatty acid pools to generate unique oils in commodity crops. In an attempt to maximize the STA content of soybean oil, a borage Δ⁶ desaturase and an Arabidopsis Δ¹⁵ desaturase were pyramided by either sexual crossing of transgenic events, re-transformation of a Δ⁶ desaturase event with the Δ¹⁵ desaturase or co-transformation of both desaturases. Expression of both desaturases in this study was under the control of the seed-specific soybean β-conglycinin promoter. Soybean events that carried only the Δ¹⁵ desaturase possessed a significant elevation of ALA content, while events with both desaturases displayed a relative STA abundance greater than 29%, creating a soybean with omega-3 fatty acids representing over 60% of the fatty acid profile. Analyses of the membrane lipids in a subset of the transgenic events suggest that soybean seeds compensate for enhanced production of polyunsaturated fatty acids by increasing the relative content of palmitic acid in phosphatidylcholine and other phospholipids.     

Characterization and expression analysis of P5CS (Δ1-pyrroline-5-carboxylate synthase) gene in two distinct populations of the Atlantic Forest native species Eugenia uniflora L.

Molecular Biology Reports - Eugenia uniflora is an Atlantic Forest native species, occurring in contrasting edaphoclimatic environments. The identification of genes involved in response to abiotic...