Effect of water stress on proline catabolism in tobacco leaves

Proline-[¹⁴C] infiltrated into leaf disks of tobacco (Nicotiana tabacum cv BY-4) in the dark was converted to glutamic acid and then metabolized through the TCA cycle. A smaller amount of proline-[¹⁴C] was metabolized when the leaf disks were wilted than when turgid. During a 6 hr period following rehydration, disks converted a larger amount of proline-[¹⁴C] to oxidized products than when wilted, although the proline content of rehydrated disks had not declined. These results indicate that proline oxidation is inhibited by water stress.     

Effect of water stress on proline content and transcript levels in Lathyrus sativus

Response of Lathyrus sativus plants to water stress showed that ABA responsive genes such as PLE 25, TAS 14 and RAB 17 are synthesized constitutively, the levels of which decline gradually with increase in water stress or ABA levels. Proline accumulation was highest in leaves (65-fold) followed by stem (56-fold), root (38-fold) and marginal increase in etiolated seedlings. Proline increase was also observed in plant parts not exposed to light.     

Inhibition of proline oxidation by water stress

The conversion of proline to glutamic acid and hence to other soluble compounds (proline oxidation) proceeds readily in turgid barley (Hordeum vulgare) leaves and is stimulated by higher concentrations of proline. This suggests that proline oxidation could function as a control mechanism for maintaining low cellular levels of proline in turgid tissue. In water-stressed tissue, however, proline oxidation is reduced to negligible rates. These results are consistent with the idea that proline accumulation results from inactivation by water stress of normal control mechanisms. It seems likely that inhibition of proline oxidation is necessary in maintaining the high levels of proline found in stressed barley leaves.     

Effect of water stress on growth and proline metabolism of Phaseolus vulgaris L.

Summary The effect of water stress on growth (fresh weight, dry weight), water relations (water saturation deficit, water potential, osmotic pressure), and proline metabolism in Phaseolus vulgaris were studied.Experimentally, water deficit was produced by reduced watering of the bean plants. This resulted in a decrease in water potential and leaf fresh and dry weight. Increases in the water saturation deficit and the osmotic pressure of the sap were, however, recorded. Water stress was also induced by treatment of the plants with polyethylene glycol, but its effects on the above mentioned parameters were different. In addition, necrosis of the foliage was observed. According to the present results, polyethylene glycol seems to be suitable only for the induction of short-termed water stress conditions.The effects of water stress on growth and on water relations of the plants were accompanied by a marked increase in the free amino acid content, especially that of the free proline content of the plants. The activities of the proline dehydrogenase and glutamate dehydrogenase were stimulated under water stress conditions, indicating that proline accumulation in water stressed plants is not attributed to an inhibited proline breakdown. The accumulated proline was metabolized rapidly once the water deficit of the plant was relieved by watering. The ability of the plant to accumulate proline might be of ecological importance for the plant and might be an adaption mechanism of the plant to overcome short periods of drought.Abbreviations -Kg -ketoglutarate - GDH glutamate dehydrogenase - PDH proline dehydrogenase - P5C -pyrroline-5-carboxylic acid - PEG polyethylene glycol - WSD water saturation deficit     

Effects of preformed proline and proline amino acid precursors (including glutamine) on collagen synthesis in human fibroblast cultures

A technique of derivatizing proline and 4-hydroxyproline with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole was used to measure the radioactivities, concentrations and specific activities of proline and hydroxyproline. The technique was used to study the conditions of procollagen synthesis in cultured human foreskin fibroblasts. Procollagen synthesis appeared to be independent of the proline concentration in the medium, in the presence of glutamine, when monitored by the assay of non-dialyzable hydroxyproline, but not when monitored by [14C]proline incorporation. In the absence of unlabelled proline added to labelled proline in the medium, the specific activity of the secreted procollagen did not reach a plateau over a 24-h period. When the medium was supplemented with glutamine, glutamic acid, or aspartic acid, both the radioactivity and concentration of intracellular free proline decreased. Pyrrolidone-2-carboxylic acid and ornithine both induced a slight increase in concentration of the intracellular free proline. Glutamine competed with [14C]proline for incorporation into prolyl-tRNA and procollagen, independently of free intracellular proline, and it stimulated the biosynthesis of procollagen (expressed as non-dialyzable hydroxyproline) by a factor of 2.3.     

Accumulation of glycinebetaine and its synthesis from radioactive precursors under salt-stress in the cyanobacterium Aphanothece halophytica

Growth in salt-stressed (2.0 M NaCl) Aphanothece halophytica was initially delayed during the first two days of cultivation and eventually attained the same growth rate as the control (0.5 M NaCl) cells. Glycinebetaine accumulation increased slightly in control cells but a dramatic increase of glycinebetaine occurred in salt-stressed cells during a growth period of six days. There was no apparent increase in the synthesis of [¹⁴C] glycinebetaine in the control cells, in contrast to the marked increase in its synthesis in the salt-stressed cells. Increasing NaCl concentration in the growth medium induced both the accumulation and the synthesis of glycinebetaine. Time course experiments provided evidence that [¹⁴C] choline was first oxidized to [¹⁴C] betaine aldehyde which was further oxidized to [¹⁴C] glycinebetaine in A. halophytica. The supporting data for such a pathway were obtained from the presence of choline and betaine aldehyde dehydrogenase activities found in the membrane and cytoplasmic fractions, respectively. The activities of these two enzymes were also enhanced upon increasing NaCl concentration in the growth medium from 0.5 M to 2.0 M. Under this condition an increaseof approximately 1.5-fold was observed for choline dehydrogenase activity as compared to 2.5-fold for betaine aldehyde dehydrogenase activity, suggesting a preferable induction of the latter enzyme by salt stress. A. halophytica was able to utilize [¹⁴C] ethanolamine and [¹⁴C] glycine for the synthesis of [¹⁴C] glycinebetaine. This revised version was published online in August 2006 with corrections to the Cover Date.     

Effect of water stress on proline accumulation of genetically modified potatoes (Solanum tuberosum L.) generating fructans

This study aimed to investigate the effect of water stress on the accumulation of proline in the leaves of transgenic potato lines generating fructans. The results of the series of bifactorial experiments in 2002 and 2003 indicated an increase of the proline level in leaves of all potato lines examined under water deficit. In addition, an increase in proline concentration during plant development was observed. The proline content was related to leaf water potential and relative water content (RWC), which indicates that proline could be involved in osmoregulation of potato plants under the experimental conditions. Surprisingly, under water deficit, the proline level was lowest in most of the transgenic SST/FFT-lines, which generate fructan molecules with a high degree of polymerization. Therefore, a pleiotropic effect can not be excluded as the reason for the divergence in behavior of these transgenic lines. The present results suggest that the modification of carbohydrate metabolism, especially the high content of soluble carbohydrates, may affect water stress-induced proline accumulation.     

Comparison of response of two C3 species to leaf water relation,proline synthesis,gas exchange and water use under periodic water stress

Water relations, proline content and gas exchange of leaf were investigated under periodic water stress for two C3 plants (eggplant and tomato) in a greenhouse to study comparative adaptive responses. Although both species showed reduced water content of leaf and increased osmolality and proline content under low soil water potential, the recovery capacity after the stress was better in eggplant than tomato. Both species over-accumulated proline under low soil water potential and returned to its initial concentration during the recovery, indicating that proline may act as an osmoprotectant during drought. Proline was directly corresponding with osmolality during stress, and dehydration stress reduced the gas exchange parameters such as transpiration rate (ET), stomatal conductance (GS), and photosynthesis rate (Pn). At the final stage of the experiment both species showed 2.6 and 3.3 times lower Pn and 27 and 19 times lower GS for eggplant and tomato, respectively, as compared to control. But after stress was relieved by rewatering, both plants increased GS for 2 to 3 times and Pn for 4.5 times. Eggplant showed better water use efficiency (WUE) in relation to fruit production under the stress than tomato. Higher biomass allocation at root and fruit parts in eggplant indicated more efficient recovery than that of tomato. These findings inferred that both C3 plants developed internal complementary drought survival mechanism by lowering relative water content, increasing proline, and decreasing stomatal conductance but eggplants withstood the periodic draughting better than tomato, mainly due to its ability to recover from a water stress condition.     

Nonspecific inhibition of proline dehydrogenase synthesis in Escherichia coli during osmotic stress

L-Proline, which is accumulated by Escherichia coli during growth in media of high osmolality, also induces the synthesis of the enzyme degrading it to glutamate. To determine if proline catabolism is inhibited during osmotic stress, proline utilization and the formation of proline dehydrogenase were examined in varying concentrations of NaCl and sucrose. Although the specific growth rate of E. coli with proline as the sole nitrogen source diminished as the solute osmolality increased, a comparable reduction in growth rate occurred with ammonium as the primary nitrogen source. Proline catabolism, as measured in whole cells by the conversion of [14C]proline to [14C]glutamate, was only slightly inhibited by solute osmolalities up to 1.0 osmol/kg; more than 50% of the initial activity was still found at 2.0 osmol/kg. By contrast, the specific activity of proline dehydrogenase in bacteria grown in the presence of added solutes decreased to less than 20% of the control level. This reduction was related to a lower rate of synthesis, but was independent of genes currently known to be involved in osmoregulation or proline metabolism. The specific activities of tryptophanase, beta-galactosidase, and histidinol dehydrogenase were also reduced under similar growth conditions. These results indicate that while proline catabolism is not directly inhibited by high solute concentrations, prolonged exposure to osmotic stress leads to its reduction as part of a more general metabolic response.     

Osmotic stress and water stress have opposite effects on putrescine and proline production in excised rice leaves

The effects of water stress and osmotic stress (sorbitol treatment) on the production of putrescine and proline in excised rice leaves were compared. Osmotic stress and water stress were found to affect differentially the levels of putrescine and proline in excised rice leaves. Putrescine accumulation is induced by osmotic stress, whereas proline accumulation is induced by water stress. The effects of ABA on the levels of proline and putrescine are similar to those of water stress, whereas the effects of jasmonic acid methyl ester (JA-Me) are similar to those of osmotic stress. Water stress results in an increase of endogenous ABA is excised rice leaves. However, neither osmotic stress nor JA-Me has effect on endogenous ABA levels in excised rice leaves. Of particular interest is the finding that proline levels increase when putrescine levels induced by osmotic stress or JA-Me are reduced by D-arginine and -methylornithine. L-arginine and L-ornithine applied exogenously also cause an increase in proline levels. It seems that L-arginine and L-ornithine are preferentially utilized as precursors for putrescine accumulation in excised rice leaves treated with osmotic stress and JA-Me, and for proline accumulation in excised rice leaves exposed to water stress and ABA.Abbreviations ABA abscisic acid - BSA bovine serum albumin - ELISA enzyme-linked immunosorbent assay - HPLC high performance chromatography - JA-Me jasmonic acid methyl ester - PVP poly-vinylpyrrolidone     

Stress-induced synthesis of proline confers tolerance to water deficit in transgenic wheat

Water deficit is one of the main abiotic factors that affect spring wheat planted in subtropical regions. Accumulation of proline appears to be a promising approach to maintain the productivity of plants under stress condition. However, morphological alterations and growth reduction are observed in transgenic plants carrying genes coding for osmoprotectants controlled by constitutive promoters. We report here the effects of water deficit on wheat plants transformed with the Vigna aconitifolia Delta(1)-pyrroline-5-carboxylate synthetase (P5CS) cDNA that encodes the key regulatory enzyme in proline biosynthesis, under the control of a stress-induced promoter complex-AIPC. Transgenic wheat plants submitted to 15 days of water shortage presented a distinct response. We have found that drought resulted in the accumulation of proline. The tolerance to water deficit observed in transgenic plants was mainly due to protection mechanisms against oxidative stress and not caused by osmotic adjustment.     

Polyamine synthesis from proline in the developing porcine placenta

Polyamines (putrescine, spermidine, and spermine) are essential for placental growth and angiogenesis. However, little is known about polyamine synthesis in the porcine placenta during conceptus development. The present study was conducted to test the hypothesis that arginine and proline are the major sources of ornithine for placental polyamine production in pigs. Placentae, amniotic fluid, and allantoic fluid were obtained from gilts on Days 20, 30, 35, 40, 45, 50, 60, 90, and 110 of the 114-day gestation (n = 6 per day). Placentae as well as amniotic and allantoic fluids were analyzed for arginase, proline oxidase, ornithine aminotransferase (OAT), ornithine decarboxylase (ODC), proline transport, concentrations of amino acids and polyamines, and polyamine synthesis using established radiochemical and chromatographic methods. Neither arginase activity nor conversion of arginine into polyamines was detected in the porcine placenta. In contrast, both proline and ornithine were converted into putrescine, spermidine, and spermine in placental tissue throughout pregnancy. The activities of proline oxidase, OAT, and ODC as well as proline transport, polyamine synthesis from proline, and polyamine concentrations increased markedly between Days 20 and 40 of gestation, declined between Days 40 and 90 of gestation, and remained at the reduced level through Day 110 of gestation. Proline oxidase and OAT, but not arginase, were present in allantoic and amniotic fluids for the production of ornithine (the immediate substrate for polyamine synthesis). The activities of these two enzymes as well as the concentrations of ornithine and total polyamines in fetal fluids were highest at Day 40 but lowest at Days 20, 90, and 110 of gestation. These results indicate that proline is the major amino acid for polyamine synthesis in the porcine placenta and that the activity of this synthetic pathway is maximal during early pregnancy, when placental growth is most rapid. Our novel findings provide a new base of information for future studies to define the role of proline in fetoplacental growth and development.     

Metabolism of [5-h]proline by barley leaves and its use in measuring the effects of water stress on proline oxidation

The objective of these experiments was to determine the fate of tritium from the 5 position of proline and to assess the validity of its loss to H₂O as a measure of proline oxidation. When [5-³H]proline was fed to barley (Hordeum vulgare) leaves, tritium was recovered in H₂O and metabolites such as glutamate, glutamine, organic acids, aspartate, asparagine, and γ-aminobutyrate. Collectively these metabolites, which are oxidation products of proline, accounted for 8% of the ³H recovered after 5 hours. In spite of the amount recovered in metabolites, the rates of proline oxidation estimated by measuring ³H₂O recovery from [5-³H]proline were only slightly lower than rates estimated by incorporation of ¹⁴C into oxidized products and loss of ¹⁴C from total proline. Therefore, ³H₂O recovery from [5-³H]proline is useful in assessing the effects of stress on proline metabolism.

Water stress inhibited proline oxidation, as reported previously. In addition, a reconversion of proline oxidation products to proline occurred in stressed leaves. This observation probably indicates a breakdown in cellular compartmentation of proline synthesis and proline oxidation.

     

水分胁迫下内生真菌感染对黑麦草叶内游离脯氨酸和脱落酸含量的影响

以黑麦草为实验对象,研究了干旱胁迫条件下内生真菌感染对植株叶片含水量和叶内游离脯氨酸含量的影响,同时对渗透胁迫条件下植株叶内ABA含量的变化进行了分析。结果表明：①内生真菌的感染有助于使叶片保持较高的含水量;②在两种形式的水分胁迫下,。前期至中期高感染种群的叶片游离脯氨酸含量低于感染种群,而在末期则有高出低感染种群的趋势;③内生真菌感染对黑麦草叶内ABA累积的正效应只发生在轻度渗透胁迫下的较短时间范围内。     

Enhanced intestinal synthesis of polyamines from proline in cortisol-treated piglets

This study was conducted to determine a role for cortisol in regulating intestinal ornithine decarboxylase (ODC) activity and to identify the metabolic sources of ornithine for intestinal polyamine synthesis in suckling pigs. Thirty-two 21-day-old suckling pigs were randomly assigned to one of four groups with eight animals each and received daily intramuscular injections of vehicle solution (sesame oil; control), hydrocortisone 21-acetate (HYD; 25 mg/kg body wt), RU-486 (10 mg/kg body wt, a potent blocker of glucocorticoid receptors), or HYD plus RU-486 for two consecutive days. At 29 days of age, pigs were killed for preparation of jejunal enterocytes. The cytosolic fraction was prepared for determining ODC activity. For metabolic studies, enterocytes were incubated for 45 min at 37 degrees C in 2 ml of Krebs-bicarbonate buffer (pH 7.4) containing 1 mM [U-(14)C]arginine, 1 mM [U-(14)C]ornithine, 1 mM [U-(14)C]glutamine, or 1 mM [U-(14)C]proline plus 1 mM glutamine. Cortisol administration increased intestinal ODC activity by 230%, polyamine (putrescine, spermidine, and spermine) synthesis from ornithine and proline by 75-180%, and intracellular polyamine concentrations by 45-83%. Polyamine synthesis from arginine was not detected in enterocytes of control pigs but was induced in cells of cortisol-treated pigs. There was no detectable synthesis of polyamines from glutamine in enterocytes of all groups of pigs. The stimulating effects of cortisol on intestinal ODC activity and polyamine synthesis were abolished by coadministration of RU-486. Our data indicate that an increase in plasma cortisol concentrations stimulates intestinal polyamine synthesis via a glucocorticoid receptor-mediated mechanism and that proline (an abundant amino acid in milk) is a major source of ornithine for intestinal polyamine synthesis in suckling neonates.     

The effect of water stress on the vacuole-extravacuole compartmentation of proline in potato cell suspension cultures

Solute compartmentation in cells is an important component of metabolic regulation. There is only little information on how stress treatment of cells effects this component. Therefore, the effect of water stress [10% (w/v) PEG 6000] on the vacuolar-extravacuolar proline compartmentation was studied in a cell suspension culture of Svlanum tuberosum L, cv, HH258, In non-stressed cells 34% of the total cellular proline was located in the vacuole. After 20 h of water stress the proline pool of the cells was increased 4-6 fold and only t6% of it was found in the vacuole. A negative correlation between the total cellular proline content and its percentage in the vacuole was observed, irrespective of the culture method (stress or non-stress culture). The stress-induced changes in proline compartmentation are discussed.     

Effect of osmotic stress and sodium nitroprusside pretreatment on proline metabolism of wheat seedlings

Effect of osmotic stress and sodium nitroprusside (SNP, NO donor) pretreatment on growth and proline metabolism of wheat seedlings was investigated. Polyethylene glycol 6000 treatment for 2, 4 and 6 d could be termed as mild, moderate and severe stress, respectively, according to decrease in the relative water content. Severe osmotic stress significantly decreased the growth and photochemical efficiency, and increased proline content due to activation of its synthesis. 0.2 mM SNP pretreatment enhanced growth of wheat seedlings, increased variable to maximum fluorescence ratio (F_v/F_m) and fluorescence yield, while decreased proline content. However, 2 mM SNP retarded the seedlings growth and chlorophyll a fluorescence, and increased proline accumulation. Our results showed that NO might be involved in the regulation of osmotic stress in a concentration-dependent manner.     

Regulation of cytoplasmic proline levels in Salmonella typhimurium: effect of osmotic stress on synthesis, degradation, and cellular retention of proline.

I investigated the effects of osmotic stress on the synthesis and catabolism of proline in Salmonella typhimurium by measuring the intracellular and extracellular proline levels in various strains. In the wild-type strain, exposure to 0.8 M NaCl did not cause a significant change in the intracellular proline level; however, it brought about a 6.5-fold increase in the intracellular glutamate pool size. These results indicate that gamma-glutamyl kinase is inhibited by proline in wild-type cells in media of normal or elevated osmolarity. I also tested whether proline is subject to turnover in cells wild type with respect to the enzymes of the proline degradation pathway. In strains that were wild type for proline biosynthesis, the loss of the proline catabolic enzymes, due to putA mutations, did not result in a statistically significant increase in the intracellular proline levels. Therefore, in the wild-type strain, proline turnover does not seem to be important for control of the intracellular proline levels. However, in a proline-overproducing mutant, a putA lesion caused a threefold increase in the intracellular proline level and a 6.5-fold increase in the extracellular proline level, indicating that proline is subject to turnover in the overproducing mutant. The proline-overproducing mutants excreted large quantities of the proline into the culture medium; osmotic stress altered the partitioning of proline such that the ratio of intracellular to extracellular levels of proline increased with increased osmotic stress. The increased cellular retention of proline in media of high osmolarity is probably due to the functioning of the ProP and ProU proline transport systems, which are stimulated under conditions of osmotic stress.