Effects of NaCl and mannitol induced stress on sugarcane (<Emphasis Type="Italic">Saccharum</Emphasis> sp.) callus cultures

The effects of NaCl and mannitol iso-osmotic stresses on calli issued from sugarcane cultivars (cvs.) R570, CP59-73 and NCo310 were investigated in relation to callus growth, water content, ion and proline concentrations. Callus growth and water content decreased under both stresses with the highest reduction under mannitol-induced osmotic stress. The ion concentration was drastically affected after exposure to NaCl and mannitol. Salt stress induced an increase in Na⁺ and Cl⁻ accumulation and a decrease in K⁺ and Ca²⁺ concentrations. Under mannitol-induced osmotic stress, K⁺ and Ca²⁺ concentrations decreased significantly while Na⁺ and Cl⁻ concentrations remained unchanged. Free proline accumulation occurred under both stresses and was more marked in stress-sensitive cv. than in stress-resistant one. Our results indicated that the physiological mechanisms operating at the plant cell level in response to salt- and osmotic-induced stress in sugarcane cvs. are different. Among the cvs., we concluded that the stress resistance is closely related to the maintain of an adequate water status and a high level of K⁺ and Ca²⁺ under both stresses and a low level of Na⁺ concentration in the presence of NaCl. Thus, sugarcane (Saccharum sp.) can be regarded as a Na⁺ excluder. We also provided evidence that proline accumulation is a stress-sensitive trait rather than a stress resistance marker.     

Cd-induced growth reduction in the halophyte <Emphasis Type="Italic">Sesuvium portulacastrum</Emphasis> is significantly improved by NaCl

The effects of Cd²⁺ and NaCl, applied together or separately, on growth and uptake of Cd²⁺ were determined for the halophyte Sesuvium portulacastrum L. Seedlings were cultivated in the presence of 50 or 100 μmol L⁻¹ Cd²⁺ alone or combined with 100 or 400 mmol L⁻¹ NaCl. Data showed that alone, Cd²⁺ induced chlorosis, necrosis, and inhibited growth. Addition of NaCl to Cd²⁺-containing medium restored growth and alleviated the toxicity, however. NaCl also enhanced the amounts of Cd²⁺ accumulated in the shoots. All Cd²⁺ treatment reduced K⁺ and Ca²⁺ uptake and transport to the shoots. Accumulation of Na⁺ in the shoots was not affected by Cd²⁺, however. Thus S. portulacastrum maintained its halophytic characteristics in the presence of Cd²⁺. We suggest this halophyte could be used for phytoextraction of Cd²⁺ from salt-contaminated sites.     

Regulated alterations in redox and energetic status are the key mediators of salinity tolerance in the halophyte <Emphasis Type="Italic">Sesuvium portulacastrum</Emphasis> (L.) L

The present work addresses the importance of antioxidant, redox and energetic parameters in regulating salt-tolerance in Sesuvium portulacastrum. Experiments were conducted on 45 days old plants subjected to 250 and 1,000 mM NaCl stress for 2–8 days. Plants showed no significant change in growth parameters (shoot length, dry weight, and water content) at 250 mM NaCl as compared to control. However, growth of plants was significantly affected at 1,000 mM NaCl. The differential growth behaviour could be attributed to a greater decline in the energetic parameters (in terms of ratios of NADP/NADPH and ATP/ADP) at 1,000 mM NaCl than at 250 mM NaCl. The osmotic stress imposed to plants at 250 mM NaCl was presumably balanced by the accumulation of sodium ions (Na⁺), an energetically favorable process, and did not require an increased synthesis of proline. In contrast, to counter osmotic stress at 1,000 mM NaCl, plants accumulated Na⁺ as well as proline and were, therefore, energetically stressed. Further, the response of enzymatic and molecular antioxidants at 1,000 mM was either close to or even lower than that at 250 mM, which resulted in oxidative damage at 1,000 mM, particularly on longer durations. In conclusion, it is suggested that altered redox and energetic status of the plants could play a key role in mediating the tolerance of Sesuvium under salinity stress.     

Effect of salinity on osmotic adjustment characteristics of <Emphasis Type="Italic">Kandelia candel</Emphasis>

To elucidate the osmotic adjustment characteristics of mangrove plants, inorganic ion and organic solute contents of intermediate leaves were investigated in 3-month-old Kandelia candel (L.) Druce seedlings during 45 days of NaCl treatments (0, 200, and 500 mM NaCl). The contents of Na⁺, Cl⁻, total free amino acids, proline, total soluble sugars, pinitol and mannitol increased to different degree by salinity, whereas, K⁺ content decreased by salinity compared with control. NaCl treatment induced an increase of inorganic ion contribution while a decrease of organic solute contribution. It was concluded that accumulating a large amount of inorganic ions was used as the main osmotic adjustment mechanism under salinity treatment. However, accumulation of organic osmolytes might be considered to play much more important role in osmoregulation under severe salinity (500 mM NaCl) than under moderate salinity (200 mM NaCl), thus the damage caused by high toxic ions (Na⁺ and Cl⁻) concentration in K. candel leaves could be avoided.     

Biochemical,physiological and growth changes in response to salinity in callus cultures of <Emphasis Type="Italic">Sesuvium portulacastrum</Emphasis> L.

In vitro-grown cells of Sesuvium portulacastrum L., an important ‘salt accumulator’ mangrove associate, were incubated on a medium containing different levels of salt, including 0, 100, 200, or 400 mM NaCl, in order to evaluate biochemical, physiological, and growth responses. A significant decrease in callus growth, water status, and cell membrane damage was observed under salt stress. Osmotic adjustment was revealed by the accumulation of inorganic ions, such as sodium (Na⁺), and organic osmolytes (proline, glycine betaine, and total soluble sugars) in NaCl-treated calli compared to control. However, accretion of osmolytes and inorganic ions did not support growth of calli under NaCl stress conditions. The observed reduced growth rate in calli subjected to stress, up to 200 mM NaCl, was coupled with lower catalase and ascorbate peroxidase activities and with a significantly higher superoxide dismutase activity. These findings suggested that S. portulacastrum cell cultures exhibited higher osmotic adjustment to salt stress.     

Ability of multicellular salt glands in <Emphasis Type="Italic">Tamarix</Emphasis> species to secrete Na<Superscript>+</Superscript> and K<Superscript>+</Superscript> selectively

The present study aimed to determine the mechanism of cation-selective secretion by multicellular salt glands. Using a hydroponic culture system, the secretion and accumulation of Na⁺ and K⁺ in Tamarix ramosissima and T. laxa under different salt stresses (NaCl, KCl and NaCl+KCl) were studied. Additionally, the effects of salt gland inhibitors (orthovanadate, Ba²⁺, ouabain, tetraethylammonium (TEA) and verapamil) on Na⁺ and K⁺ secretion and accumulation were examined. Treatment with NaCl (at 0–200 mmol L⁻¹ levels) significantly increased Na⁺ secretion, whereas KCl treatment (at 0–200 mmol L⁻¹ levels) significantly increased K⁺ secretion. The ratio of secretion to accumulation of Na⁺ was higher than that of K⁺. The changes in Na⁺ and K⁺ secretion differed after adding different ions into the single-salt solutions. Addition of NaCl to the KCl solution (at 100 mmol L⁻¹ level, respectively) led to a significant decrease in K⁺ secretion rate, whereas addition of KCl to the NaCl solution (at 100 mmol L⁻¹ level, respectively) had little impact on the Na⁺ secretion rate. These results indicated that Na+ secretion in Tamarix was highly selective. In addition, Na⁺ secretion was significantly inhibited by orthovanadate, ouabain, TEA and verapamil, and K⁺ secretion was significantly inhibited by ouabain, TEA and verapamil. The different impacts of orthovanadate on Na⁺ and K⁺ secretion might be the primary cause for the different Na⁺ and K⁺ secretion abilities of multicellular salt glands in Tamarix.     

Alleviation of phosphorus deficiency stress by moderate salinity in the halophyte <Emphasis Type="Italic">Hordeum maritimum</Emphasis> L.

Hordeum maritimum (Poacea) is a facultative halophyte potentially useful for forage production in saline zones. Here, we assessed whether moderate NaCl-salinity can modify the plant response to phosphorus (P) shortage. Plants were cultivated for 55 days under low or sufficient P supply (5 or 60 μmol plant⁻¹ week⁻¹ KH₂PO₄, respectively), with or without 100 mM NaCl. When individually applied, salinity and P deficiency significantly restricted whole-plant growth, with a more marked effect of the latter stress. Plants subjected to P deficiency showed a significant increase in root growth (as length and dry weight) and root/shoot DW ratio. Enhanced root growth and elongation presumably correspond to the well-known root adaptive response to mineral deficiency. However, leaf relative water content, leaf P concentration, and leaf gas exchange parameters were significantly restricted. The interactive effects of salinity and P deficiency were not added one to another neither on whole plant biomass nor on plant nutrient uptake. Indeed, 100 mM NaCl-addition to P-deficient plants significantly restored the plant growth and improved CO₂ assimilation rate, root growth, K⁺/Na⁺ ratio and leaf proline and soluble sugar concentrations. It also significantly enhanced leaf total antioxidant capacity and leaf anthocyanin concentration. This was associated with significantly lower leaf osmotic potential, leaf Na⁺ and malondialdehyde (MDA) concentration. Taken together, these results suggest that mild salinity may mitigate the adverse effects of phosphorus deficiency on H. maritimum by notably improving the plant photosynthetic activity, the osmotic adjustment capacity, the selective absorption of K⁺ over Na⁺ and antioxidant defence.     

Effects of salinity on growth and compatible solutes of callus induced from <Emphasis Type="Italic">Populus euphratica</Emphasis>

Summary The present study aimed to evaluate the response to salinity of Populus euphratica, which is more salt-resistant than other poplar cultivars, at the cellular level. To this purpose, callus was induced from shoot segments of P. euphratica on Murashige and Skoog (MS) medium supplemented with 0.5 mg l⁻¹ (2.2 μM) 6-benzyladenine (BA) and 0.5 mg l⁻¹ (2.7 μM 1-naphthaleneacetic acid (NAA). Callus was transferred to MS medium supplemented with 0.25 mg l⁻¹ (1.1 μM) BA and 0.5 mg l⁻¹ NAA. The relative growth rate of callus reached a maximum in the presence of 50 mmol l⁻¹ NaCl and growth was inhibited with increasing NaCl concentrations. Examination of the changes of osmotic substances under salt stress showed that accumulation of proline, glycine betaine, and total soluble sugars increased with increasing salt concentrations. The results indicate that the response of the callus of P. euphratica to salt stress is similar to that of the whole plant.     

Effects of silicon on photosynthesis,water relations and nutrient uptake of <Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> under NaCl stress

A greenhouse experiment was conducted to investigate the effects of silicon application on Phaseolus vulgaris L. under two levels of salt stress (30 and 60 mM NaCl in the irrigation water). Salinity significantly reduced growth, stomatal conductance and net photosynthetic rate, and increased Na⁺ and Cl⁻ content mainly in roots. Silicon application enhanced growth of salt stressed plants, significantly reduced Na⁺ content especially in leaves and counterbalanced the effects of NaCl on gas exchange; the effect was more evident at 30 mM NaCl. Cl⁻ content in shoots and roots was not significantly modified by silicon application; the drop in K⁺ content caused by salinity was partially counterbalanced by silicon, especially in roots.     

Changes in water relations,photosynthetic activity and proline accumulation in one-year-old olive trees (<Emphasis Type="Italic">Olea europaea</Emphasis> L. cv. Chemlali) in response to NaCl salinity

The comparative responses of young olive trees (Olea europaea L. cv “Chemlali”) to different NaCl salinity levels were investigated over 11 months. One-year-old own rooted plants were grown in 10-L pots containing sand and perlite mixture (1:3 v/v). Trees were subjected to three irrigation treatments: CP (control plants that were irrigated with fresh water); SS1 (salt stressed plants irrigated with water containing 100 mM NaCl) and SS2 plants (salt stressed plants irrigated with water containing 200 mM NaCl). Shoot elongation rate, relative water content, leaf water potential and net carbon dioxide exchange rates decreased significantly with increased NaCl salinity level. Under stressed conditions, the increase of Na⁺ and Cl⁻ ions in both leaves and roots was accompanied with that of proline and soluble sugars. The above results show that the accumulation of proline and sugars under stressed conditions could play a role in salt tolerance. The absence of toxicity symptoms under both stress treatments and the superior photosynthetic activity recorded in SS1-treated plants suggest that cv Chemlali is better able to acclimatize to 100 mM NaCl than at 200 mM NaCl. Our findings indicate that saline water containing 100 mM NaCl, the most available water in arid region in Tunisia, can be recommended for the irrigation of cv Chemlali in the arid south of Tunisia.     

<Emphasis Type="Italic">In vitro</Emphasis> selection of NaCl-tolerant callus lines and regeneration of plantlets in a bamboo (<Emphasis Type="Italic">Dendrocalamus strictus</Emphasis> Nees)

Summary Sodium chloride-tolerant plantlets of Dendrocalamus strictus were regenerated successfully from NaCl-tolerant embryogenic callus via somatic embryogenesis. The selection of embryogenic callus tolerant to 100 mM NaCl was made by exposing the callus to increasing (0–200 mM) concentrations of NaCl in Murashige and Skoog medium having 3% (w/v) sucrose, 0.8% (w/v) agar, 3.0 mg l⁻¹ (13.6 μM) 2,4-dichlorophenoxyacetic acid (2,4-D), and 0.5mg l⁻¹ (2.3μM) kinetin (callus initiation medium). The tolerance of the selected embryogenic callus to 100 mM NaCl was stable through three successive transfers on NaCl-free callus initiation medium. The tolerant embryogenic callus had high levels of Na⁺, sugar, free amino acids, and proline but a slight decline was recorded in K⁺ level. The stable 100 mM NaCl-tolerant embryogenic callus differentiated somatic embryos on maintenance medium [MS medium +3% sucrose +0.8% agar +2.0 mg l⁻¹ (9.0 μM) 2,4-D+0.5 mg l⁻¹ (2.3 μM) kinetin] supplemented with different (0–200 mM) concentrations of NaCl. About 39% of mature somatic embryos tolerant to 100 mM NaCl germinated and converted into plantlets in germination medium [half-strength MS+2% sucrose+0.02 mg l⁻¹ (0.1 μM) α-naphthaleneacetic acid +0.1 mg l⁻¹ (0.49 μM) indole-3-butyric acid] containing 100 mM NaCl. Of these plantlets about 31% established well on transplantation into a garden soil and sand (1:1) mixture containing 0.2% (w/w) NaCl.     

Salt-induced modulation in growth,photosynthetic capacity,proline content and ion accumulation in sunflower (<Emphasis Type="Italic">Helianthus annuus</Emphasis> L.)

Salt-induced changes in growth, photosynthetic pigments, various gas exchange characteristics, relative membrane permeability (RMP), relative water content (RWC) and ion accumulation were examined in a greenhouse experiment on eight sunflower (Helianthus annuus L.) cultivars. Sunflower cultivars, namely Hysun-33, Hysun-38, M-3260, S-278, Alstar-Rm, Nstt-160, Mehran-II and Brocar were subjected to non-stress (0 mM NaCl) or salt stress (150 mM NaCl) in sand culture. On the basis of percent reduction in shoot biomass, cvs. Hysun-38 and Nstt-160 were found to be salt tolerant, cvs. Hysun-33, M-3260, S-278 and Mehran-II moderately tolerant and Alstar-Rm and Brocar salt sensitive. Salt stress markedly reduced growth, different gas exchange characteristics such as photosynthetic rate (A), water-use efficiency (WUE) calculated as A/E, transpiration rate (E), internal CO₂ concentration (C _i) and stomatal conductance (g _s) in all cultivars. The effect of 150 mM NaCl stress was non-significant on chlorophyll a and b contents, chlorophyll a/b ratio, RWC, RMP and leaf and root Cl⁻, K⁺ and P contents; however, salt stress markedly enhanced C _i /C _a ratio, free proline content and leaf and root Na⁺ concentrations in all sunflower cultivars. Of all cultivars, cv. Hysun-38 was higher in gas exchange characteristics, RWC and proline contents as compared with the other cultivars. Overall, none of the earlier-mentioned physiological attributes except leaf K⁺/Na⁺ ratio was found to be effective in discriminating the eight sunflower cultivars as the response of each cultivar to salt stress appraised using various physiological attributes was cultivar-specific.     

Salicylic acid-mediated changes in photosynthesis,nutrients content and antioxidant metabolism in two mustard (<Emphasis Type="Italic">Brassica juncea</Emphasis> L.) cultivars differing in salt tolerance

Mustard (Brassica juncea L.) cultivars Alankar (salt-tolerant) and PBM16 (salt-sensitive) plants were grown with 50 mM NaCl and were sprayed with 0.1, 0.5, and 1.0 mM salicylic acid (SA) to study the physiological processes determining salt tolerance and to observe the influence of SA application on the alleviation of NaCl-induced adverse effects. The content of leaf Na⁺, Cl⁻, H₂O₂, TBARS, and electrolyte leakage and the activity of SOD were higher in PBM16 than Alankar. In contrast, nutrients content, activity of APX and GR, glutathione content, photosynthetic and growth characteristics were higher in Alankar. Treatment of 50 mM NaCl resulted in increase of Na⁺ and Cl⁻, oxidative stress, activity of antioxidant enzymes and glutathione content, while nutrients content, photosynthetic, and growth characteristics decreased in both the cultivars. Application of 0.5 mM SA alleviated the negative effects of 50 mM NaCl maximally, but 1.0 mM SA proved inhibitory. The effect of SA was more conspicuous in Alankar than PBM16. It is concluded that the higher tolerance of Alankar was due to its lower leaf Na⁺ and Cl⁻ content, higher nutrients content, and efficient antioxidant metabolism. The application of 0.5 mM SA substantially alleviated salt-induced adverse effects in Alankar.     

Hormones-induced modifications in the response of wheat flag leaf to NaCl

A field experiment was carried out to investigate the effects of presoaking the wheat grains (Triticum aestivum L.) in 33 or 66 mM NaCl and indolyl-3-acetic acid (IAA at 50 g m⁻³), gibberellic acid (GA₃ at 100 g m⁻³) or kinetin (100 g m⁻³) on some tolerance criteria in wheat flag leaf at different stages of development. At various stages of flag leaf development pretreatment with 33 or 66 mM NaCl decreased degree of succulence (particularly 66 mM), relative growth rate, net assimilation rate, relative water content, K⁺ content and K⁺/Na⁺ ratio and at the same time induced accumulation of abscisic acid and Na⁺. In the majority of cases grain pretreatment with GA₃ or kinetin and to a lesser extent with IAA alleviated either partially or completely the deleterious effect of salinity on the above mentioned parameters.     

Leaf gas exchange,water relations,nutrient content and growth in citrus and olive seedlings under salinity

The effects of salinity on growth, leaf nutrient content, water relations, gas exchange parameters and chlorophyll fluorescence were studied in six-month-old seedlings of citrus (Citrus limonia Osbeck) and rooted cuttings of olive (Olea europaea L. cv. Arbequina). Citrus and olive were grown in a greenhouse and watered with half strength Hoagland’s solution plus 0 or 50 mM NaCl for citrus, or plus 0 or 100 mM NaCl for olive. Salinity increased Cl⁻ and Na⁺ content in leaves and roots in both species and reduced total plant dry mass, net photosynthetic rate and stomatal conductance. Decreased growth and gas exchange was apparently due to a toxic effect of Cl⁻ and/or Na⁺ and not due to osmotic stress since both species were able to osmotically adjust to maintain pressure potential higher than in non-salinized leaves. Internal CO₂ concentration in the mesophyll was not reduced in either species. Salinity decreased leaf chlorophyll a content only in citrus.     

Localization of aquaporin-2, renal morphology and urine composition in the bottlenose dolphin and the Baird’s beaked whale

This study examined the distribution pattern of aquaporin-2 (AQP2), relative medullary thickness (RMT) and urine properties in the bottlenose dolphin Tursiops truncatus and Baird’s beaked whale Berardius bairdii. Immunohistochemical studies revealed that AQP2 was localized in the collecting tubules/ducts of both species’ renicules, as in terrestrial mammals. The collecting ducts with AQP2 were thinner and arranged more densely in the dolphin than in the whale. RMT values in the renicule were moderate in both species, but were significantly higher in the dolphin (6.0 ± 0.9) than the whale (4.9 ± 0.7). Urine of the bottlenose dolphin is comparatively concentrated (osmolality: 1715.7 ± 279.4 mOsm kg⁻¹, Na⁺: 490.1 ± 87.9 mmol l⁻¹, Cl⁻: 402.7 ± 79.6 mmol l⁻¹, K⁺: 80.7 ± 25.8 mmol l⁻¹, urea nitrogen: 703.5 ± 253.9 mmol l⁻¹), while urine of the dead Baird’s beaked whale is less concentrated (osmolality: 837.5 ± 293.8 mOsm kg⁻¹, Na⁺: 192.9 ± 81.5 mmol l⁻¹, Cl⁻: 159.9 ± 71.4 mmol l⁻¹, K⁺: 44.3 ± 29.5 mmol l⁻¹, urea nitrogen: 270.7 ± 120.3 mmol l⁻¹). These data suggest it is possible that the differences in these renal morphological features may be related in some way to the difference in urine composition between the species, although further studies are necessary. M. Suzuki and N. Endo are equal contributors to this study.     

Osmotic adjustment,water relations and growth attributes of the xero-halophyte <Emphasis Type="Italic">Reaumuria vermiculata</Emphasis> L. (Tamaricaceae) in response to salt stress

Reaumuria vermiculata (L.), a perennial dwarf shrub in the family of Tamaricaceae, is a salt-secreting xero-halophyte found widely in arid areas of Tunisia. In the present study, physiological attributes of R. vermiculata were investigated under salt stress. Four-month-old plants were subjected to various salinity levels (0, 100, 200, 300, 400 or 600 mM NaCl) for 30 days under greenhouse conditions. Results showed that plants grew optimally when treated with standard nutrient solution without NaCl supply. However, increasing osmolality of nutrient solutions caused a significant reduction in biomass production and relative growth rate. This reduction was more pronounced in roots than in shoots. In addition, this species was able to maintain its shoot water content at 30% of the control even when subjected to the highest salt level, whereas root water content seemed to be unaffected by salt. Shoot water potential declined significantly as osmotic potential of watering solutions was lowered and the more negative values were reached at 600 mM NaCl (−3.4 MPa). Concentrations of Na⁺ and Cl⁻ in the shoots of R. vermiculata were markedly increased with increasing osmolality of nutrient solutions, whereas concentration of K⁺ was not affected by NaCl supply. Salt excretion is an efficient mechanism of Na⁺ exclusion from the shoots of this species exhibiting high K⁺/Na⁺ selectivity ratio over a wide range of NaCl salinity. Proline accumulation in shoots was significantly increased with increase in salt level and may play a role in osmoregulation.     

Salt stress responses of a halophytic grass <Emphasis Type="Italic">Aeluropus lagopoides</Emphasis> and subsequent recovery

To investigate the salt tolerance mechanisms, Aeluropus lagopoides as a halophytic plant was used. Plants were treated with 0, 150, 450, 600, and 750 mM NaCl and harvested at 0, 4, 8, and 10 days after treatment and 1 day and 1 week after recovery. Optimal growth, measured as fresh and dry weights, occurred at 150 mM NaCl, but it was suppressed by 450, 600, and 750 mM NaCl. Recovery significantly increased fresh and dry weights only in 750 mM NaCl-treated plants. Water content was decreased after NaCl treatment and increased after recovery. Na⁺ and proline contents and activity of superoxide dismutase (SOD) were increased after NaCl treatment and decreased after recovery in all treated plants. In contrast, K⁺ content and ascorbate peroxidase activity decreased after NaCl treatment and increased after recovery in all treated plants. Catalase (CAT) was activated only in 750 mM NaCl-treated plants. Total content of soluble protein was slightly changed after NaCl treatment. It was concluded that proline accumulation for osmotic adjustment, SOD activation for O₂^·− scavenging, and CAT activation at the higher level of salt stress to detoxify produced H₂O₂ were main A. lagopoides strategies under salt stress. A. lagopoides salt tolerance was not based on the restriction of Na⁺ uptake.     

<Emphasis Type="SmallCaps">l</Emphasis>-Proline nutrition and catabolism in <Emphasis Type="Italic">Staphylococcus saprophyticus</Emphasis>

Staphylococcus saprophyticus strains ATCC 15305, ATCC 35552, and ATCC 49907 were found to require l-proline but not l-arginine for growth in a defined culture medium. All three strains could utilize l-ornithine as a proline source and contained l-ornithine aminotransferase and Δ¹-pyrroline-5-carboxylate reductase activities; strains ATCC 35552 and ATCC 49907 could use l-arginine as a proline source and had l-arginase activity. The proline requirement also could be met by l-prolinamide, l-proline methyl ester, and the dipeptides l-alanyl-l-proline and l-leucyl-l-proline. The bacteria exhibited l-proline degradative activity as measured by the formation of Δ¹-pyrroline-5-carboxylate. The specific activity of proline degradation was not affected by addition of l-proline or NaCl but was highest in strain ATCC 49907 after growth in Mueller–Hinton broth. A membrane fraction from this strain had l-proline dehydrogenase activity as detected both by reaction of Δ¹-pyrroline-5-carboxylate with 2-aminobenzaldehyde (0.79 nmol min⁻¹ mg⁻¹) and by the proline-dependent reduction of p-iodonitrotetrazolium (20.1 nmol min⁻¹ mg⁻¹). A soluble fraction from this strain had Δ¹-pyrroline-5-carboxylate dehydrogenase activity (88.8 nmol min⁻¹ mg⁻¹) as determined by the NAD⁺-dependent oxidation of dl-Δ¹-pyrroline-5-carboxylate. Addition of l-proline to several culture media did not increase the growth rate or final yield of bacteria but did stimulate growth during osmotic stress. When grown with l-ornithine as the proline source, S. saprophyticus was most susceptible to the proline analogues L-azetidine-2-carboylate, 3,4-dehydro-dl-proline, dl-thiazolidine-2-carboxylate, and l-thiazolidine-4-carboxylate. These results indicate that proline uptake and metabolism may be a potential target of antimicrobial therapy for this organism.     

The role of ion regulation in the control of the distribution of Gammarus tigrinus (Sexton) in salt-polluted rivers

Fluctuating salinities at different sites on the German salt-polluted rivers Werra and Weser were compared with extracellular ion levels of specimens of Gammarus tigrinus (Sexton; Amphipoda, Crustacea), collected at the same sites. G. tigrinus regulated haemolymph concentrations of inorganic anions (Cl⁻, SO²⁻ ₄, PO³⁻ ₄) and cations (Na⁺, K⁺, Mg²⁺, Ca²⁺) during fluctuations of salt pollution in the upper Weser. This capacity to regulate varying levels of salt pollution in the upper Weser, correlated well with the distribution of the brackish amphipods in this river ecosystem. G. tigrinus tolerated periods of Na⁺ and Cl⁻ stress (>380 mmol l⁻¹) without compensating these maxima by regulating extracellular Na⁺ and Cl⁻. However, during such bursts of Na⁺ and Cl⁻ stress in Werra and Weser, the ability to regulate extracellular [K⁺] at river water K⁺ stress of ≥6.0 mmol l⁻¹ may explain why this brackish species has been more successful in these rivers than its competitors like Gammarus pulex. The present investigation demonstrates that the water salinity affects the [NO⁻ ₃] in the haemolymph of G. tigrinus. With increasing hypo-osmotic stress the animals accumulate increasing amounts of NO⁻ ₃. A simultaneous increase in stream water [NO⁻ ₃] causes an additional accumulation of NO⁻ ₃ in the haemolymph. The high extent of accumulation indicates that active ion transport systems may be involved. The accumulation of NO⁻ ₃ in the haemolymph has low physiological consequences to G. tigrinus, but when hypo-osmotically stressed under anoxic conditions, nitrite formed by the reduction of nitrate may have an adverse affect on the metabolism of G. tigrinus. Accepted: 4 October 1999