Characterization of a gamma-glutamyl kinase from Escherichia coli that confers proline overproduction and osmotic tolerance.

Mutation(s) in the proBA operon of Escherichia coli confers proline overproduction and enhanced osmotic tolerance in enteric bacteria (L. N. Csonka, Mol. Gen. Genet. 182:82-86, 1981; M. J. Mahan and L. N. Csonka, J. Bacteriol. 156:1249-1262, 1983). A glutamate-dependent ATPase assay was developed and used to determine proB-encoded gamma-glutamyl kinase activity in the absence of glutamate-gamma-semialdehyde dehydrogenase. This assay indicated that the feedback insensitivity of mutant gamma-glutamyl kinase was independent of glutamate-gamma-semialdehyde dehydrogenase. However, the capacity of glutamate-gamma-semialdehyde dehydrogenase from the osmotolerant mutant to interact with the kinase was altered in thermal stability, suggesting that mutations in both proB and proA may be required for osmotolerance.     

Expression of Bacillus subtilis proBA genes and reduction of feedback inhibition of proline synthesis increases proline production and confers osmotolerance in transgenic Arabidopsis

Proline accumulation has been shown to correlate with tolerance to drought and salt stresses in plants. We attempt to introduce the wild-type, mutant, and fusion proBA genes derived from Bacillus subtilis into Arabidopsis thaliana under the control of a strong promoter cauliflower mosaic virus 35S (CaMV35S). The transgenic plants produced higher level of free proline than control and the overproduction of proline resulted in the increased tolerance to osmotic stress in transgenic plants. Besides, the mutation in proBA genes, which were proved to lead gamma-glutamyl kinase (gamma-GK) reduces sensitivity to the end-product inhibition and the fusion of proB and proA also result in increasing proline production and confer osmotolerance in transgenic lines.     

Salt Tolerance in the Halophyte, Suaeda maritima (L.) Dum.: Properties of Malic Enzyme and PEP carboxylase

Malic enzyme and phosphenol pyruvate carboxylase activitieshave been isolated and characterized from the shoots of Suaedamaritima plants grown in culture solution (with and withoutNaCl) or in tap water. The enzymes isolated from the lattershowed increases in both specific activity and K_m values incomparison with plants grown in culture solution: however, theaddition of NaCl to the culture solution had no significanteffect on either enzyme. Malate levels were high in plants grownin tap water, reduced by an ordeT of magnitude by the additionof culture solution and then halved by the addition of NaCl. Both enzymes were inhibited in vitro by NaCl, although the additionof high concentrations of betaine and proline to the assay mediumdid not further inhibit enzyme activity. The significance ofthese results is discussed in relation to the proposed roleof betaine and proline as cytoplasmic osmoregulators. Suaeda maritima, halophyte, salt tolerance, malic enzyme, PEP carboxylase     

Salt Tolerance in Simmondsia chinensis: Water Balance and Accumulation of Chloride, Sodium and Proline Under Low and High Salinity

The response of jojoba [Simmondsia chinensis (Link) Schneid]plants to salinity was studied in solution culture. At concentrationsof 0, 100, 200 and 600 m-mol l^–1 NaCI it was found thatjojoba plants have high tolerance to NaCl. The growth of theseplants was not affected by salinity. They accumulated largeamounts of Cl^–, Na⁺ and proline. These amounts decreasedrapidly in plants transferred back to control medium. Potassiumcontent decreased in NaCl-treated plants and tended to increaserapidly to the control level in plants transferred to controlmedium. The effect of salinity on water balance was not appreciable.As suggested for other xerophytic species, it could be assumedthat the high tolerance of jojoba to salinity plays an importantrole in its ability to endure periods of drought. The role ofproline during or after stress remains an open question. Simmondsia chinensis (Link) Schneid, jojoba, salt tolerance, sodium accumulation, chloride ion accumulation, proline accumulation, xerophytism, drought tolerance     

Growth and nitrogen assimilation in nodules in response to nitrate levels in Vicia faba under salt stress

This study analyses the effects of salt on the effective symbiosisof faba bean (Vicia faba L. var. minor cv. Alborea) and salt-tolerantRhizobium leguminosarum biovar. viciae strain GRA19 grown withtwo KNO₃ levels (2 and 8 mM). The addition of 8 mM KNO₃ to thegrowth medium increases plant tolerance to salinity even witha concentration of 100 mM NaCl. This KNO₃ level in control plantsreduced the N₂ fixation. For 2 and 8 mM KNO₃ the plants treatedwith NaCl reduced N₂ fixation to identical values. The activityof the enzymes mediating ammonium assimilation in nodules (GS,NADH-GOGAT and NADH-GDH) was decreased by high KNO₃ levels.The results show that NADH-GOGAT activity was more markedlyinhibited than was GS activity by salinity, therefore NADH-GOGATlimits the ammonium assimilation by nodules in V. faba undersalt stress. The total proline content in the nodule was notrelated to salt tolerance and thus does not serve as a salttoleranceindex for V. faba. Key words: Glutamate synthase, glutamine synthetase, N₂ fixation, nitrate, salinity     

Identification and Disruption of the proBA Locus in Listeria monocytogenes: Role of Proline Biosynthesis in Salt Tolerance and Murine Infection

Intracellular accumulation of the amino acid proline has previously been linked to the salt tolerance and virulence potential of a number of bacteria. Taking advantage of the proBA mutant Escherichia coli CSH26, we identified a listerial proBA operon coding for enzymes functionally similar to the glutamyl kinase (GK) and glutamylphosphate reductase (GPR) enzyme complex which catalyzes the first and second steps of proline biosynthesis in E. coli. The first gene of the operon, proB, is predicted to encode GK, a 276-residue protein with a calculated molecular mass of 30.03 kDa and pl of 5.2. Distal to the promoter and overlapping the 3′ end of proB by 17 bp is proA, which encodes GPR, a 415-residue protein with a calculated molecular mass of 45.50 kDa (pl 5.3). Using this information, we created a chromosomal deletion mutant by allelic exchange which is auxotrophic for proline. This mutant was used to assess the contribution of proline anabolism to osmotolerance and virulence. While inactivation of proBA had no significant effect on virulence in mouse assays (either perorally or intraperitoneally), growth at low (2 to 4% NaCl) and high (>6% NaCl) salt concentrations in complex media was significantly reduced in the absence of efficient proline synthesis. We conclude that while proline biosynthesis plays little, if any, role in the intracellular life cycle and infectious nature of Listeria monocytogenes, it can play an important role in survival in osmolyte-depleted environments of elevated osmolarity.     

Influence of Proline and Glycinebetaine on Salt Tolerance of Cultured Barley Embryos

Lone, M. I., Kueh, J. S. H., Wyn Jones, R. G. and Bright, S.W. J. 1987. Influence of proline and glycinebetaine on salttolerance of cultured barley embryos.—J. exp. Bot. 38:479–490. The addition of exogenous proline and glycinebetaine to culturedbarley (Hordeum vulgare L. cv. Maris Mink) embryos increasedshoot elongation under saline conditions. Inhibition of shootelongation by NaCl was relieved by proline when plantlets weregrown in deep crystallizing dishes but not in Petri dishes whereshoots come into direct contact with the medium. The effectof proline could be related to a decrease in shoot Cl^–and Na⁺ accumulation which was only observed in plantlets grownin crystallizing dishes. Proline but not betaine uptake intocultured plantlets was stimulated by NaCl while each organicsolute inhibited the endogenous synthesis of the other soluteunder salt stress. Comparison of the effects of exogenously supplied proline withenhanced endogenous proline accumulation in the mutant lineR5201 suggested that the increased proline accumulation in themutant is an order of magnitude too low to have a significantphysiological effect. The implications of the effect of prolineon ion transport, discrimination and accumulation are discussed. Key words: Salt tolerance, proline, ion transport, barley embryo culture     

Recombinant plasmid conferring proline overproduction and osmotic tolerance

A recombinant plasmid carrying the proBA (pro-74) mutant allele which governs osmotic tolerance and proline overproduction was constructed by using the broad-host-range plasmid vector pQSR49. The physiological, biochemical, and genetic properties of strains carrying the pQSR49 derivatives pMJ101 and pMJ1, mutant and wild type, respectively, were investigated. pMJ101 conferred enhanced osmotolerance compared with strains carrying the wild type, pMJ1. These results are in contrast to those obtained previously with strains carrying recombinant plasmids based on pBR322 that failed to confer the osmotic tolerance phenotype. gamma-Glutamyl kinase (first step in proline biosynthesis) from strains carrying pMJ101 was 200-fold less sensitive to feedback inhibition than was the wild-type enzyme. As expected, the intracellular proline levels of strains carrying pMJ101 were more than an order of magnitude higher than those of the wild type. An analysis of copy number revealed that the pQSR49 constructs were present in the cell at a level six- to eightfold lower than those of the pBR322 recombinants, which may account for the difference in phenotype. We found that the genetic stability of the pQSR49 derivative in a variety of gram-negative bacteria was dependent on the insert orientation and the presence of foreign DNA on the plasmid. These factors may be significant in future studies aimed at expanding the osmotolerance phenotype to a broad range of gram-negative bacteria.     

Recombinant plasmid conferring proline overproduction and osmotic tolerance.

A recombinant plasmid carrying the proBA (pro-74) mutant allele which governs osmotic tolerance and proline overproduction was constructed by using the broad-host-range plasmid vector pQSR49. The physiological, biochemical, and genetic properties of strains carrying the pQSR49 derivatives pMJ101 and pMJ1, mutant and wild type, respectively, were investigated. pMJ101 conferred enhanced osmotolerance compared with strains carrying the wild type, pMJ1. These results are in contrast to those obtained previously with strains carrying recombinant plasmids based on pBR322 that failed to confer the osmotic tolerance phenotype. gamma-Glutamyl kinase (first step in proline biosynthesis) from strains carrying pMJ101 was 200-fold less sensitive to feedback inhibition than was the wild-type enzyme. As expected, the intracellular proline levels of strains carrying pMJ101 were more than an order of magnitude higher than those of the wild type. An analysis of copy number revealed that the pQSR49 constructs were present in the cell at a level six- to eightfold lower than those of the pBR322 recombinants, which may account for the difference in phenotype. We found that the genetic stability of the pQSR49 derivative in a variety of gram-negative bacteria was dependent on the insert orientation and the presence of foreign DNA on the plasmid. These factors may be significant in future studies aimed at expanding the osmotolerance phenotype to a broad range of gram-negative bacteria.     

Salinity Stress and the Content of Proline in Roots of Pisum sativum and Tamarix tetragyna

Amino acid composition of the free amino acid pool and the TCA-insolubleprotein fraction were investigated in root tips of pea and Tamarixtetragyna plants grown at various levels of NaCl salinity. Salinitystress induced an increase of proline content, mainly in thefree amino acid pool in both plants, and of proline or hydroxyprolinecontent in the protein. Externally-supplied proline was absorbedand incorporated into protein, by pea roots, more effectivelythan by Tamarix roots. Salinity stress, apparently, stimulatedthe metabolism of externally-supplied labelled proline. Pearoots have a very large pool of free glutamic acid; however,70 per cent of the ¹⁴C from externally-supplied ¹⁴C-U-glutamicacid was released as CO₂. Very small amounts of it were incorporatedinto protein. No measurable amount of radioactivity could bedetected in any one of the individual amino acids, either ofprotein hydrolysate or the free amino acid pool. Proline very effectively counteracted the inhibitory effectof NaCl on pea seed germination and root growth. A similar effectbut to a lesser degree was achieved with phenylalanine and asparticacid. The feasibility of proline being a cytoplasmic osmoticumis discussed.     

Proline over-production results in enhanced osmotolerance in Salmonella typhimurium

Summary Mutants of S. typhimurium with enhanced osmotolerance were isolated. These mutants were obtained as strains which over-produced proline due to regulatory mutations affecting proline biosynthesis. The mutations are located on FproBA and upon transfer to other S. typhimurium strains, they confer enhanced osmotolerance on the recipients. The osmotolerant mutants not only have higher intracellular proline levels than the osmosensitive parental strain, but the proline levels in the osmotolerant mutants are regulated such that they increase in response to osmotic stress. Possible reasons why elevated proline levels lead to enhanced osmotolerance are discussed.     

Nitrogen fixaton in Klebsiella pneumoniae during osmotic stress effect of exogenous proline or a proline overproducing plasmid

Osmotic stress, imposed by 0.5 M NaCl or other electrolytes and non-electrolytes, caused over a 100-fold reduction in the whole-cell nitrogen fixation activity in Klebsiella pneumoniae, wild-type strain M₅A₁. This reduction of nitrogen fixation activity could be reversed by the addition of proline to the culture medium at 0.5 mM concentration. With 0.5 M NaCl, in the presence of proline, nitrogenase activity was 47-fold greater than in the absence of proline. A mutation, originally isolated in Salmonella typhimurium, which resulted in proline over-production and enhanced osmotolerance, was transferred into K. pneumoniae by F′ conjugation. Intracellular proline, synthesized at high levels because of the mutation, had similar stimulatory effects on nitrogen fixation under osmotic stress as proline provided exogenously. In the overproducing strain, the cellular level of proline is elevated as much as 125-fold during stress over that seen in the control strain. To determine the mechanism of stimulation of nitrogen fixaton by proline during stress, the biosynthesis of nitrogenase polypeptides was studied. Net nitrogenase biosynthesis and the biosynthesis of other unidentified peptides, is strongly inhibited during osmotic stress; proline reverses the inhibition. The role of proline in enhancing nitrogen fixation during osmotic stress is discussed.     

Isolation and Characterization of Sodium Chloride-Resistant Callus Culture of Vigna radiata (L.) Wilczek var. radiata

Callus cultures were initiated from seedling root segments ofmungbean (Vigna radiata (L.) Wilczek var. radiata) cv. K 851on modified PC-L2 basal medium. Growing cells were exposed toincreasing concentrations of NaCl in the medium. A concentrationof 300 mol m^–3 NaCl proved completely inhibitory to growthof the calli. On incubation for 25 d, cells which could toleratethis concentration of NaCl grew to form cell clones. Selectedclones were characterized with regard to their growth behaviour,K⁺, Na⁺ and free proline content when grown under stress aswell as on normal media and were compared with the normal sensitivecallus. The selected callus was capable of growing on mediumcontaining NaCl at the inhibitory concentration. The K⁺ contentof the selected callus was lower in the case of the NaCl mediumthan for the normal medium. However, the selected clones maintainedhigher K⁺ and Na⁺ levels, with increased salinization comparedwith the wild-type cells. Salt-selected cells accumulated higherlevels of free proline under NaCl stress compared to wild-typecells. Under normal conditions, however, the amounts of freeproline in selected and non-selected calli were comparable. Key words: Vigna radiata, callus culture, NaCl stress     

A change in a single gene of Salmonella typhimurium can dramatically change its buoyant density.

The growth rates and buoyant densities of a Salmonella typhimurium mutant, TL126 (proB74A+), with enhanced osmotolerance caused by proline overproduction were measured and compared with the growth rates and buoyant densities of an isogenic (wild-type) strain, TL128 (proB+ A+), with normal control of proline production. Growth rates were determined in a rich medium (Luria broth) with added NaCl to produce various osmotic strengths ranging from 300 to 2,000 mosM. At low concentrations of NaCl, there was little variation in doubling times between the two strains. However, as the osmotic strength of the medium approached and exceeded 1,300 mosM, the doubling times of TL126 (osmotolerant) were 1.5 to 2 times faster than those of TL128 (wild type), confirming the osmotolerance of TL126. Buoyant densities were determined by equilibrium sedimentation in a Percoll gradient of osmotic strength equal to that of the growth medium. The osmolarity of the Percoll gradient was adjusted by the addition of NaCl. At low osmolarities (300 to 500 mosM), the buoyant density of TL126 (osmotolerant) was slightly but consistently lower than that of TL128 (wild type). As the osmotic strength was increased, the buoyant density of TL126 (osmotolerant) increased in proportion to the osmotic strength. In contrast, the buoyant density of strain TL128 (wild type) did not increase as much. At high osmolarities (1,600 to 2,000 mosM), the buoyant density of TL126 (osmotolerant) was consistently higher than that of TL128 (wild type). These results suggest that the intracellular accumulation of proline by TL126, the osmotolerant strain, increases both the growth rates and buoyant densities at osmolarities of 1,300 mosM and above.     

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

The effect of salt stress was studied on proline accumulationand the activities of proline metabolic pathway enzymes in seedlingand leaf tissue of two genetically stable lines (SR2P1-2 andSR3P6-2) of in vitro selected NaCl-tolerant plants and parentcultivar Prakash of Brassica juncea L. Salt stress caused differentialenhancement in proline level in both seedlings and leaf tissueof plants at different developmental stages. The magnitude ofincrease in proline content was higher in SR3P6-2 line in seedlings(34 fold at 140 meq^-1 NaCl) as well as leaves (16 fold at 40d after sowing at 100 meq^-1 NaCl) compared to the parent cv.Prakash (29 fold in seedlings and five fold in leaves) and SR2P1-2(21 fold in seedlings and five fold in leaves) at similar stresslevels. Salt stress also resulted in changes in the activitiesof enzymes of proline metabolism. The activities of prolinebiosynthetic enzymes, pyrroline-5-carboxylate reductase andornithine aminotransferase, increased under salt stress bothin the seedlings and leaves. The range of increase in the activitiesof the two enzymes was relatively higher in SR3P6-2 (3·3-3·9fold) compared to the SR2P1-2 (1·8-2·8 fold) andparent cv. Prakash (1·5-2·8 fold). The activityof proline degrading enzyme, proline oxidase, decreased undersalt stress in both the tissues of all the lines; the reductionin activity was relatively greater in SR3P6-2 compared to SR2P1-2or cv. Prakash. The trend of changes in the enzyme activitieswas in tune with the increase in proline level, the magnitudeof change did not match the extent of increase in proline level.Copyright1995, 1999 Academic Press Brassica juncea L., NaCl-tolerant somaclones, proline content, ornithine aminotransferase, proline oxidase, pyrroline 5-carboxylate reductase     

Interactive effects of salinity, nitrogen and sulphur on the organic solutes in Spartina alterniflora leaf blades

Glycinebetaine, proline, asparagine, sucrose, glucose, and dimethylsulphoniopropionate(DMSP) were the major organic solutes in Spartina alternifloraleaf blades. To investigate the physiological role(s) of thesesolutes, the effects of salinity, nitrogen, and sulphur treatmentson leaf blade solute levels were examined. Glycinebetaine wasthe major organic solute accumulated in leaf blades grown at500 mol m^–3 NaCl, although asparagine and proline alsoaccumulated when the supply of nitrogen was sufficient. Thesesolutes may play a role in osmotic adjustment. In contrast,DMSP levels either did not change or were reduced in responseto the 500 mol m^–3 NaCl treatment. Furthermore, elevatednitrogen supply decreased leaf blade DMSP levels, which wasopposite to the response of glycinebetaine, proline, and asparagine.A 1000-fold increase in external sulphate concentration hadno effect on the leaf blade levels of DMSP, glycinebetaine,proline, or asparagine. These findings suggest that the majorphysiological role of DMSP in S. alterniflora leaf blades isnot for osmotic adjustment, even under conditions of nitrogendeficit and excess sulphur. Instead, DMSP which was presentat 45—130 µmol g^–1 dry weight, may play arole as a constitutive organic osmoticum. Key words: Spartina alterniflora, dimethylsulphoniopropionate, glycinebetaine, nitrogen, salinity     

In vitro salt tolerance of cell wall enzymes from halophytes and glycophytes

The halophyte Suaeda maritima grows optimally in high concentrations(40–60% seawater) of salt. In these conditions the concentrationof salt in the apoplast of the leaves is at least 500 mM, aconcentration which severely inhibits the activity of cytoplasmicenzymes of both glycophytes and halophytes. The in vitro salttolerance of a number of cell wall enzymes was assayed in thepresence of a range of concentrations of NaCl. There was nosignificant inhibition of the activity of galactosidase, glucosidase,peroxidase or xyloglucan endo-transglycosylase extracted fromSuaeda maritima by in vitro concentrations of NaCl up to atleast 1 M. In vitro salt tolerance of cell wall enzymes wasnot restricted to the halophyte, similar enzymes from the non-halophilicrelative Kochia tricophylla, and from the glycophytes Vignaradiata and Cicer arietinum, were inhibited little, or not atall, by the same concentrations of salt. Pectin esterase wassomewhat less tolerant, but activity at 500 mM NaCl was stillgreater than at 0 mM NaCl in both Suaeda and Vigna. It is concludedthat these enzymes of the cell wall compartment are much moresalt-tolerant than cytoplasmic enzymes of higher plants. Theresults are discussed in relation to conditions thought to pertainin the apoplast. Key words: Apoplast, cell wall enzymes, halophyte, salt tolerance, Suaeda maritima     

Salt Tolerance in Cell Suspension Cultures of the Halophyte Kosteletzkya virginica

Tolerance to NaCl was studied in cell suspension cultures ofKosteletzkya virginica (L.) Presl. (Malvaceae), a dicotyledonoushalophyte that grows in tidal marshes of the eastern UnitedStates. Growth of salinized cultures was significantly inhibitedat high (255 mol m^–3 NaCl), but not at lower externalsalinities. Adjustment of cell suspensions to Nacl was rapid,with the duration of the normal growth cycle unaffected by salinity.Maximum biomass was attained when cultures were exposed to NaClduring early log growth. Patterns of inorganic ion accumulationreflected the utilization of both Na⁺ and K⁺ as osmotica, withNa⁺ content substantially increasing when cells were grown atan external salinity sufficient to reduce growth. K⁺ uptakeselectivity was high and Na⁺/K⁺ ratios were low in salt-treatedcultures even though K⁺ content was somewhat lower comparedto unsalinized cultures. Free proline and microsomal lipid contentincreased in salt-treated cell cultures. Key words: Kosteletzkya virginica, halophyte, salt tolerance, cell suspension culture     

Proline Accumulation during Water Stress in Resurrection Plants

In leaves of resurrection plants dried intact, free prolineincreased in nine species and remained unchanged or declinedin two other tolerant species. Infiltration of proline into hydrated resurrection leaves oftwo species (a relatively high accumulator of proline, Borya,and low accumulator, Myrothamnus) prior to dehydration did notimprove the cells' survival rate, and in some cases caused adecrease in survival. These data indicate that the extreme drought tolerance of resurrectionplants is not related per se to proline levels.