Proline accumulation and sodium sulfate tolerance in callus cultures of Brassica napus L. cv. Westar

Callus cultures of Brassica napus L. cv. Westar were selected which contained 5 – 6 times more proline than unselected callus. Callus pieces from these cultures were able to survive much better after subculture to medium containing 105 mM Na₂SO₄ than unselected callus, or unselected callus cultured on exogenous proline before or during transfer to the salt. Exogenous proline was rapidly absorbed. In unselected callus there was a peak in proline accumulation ca. 2 days after transfer to Na₂SO₄, followed by a decline. In contrast proline accumulation in tolerant callus was linear with time, reaching maximum levels at 8 days. Proline levels induced by exposure to salt were maintained in the absence of stress.Abbreviations DW Dry weight - FW Fresh weight     

Stable sodium sulfate tolerance inBrassica napus cv. Westar callus cultures

Summary Sodium-sulfate-tolerant callus ofBrassica napus cv. Westar, selected on medium containing 105 mM Na₂SO₄, was maintained on medium without the salt to test for stability of tolerance. Tolerance to Na₂SO₄ was retained even after 18 subcultures on no salt. This tissue also showed tolerance to K₂SO₄, NaCl, and KCl. However, with the exception of callus grown on KCl fresh weight yields were less than that of tolerant callus maintained continuously on Na₂SO₄. Tolerant callus maintained on no salt had a mixture of the compact morphology of unselected callus and the friable morphology of tolerant callus. Both callus types expressed salt tolerance. Sucrose, reducing sugars and proline concentrations were measured in unselected callus, tolerant callus maintained continuously on Na₂SO₄, and tolerant callus maintained on no salt. Sucrose levels were similar in all cases. Whether maintained on or off Na₂SO₄., tolerant callus had reducing sugars levels three to fou times greater than unselected callus. Tolerant callus maintained on no salt had twice the amount of proline found in the unselected callus. Tolerant callus maintained in the absence of salt had an ash content, sodium concentration, and potassium concentration significantly lower than that of unselected callus. Supported by the Natural Sciences and Engineering Research Council of Canada Strategic Grant nos. G 0949 and G 1642 to T. A. Thorpe, D. M. Reid, and E.C. Yeung.     

Some morphogenic effects of sodium sulfate on tobacco callus

Callus cultures of Nicotiana tabacum L cv. Wisconsin 38 were initiated and grown on shoot-forming (SF) and callus proliferation (CP) medium with or without Na₂SO₄. Two cultures were maintained on SF medium with 0, 0.75, 1 or 1.5% Na₂SO₄ for 2.5 and 3.5 years. In the older culture only callus grown on salt formed shoots throughout the maintenance period, while in the younger culture the control responded best and Na₂SO₄ was inhibitory. Callus from the older culture which had been grown on salt continued to form shoots in the absence of salt. Na₂SO₄ caused adventitious shoot formation in three cultures on CP medium. These shoots were present for 7 subcultures after removal of Na₂SO₄; but established, control callus, did not form shoots when transferred to Na₂SO₄. Callus initiated and maintained on NaCl or mannitol showed a slight increase in shoot initiation. On NaCl, Na₂SO₄ or mannitol, the tissue osmotic potential became more negative and proline concentration increased.     

Selection and characterization of mannitol-tolerant callus lines of Vigna radiata (L.) Wilczek

An osmotically (mannitol) tolerant callus line of Vigna radiata (L.) Wilczek has been isolated from callus cultures grown on modified PC-L2 medium supplemented with increasing concentrations of mannitol. The tolerance was stable and retained after growth in the absence of mannitol selection for 2 months. The growth of the tolerant line, in the presence of mannitol (540 mol m^-3) was comparable to that of a sensitive callus line growing in the absence of mannitol. This line not only grew well on media containing up to 720 mol m^-3 mannitol, but also required 450 mol m^-3 mannitol for its optimal growth. Osmotically tolerant callus also showed increased tolerance to NaCl (0–250 mol m^-3) stress as compared to sensitive callus. Accumulation of Na⁺ was lower, and the level of K⁺ was more stable in osmotically tolerant than in sensitive calli, when both were exposed to salt. The free proline content of both tolerant and sensitive calli increased on media supplemented with mannitol or NaCl. However, the proline content of sensitive callus was higher than in tolerant callus in the presence of same concentrations of mannitol or NaCl.Abbreviations NAA -naphthaleneacetic acid - 2,4-d 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine     

Growth, proline accumulation and ion content in sodium chloride-stressed callus of date palm

Summary Growth and physiological responses of date palm. Phoenix dactylifera L. cv. Barhee, callus to salinity stress were examined. Callus induced from shoot tips of offshoots was cultured on Murashige and Skoog medium supplemented with NaCl at concentrations ranging from 0 to 225 mM, in consective increments of 25 mM. Data obtained after 6 wk of exposure to salt have shown a significant increase in callus proliferation in response to 25 mM NaCl the lowest level tested, beyond which callus weight decreased. At 125 mM NaCl and higher, callus growth was nearly completely inhibited. Physiological studies on callus exposed to salt stress have shown an increase in proline accumulation in response to increased salinity. Proline accumulation was correlated to callus growth inhibition. Furthermore, increasing the concentration of NaCl in the culture medium generally resulted in a steady increase in Na⁺ and reduction in K⁺ concentrations. However, at 25 mM NaCl, the only level at which callus growth was significantly enhanced, an increase in K⁺ content was noted, in comparison to the NaCl free control. In response to increasing external NaCl level, the Na⁺/K⁺ ratio increased The Na⁺/K⁺ ratio was positively correlated to proline accumulation and hence callus growth inhibition. This study provides, an understanding of the response of date palm callus to salinity, which is important for future studies aimed at developing strategies for selecting and characterizing somaclonal variants tolerant to salt stress.     

The effect of sodium chloride on solute potential and proline accumulation in soybean leaves

Two cultivars of soybean (Glycine max [L.] Merr.) were grown in solution with up to 100 millimolar NaCl. Leaf solute potential was −1.1 to −1.2 megapascals in both cultivars without NaCl. At 100 millimolar NaCl leaf solute potential was −3.1 to −3.5 megapascals in Bragg and −1.7 megapascals in Ransom. The decrease in solute potential was essentially proportional to the concentration of NaCl. In both salt susceptible Bragg and salt semitolerant Ransom, leaf proline was no more than 0.4 micromole per gram fresh weight at or below 20 millimolar NaCl. At 40 and 60 millimolar NaCl, Bragg leaf proline levels were near 1.2 and 1.9 micromoles per gram fresh weight, respectively. Proline did not exceed 0.5 micromole per gram fresh weight in Ransom even at 100 millimolar NaCl. Proline accumulated in Bragg only after stress was severe enough to induce injury; therefore proline accumulation is not a sensitive indicator of salt stress in soybean plants.     

Proline accumulation and its implication in cold tolerance of regenerable maize callus

Embryogenic callus of maize (Zea mays L.) inbreds B37wx, H99, H99³H95, Mo17, and Pa91 accumulated proline to levels 2.1 to 2.5 times that of control callus when subjected to mannitol-induced water stress, cool temperatures (19°C) and abscisic acid (ABA). A combination of 0.53 molar mannitol plus 0.1 millimolar ABA induced a proline accumulation to about 4.5 times that of control callus, equivalent to approximately 0.18 millimoles proline per gram fresh weight of callus. Proline accumulation was directly related to the level of mannitol in the medium. Levels of ABA greater than 1.0 micromolar were required in the medium to induce proline accumulation comparable to that induced by mannitol. Mannitol and ABA levels that induced maximum accumulation of proline also inhibited callus growth and increased tolerance to cold. Proline (12 millimolar) added to the culture media also increased the tolerance of callus to 4°C. The increased cold tolerance induced by the combination of mannitol and ABA has permitted the storage of the maize inbreds A632, A634Ht, B37wx, C103DTrf, Fr27rhm, H99, Pa91, Va35, and W117Ht at 4°C for 90 days which is more than double the typical survival time of callus. These studies show that proline and conditions which induce proline accumulation increase the cold tolerance of regenerable maize callus.     

Proline is not the primary determinant of chilling tolerance induced by mannitol or abscisic Acid in regenerable maize callus cultures

Chilling sensitive regenerable maize (Zea mays L.) callus cultures can be induced to survive prolonged exposure to 4°C by treatments with mannitol, abscisic acid (ABA), and/or high levels of proline. Maize callus with a free proline content of about 122 micromoles/grain fresh weight survived longer exposures to 4°C than did callus with a free proline content of about 68 micromoles/grain fresh weight. The addition of 0.53 molar mannitol or 0.1 millimolar ABA to culture medium produced a free proline content in maize callus of about 136 and 145 micromoles/grain fresh weight, respectively, if the medium contained 12 millimolar proline or about 36 and 1 micromoles/grain fresh weight, respectively, if no proline was in the medium. Although these mannitol and ABA treatments produced drastically different free proline levels in maize callus, callus grown on these media survived longer exposures to 4°C than did maize callus grown on any proline treatment alone. Thus, the internal free proline level of treated callus is not the primary factor conferring chilling tolerance on these tissues.     

Retention of shoot regeneration capacity of tobacco callus by Na2SO4

Callus of tobacco (Nicotiana tabacum L. cv. Wisconsin 38) was grown in the light on shoot-forming medium in the presence of Na₂SO₄ for over a year. An increase in Na₂SO₄ concentration resulted in decreasing callus growth, decreasing percentage of calli producing shoots and number of shoots per callus, and increasing callus percent dry weight. Regeneration of shoots from callus grown in the absence of Na₂SO₄ began to decline after 14 months in culture, and shoot regeneration capacity was completely lost after 18 months. In contrast, 18-month old callus continuously grown in the presence of Na₂SO₄ retained the ability to form shoots. The highest percent of callus pieces that formed shoots and the maximum number of shoots per callus occurred at 70 mM (1%) Na₂SO₄. All plants arising from the 18-month old callus were polyploid. Both 9- and 18-month old callus exhibited more negative water and osmotic potentials in the presence of increasing Na₂SO₄ concentration.Abbreviations water potential - _S osmotic potential - CGI callus growth index     

Differential Na2SO4 tolerance in tobacco plants regenerated from Na2SO4-grown callus

Abstract The regenerated shoots from sodium sulphate (Na₂SO₄) grown callus of tobacco (Nicotiana tabacum L. cv. Wisconsin 38) were evaluated for Na₂SO₄ tolerance based on shoot proliferation and rooting in vitro, and seed germination in vivo in response to Na₂SO₄. An increase in Na₂SO₄ concentration resulted in significantly decreasing shoot fresh weight, number of shoots, shoot length and leaf size, and increasing per cent shoot dry weight of both control and Na₂SO₄-grown cultures. In rooting, shoots of Na₂SO₄-grown cultures exhibited the highest per cent rooting (85%) in the presence of 1% w/v Na₂SO₄. However, per cent rooting, root number per rooted cutting and root fresh weight decreased significantly with increasing Na₂SO₄ concentration when shoots were transferred to the medium in the absence of Na₂SO₄ for 4-monthly passages. Following acclimatization of the rooted shoots of Na₂SO₄-grown cultures, phenotypic variation was observed during growth and development. There were 13.2% sterile plants. Fertile plants were sorted into normal (N), tolerant (T), and sensitive (S) categories and the respective percentages of plants were 31.6, 44.7 and 10.5, based on per cent germination, germination velocity index and seedling survival to Na₂SO₄. The response of N, T and S types to Na₂SO₄ in subsequent shoot proliferation was similar to that of seed germination.     

Effects of NaCl on Proline Synthesis and Utilization in Excised Barley Leaves

Proline accumulation in NaCl-treated excised barley (Hordeum vulgare var Larker) leaves was studied. Leaves were treated by placing the cut end in NaCl solutions and allowing the salt to enter the leaf via the transpiration stream. Leaves treated this way maintained turgor while the sodium content increased and the osmotic potential decreased. Proline began accumulating after 12 hours and continued accumulating over the subsequent 12-hour period at an average rate of 0.6 micromoles per hour per gram fresh weight.

During the time proline was accumulating, [¹⁴C]glutamate was added to measure the effects of salt on proline synthesis from glutamate and [¹⁴C] proline was added in separate experiments to determine the effect of salt on proline utilization. Salt treatment dramatically increased proline synthesis from glutamate. Proline utilization by oxidation and for protein synthesis was decreased by 50 and 60%, respectively, by the salt treatment.

These effects are similar to the effects of drought and abscisic acid in barley leaves. The results indicate that common mechanisms cause proline to accumulate under these different stresses.

     

Effect of sodium and calcium chlorides,abscisic acid and proline on callus cultures ofArachis hypogaea L.

Callus cultures ofArachis hypogaea L. cv. JL-24 adapted to 200 mM NaCl (otherwise lethal to cells) were used for the study. Calli grew slowly when transferred to 250 mM NaCl, but the growth was enhanced when ABA was included in the medium. ABA induced increase in growth of callus was not accompanied by corresponding increase in internal free proline levels. 0.5 mM of CaCl₂ ameliorated the negative effect of NaCl indicating that cells require a specific Ca²⁺/Na⁺ ratio for their growth. Proline content also increased at this ratio thereby suggesting that increase in growth at 0.5 mM Ca²⁺ may be due to an increase in proline content. However, exogenous proline did not increase the growth of callus (adapted to 200 mM), and higher concentrations even inhibited the growth. This shows that proline is not required for growth or adaptation of cells to salt stress, but is produced as a consequence of stress.     

In vitro characterization of salt stress effects and the selection of salt tolerant plants in rice (Oryza sativa L.)

Summary The response of plant cells to salt stress was studied on embryo derived calli of rice (Oryza sativa L.) in order to identify cellular phenotypes associated with the stress. The feasability of selecting salt tolerant callus and its subsequent regeneration to plants was also studied. Callus was grown on agar-solidified media containing 0%, 1% and 2% (w/v) NaCl for 24 days. Parameters such as fresh weight, dry weight, soluble protein and proline content were measured. The callus growth decreased markedly with increasing NaCl concentration in the medium. The proline content was enhanced several fold in salt stressed calli. A prolonged exposure of callus to the salt environment led to discolouration and arrested growth in the majority of the calli and only a small number of callus cells maintained healthy and stable growth. These variants were subcultured every three weeks for a period of four months onto medium containing 1% NaCl to identify tolerant lines. At the end of the third cell passage, the tolerant calli were transferred to regeneration medium to regenerate plants. The regeneration frequency in the salt-selected lines was enhanced when compared to unselected lines.     

Osmoregulation inThiobacillus ferrooxidans: Stimulation of iron oxidation by proline and betaine under salt stress

The osmoregulatory mechanisms of chemolithotrophs have not previously been investigated. We tested glutamic acid, proline, and betaine as potential osmorprotectants for the acidic chemolithotrophThiobacillus ferrooxidans. Salt stresses were imposed by NaCl, KCl, Na₂SO₄, and K₂SO₄ in separate experiments. Proline enhanced rates of iron oxidation in all saltstressed cultures, whereas betaine acted as an osmoprotectant only in sulfate-salt-stressed cultures and in cultures severely stressed by NaCl (0.3–0.4M). Glutamic acid inhibited iron oxidation in all cases.     

Iso-osmotic effect of NaCl and PEG on growth, cations and free proline accumulation in callus tissue of two indica rice (Oryza sativa L.) genotypes

One-month old calli of two indica rice genotypes, i.e., Basmati-370 and Basmati-Kashmir were subjected to two iso-osmotic concentrations (−0.57 MPa and −0.74 MPa) created with 50 and 100 mol m⁻³ NaCl or 10 and 18% solutions of PEG-8000. Both genotypes tolerated only low levels of stress and showed severe growth suppression at −0.74 MPa. The degree of stress tolerance of both genotypes was greater for PEG induced stress than for NaCl induced stress. The relative growth rate of callus was reduced under both stresses, however, the reverse was true for callus dry weight. Sodium (Na⁺) content of the callus tissue was increased only under NaCl induced stress. Salt induced stress reduced K⁺ and Ca²⁺ contents, but the PEG induced stress increased them. Higher levels of stress increased the proline content many folds with more increase being under PEG stress than that under NaCl. Water and osmotic potentials of the callus tissue decreased, whereas turgor potential increased under both abiotic stresses. Overall, Basmati-370 was more tolerant to both NaCl and PEG induced stresses than Basmati-Kashmir, because of less reduction in growth and more dry weight. Moreover, Basmati-370 accumulated higher amounts of cations, free proline, and maintained maximum turgor as compared to Basmati-Kashmir. In conclusion, at cellular level, mechanism of NaCl induced osmotic stress tolerance was found to be associated with more ionic accumulation of inorganic solutes and that of PEG induced osmotic stress tolerance with the accumulation of free proline, as an important osmolyte in the cytosol.     

Callus culture from hypocotyls of Kosteletzkya virginica (L.) seedlings

It was shown that callus established from Kosteletzkya virginica (L.) Presl. (Malvaceae) can grow in salinities higher than 200 mM NaCl if previously accomodated stepwise. Callus lines developed from seedlings of different harvests or of the same harvest at different times, all showed the same pattern of growth and sensitiviy to salinity. The absorption of Na⁺ into the callus increased with increasing external NaCl concentration. In the callus, Na⁺ was apparently distributed outside and inside a cellular membrane (possibly the plasmalemma). This membrane was, apparently, capable of regulating the Na⁺ concentration in the protoplast. Outside this membrane Na⁺ accumulated to concentrations higher than in the external growth medium. Exogenously supplied proline or glycine-betaine did not affect the growth of the callus. Externally applied ABA stimulated growth under saline conditions and increased the accumulation of proline. Growth and proline content were positively correlated in callus exposed to salinity, but in the presence of ABA they were negatively correlated. ABA was involved in both growth and proline accumulation, but there was no clear relationship between these two effects. Both ABA and proline, if added to the growth medium, improved the appearence of the callus.Abbreviations ABA abscisic acid - B₅ Gamborg's medium - BA benzylalanine - 2,4-d 2,4-dichlorophenoxy acetic acid - FW fresh weight - G B₅ medium without growth regulators - GH B₅ medium supplemented with growth regulators - NAA naphthalene acetic acid - PGR plant growth regulators - Q _T total amount of a certain ion in the tissue - Q _s amount of the ion that has leaked out - QAC Quaternarty Ammonium Compounds - RGR mean relative growth rate - W₁ and W₂ fresh weight at times t₁ and t₂     

Relationship between Stress-Induced ABA and Proline Accumulations and ABA-Induced Proline Accumulation in Excised Barley Leaves

When excised second leaves from 2-week-old barley (Hordeum vulgare var Larker) plants were incubated in a wilted condition, abscisic acid (ABA) levels increased to 0.6 nanomole per gram fresh weight at 4 hours then declined to about 0.3 nanomole per gram fresh weight and remained at that level until rehydrated. Proline levels began to increase at about 4 hours and continued to increase as long as the ABA levels were 0.3 nanomole per gram fresh weight or greater. Upon rehydration, proline levels declined when the ABA levels fell below 0.3 nanomole per gram fresh weight.

Proline accumulation was induced in turgid barley leaves by ABA addition. When the amount of ABA added to leaves was varied, it was observed that a level of 0.3 nanomole ABA per gram fresh weight for a period of about 2 hours was required before proline accumulation was induced. However, the rate of proline accumulation was slower in ABA-treated leaves than in wilted leaves at comparable ABA levels. Thus, the threshold level of ABA for proline accumulation appeared to be similar for wilted leaves where ABA increased endogenously and for turgid leaves where ABA was added exogenously. However, the rate of proline accumulation was more dependent on ABA levels in turgid leaves to which ABA was added exogenously than in wilted leaves.

Salt-induced proline accumulation was not preceded by increases in ABA levels comparable to those observed in wilted leaves. Levels of less than 0.2 nanomole ABA per gram fresh weight were measured 1 hour after exposure to salt and they declined rapidly to the control level by 3 hours. Proline accumulation commenced at about 9 hours. Thus, ABA accumulation did not appear to be involved in salt-induced proline accumulation.

     

Sucrose Phosphate Synthase, Sucrose Synthase, and Invertase Activities in Developing Fruit of Lycopersicon esculentum Mill. and the Sucrose Accumulating Lycopersicon hirsutum Humb. and Bonpl

The green-fruited Lycopersicon hirsutum Humb. and Bonpl. accumulated sucrose to concentrations of about 118 micromoles per gram fresh weight during the final stages of development. In comparison, Lycopersicon esculentum Mill. cultivars contained less than 15 micromoles per gram fresh weight of sucrose at the ripe stage. Glucose and fructose levels remained relatively constant throughout development in L. hirsutum at 22 to 50 micromoles per gram fresh weight each. Starch content was low even at early stages of development, and declined further with development. Soluble acid invertase (EC 3.2. 1.26) activity declined concomitant with the rise in sucrose content. Acid invertase activity, which was solubilized in 1 molar NaCl (presumably cell-wall bound), remained constant throughout development (about 3 micromoles of reducing sugars (per gram fresh weight) per hour. Sucrose phosphate synthase (EC 2.4.1.14) activity was present at about 5 micromoles of sucrose (per gram fresh weight) per hour even at early stages of development, and increased sharply to about 40 micromoles of sucrose (per gram fresh weight) per hour at the final stages of development studied, parallel to the rise in sucrose content. In comparison, sucrose phosphate synthase activity in L. esculentum remained low throughout development. The possible roles of the sucrose metabolizing enzymes in determining sucrose accumulation are discussed.     

Osmotic adaption in Australian mangroves

Young and old leaves of twenty-three mangrove species from northern Queensland (Australia) were investigated for their mineral ion and organic solute content. With a few exceptions, the Na⁺ and the Cl-concentrations calculated on the basis of plant water (p.w.) were close to that of seawater and showed little age-induced changes. In some species, especially in Ceriops tagal, SO₄ ^2- accumulated with increasing leaf age. The most widely distributed organic solutes were pinitol and mannitol, which were stored up to 280 mM plant water. A negative correlation between pinitol and SO₄ ^2- was found in the case of Ceriops tagal. Other compatible solutes known for halophytes, such as proline and methylated quaternary ammonium compounds (MQAC), were present only in a few mangrove species. Proline occurred in two Xylocarpus species, while MQAC were accumulated by Avicennia eucalyptifolia, A. marina, Acanthus ilicifolius, Heritiera littoralis and Hibiscus tiliaceus. In all other species, low molecular weight carbohydrates (LMWC) were the main organic solutes.     

Changes in growth and water-soluble solute concentrations in Sorghum bicolor stressed with sodium and potassium salts

Sorghum bicolor L. Moench, RS 610, was grown in liquid media salinized with NaCl, KCl, Na₂SO₄, K₂SO₄ or with variable mixtures of either NaCl/KCl or Na₂SO₄/K₂SO₄ at osmotic potentials ranging from 0 to -0.8 MPa. The purpose was to study the effects of different types and degrees of salinity in growth media on growth and solute accumulation. In 14-day-old plants the severity of leaf growth inhibition at any one level of osmotic potential in the medium increased according to the following order: NaCl < Na₂SO₄ < KCl = K₂SO₄. Inhibition of growth by mixtures of Na⁺ and K⁺ salts was the same as by K⁺ salts alone. Roots responded differently. Root growth was not affected by Na⁺ salts in the range of 0 to -0.2 MPa while it was stimulated by K⁺ salts. The major cation of leaves was K⁺ because S. bicolor is a Na⁺-excluder, while Na⁺ was the major cation in roots except at low Na⁺/K⁺ ratios in media. Anions increased in tissues linearly in relation to total monovalent cation, but not with a constant anion/cation ratio. This ratio increased as the cation concentrations in tissues increased. Sucrose in leaf tissue increased 75 fold in Chloride-plants (plants growing in media in which the only anion of the salinizing salts was Cl^?) and 50 fold in Sulphate-plants (the only anion of the salinizing salts was SO₄^2-). Proline increased 60 and 18 fold in Chloride- and Sulphate-plants, respectively, as growth media potentials decreased from 0 to -0.8 MPa. The concentrations of both sucrose and proline were directly proportional to the amount of total monovalent cation in the tissue. Sucrose concentrations began increasing when total monovalent cations exceeded 100 μmol (g fresh weight)^?1 (the monovalent cation level in non-stressed plants), but proline did not start accumulating until monovalent cation concentrations exceeded 200 μmol (g fresh weight)^?1. Therefore, sucrose seemed to be the solute used for osmotic adjustment under mild conditions of saline stress while proline was involved in osmotic adjustment under more severe conditions of stress. Concentrations of inorganic phosphate, glucose, fructose, total amino acids and malic acid fluctuated in both roots and leaves in patterns that could be somewhat correlated with saline stress and, sometimes, with particular salts in growth media. However, the changes measured were too small (at most a 2–3 fold increase) to be of importance in osmotic adjustment.