Uptake and use of the osmoprotective compounds trehalose, glucosylglycerol, and sucrose by the cyanobacterium Synechocystis sp. PCC6803

Accumulation of exogenously supplied osmoprotective compounds was analyzed in the cyanobacterium Synechocystis sp. PCC6803, which synthesizes glucosylglycerol as the principal osmoprotective compound. Glucosylglycerol and trehalose were accumulated to high levels and protected cells of a mutant unable to synthesize glucosylglycerol against the deleterious effects of salt stress. In the wild-type, uptake of trehalose repressed the synthesis of glucosylglycerol and caused metabolic conversion of originally accumulated glucosylglycerol. Trehalose cannot be synthesized by Synechocystis and was not or only insignificantly metabolized. Sucrose, which can be synthesized in low quantities by Synechocystis, was also taken up, as indicated by its disappearance from the medium. Sucrose was not accumulated to high levels, probably due to a sucrose-degrading activity found in cells adapted to both low- and high-salt conditions. Despite its low intracellular concentration, sucrose showed a weak osmoprotective effect in salt-shocked cells of a mutant unable to synthesize glucosylglycerol. Received: 4 September 1996 / Accepted: 18 November 1996     

Glucosylglycerol-phosphate synthase: target for ion-mediated regulation of osmolyte synthesis in the cyanobacterium Synechocystis sp. strain PCC 6803

The response of cyanobacteria to a changing osmotic environment includes the accumulation of organic osmolytes such as glucosylglycerol. The activation of the enzymes involved in glucosylglycerol synthesis [glucosylglycerol-phosphate synthase (GGPS) and glucosylglycerol-phosphate phosphatase (GGPP)] in Synechocystis sp. strain PCC 6803 by various salts and salt concentrations was investigated in vitro. GGPS seemed to be the target for salt-mediated regulation of glucosylglycerol synthesis in vitro. GGPS activation was dependent on the concentration of NaCl, and a sigmoidal plot was obtained. Sensitivity to NaCl was markedly enhanced by low Mg⁺² concentrations (optimal at 4 mM), but Mg²⁺ was not absolutely necessary for the Na⁺ stimulation. As in the case of NaCl, other salts (including MgCl₂) stimulated GGPS. The relative order of GGPS activation in the presence of chloride by the cations at constant ionic strength was Li⁺ > Na⁺ > K⁺, Mg²⁺ Mn²⁺. No absolute dependence on ionic strength was observed in Mg²⁺/Na⁺-exchange experiments. The degree of activation by ions at various concentrations was positively related to the increasing destabilizing properties of the cations according to the Hofmeister rule, where chaotropic cations are most efficient. Cations were responsible for activation since chaotropic anions counteracted the activating effect of cations. Received: 10 August 1998 / Accepted: 11 November 1998     

Development of callus and suspension cultures of potato resistant to NaCl and mannitol and their response to stress

Callus and suspension cultures adapted to various concentrations of NaCl or mannitol were developed from the cultivated potato Solanum tuberosum cv. Desire. Growth of the calli was less inhibited by mannitol than by iso-osmotic concentrations of NaCl. Reduction of growth by both NaCl and mannitol was considerably lower in osmotically adapted calli than in non-adapted ones. Salt-adapted suspension cultures that grew in the medium to which they had been originally adapted had a shorter lag in growth as well as a shorter time required to achieve the maximum growth, as compared with non-adapted cells. Suspension cultures adapted to NaCl concentrations higher than 150 mM were obtained only after preadaptation to osmotic stress. Adaptation of these cells was found to be stable. Accumulation of Na⁺ was lower and level of K⁺ was more stable in osmotically adapted than in non-adapted calli, when both were exposed to salt. Potassium level in NaCl-adapted calli exposed to saline medium was lower than that in non-adapted calli in standard medium. The maximum of Cl^– and Na⁺ accumulation was reached at higher external salt concentration in salt-adapted than in non-adapted suspension cultures. In both callus and suspension cultures, Cl^– accumulated more than Na⁺. Potassium level decreased more in non-adapted than in NaCl-adapted suspension cultures. The decrease of osmotic potential in osmotically adapted calli exposed to mannitol and in salt-adapted calli and suspension cultures exposed to salt was correlated to the increase of the external concentration. Such a correlation was not found in osmotically adapted calli exposed to salt. Non-electrolytes were found to be the main contributors to the decrease is osmotic potential in both callus and suspension cultures.     

Selection and characterization of mutants of the cyanobacterium Synechocystis sp. PCC 6803 unable to tolerate high salt concentrations

Three mutants of the cyanobacterium Synechocystis sp. PCC 6803 unable to tolerate high salt concentrations were generated using random cartridge mutagenesis. Analysis of the phenotypes revealed that the salt sensitivity of one mutant (6803/143) is caused by a block in the synthesis of the osmoprotective substance glucosylglycerol, while in the two other mutants no physiological defect could be detected which was responsible for the loss of salt tolerance. Southern hybridization analyses and cloning of the integration sites of the resistance marker demonstrated that different genes are affected in each of the three mutants.Abbreviations aphII aminoglycoside phosphotransferase II - kb kilobasepairs - Km kanamycin - Km^r kanamycin-resistance     

Respiratory activity in the marine cyanobacterium Spirulina subsalsa and its role in salt tolerance

Intracellular ion concentration and respiratory activity in the marine cyanobacterium Spirulina subsalsa was analyzed during cell transition from saline to hypersaline medium. During salt upshock, an early phase of Na⁺ and Cl^- influx was observed, followed by an adaptation phase where both Na⁺ and Cl^- were excluded from the cell. Respiration in intact cells was enhanced during salt upshock. S. subsalsa spheroplasts exhibited a high rate of O₂ uptake, which was further enhanced in cells grown in hypersaline medium, upon addition of NaCl to the assay mixture. This effect was found to be specific to sodium ions. Plasma membrane fractions from cells grown in hypersaline medium exhibited a high rate of cytochrome oxidase activity, which was further stimulated by NaCl, and was sensitive to DCCD. Immunoblot analysis of Spirulina plasma membrane polypeptides with anti-cytochrome oxidase serum demonstrated high content of 53.4 kDa polypeptide of cytochrome oxidase, which was enriched in membranes obtained from hypersaline Spirulina cells. The enhanced respiration, and more specifically the enrichment of cytochrome oxidase activity in salt-adapted cells in situ, as well as its stimulation by NaCl in vitro and inhibition by DCCD, suggest that cytochrome oxidase is involved in the extrusion of sodium ions from cells of the salt-tolerant Spirulina subsalsa.Abbreviations DCCD dicyclohexylcarbodiimide - CCCP carbonylcyanide m-chlorophenyl hydrazone - TMPD N, N, N, N, tetramethyl p-phenylenediamine dichloride     

Incorporation of 14C in the cyanobacterium Synechococcus PCC 6301 following salt stress

Synechococcus PCC 6301 synthesized sucrose as a compatible solute following hyperosmotic shock induced by NaCl. Initial rates of photosynthetic ¹⁴C incorporation were reduced following salt shock. Photosynthetic rates were comparable in cells enriched for glycogen (by growth in NO ₃ ^- -deficient medium) and cells grown in NO ₃ ^- -sufficient medium in the absence of osmotic shock. Incorporation of ¹⁴C was predominantly into the NaOH fraction and the residual acidic fraction in cells grown in NO ₃ ^- -sufficient medium, whereas incorporation was predominantly into the residual acidic fraction in cells grown in NO ₃ ^- -deficient medium. Following salt stress, ¹⁴C incorporation was initially into the ethanol-soluble fraction and the majority of tracer was recovered in sucrose. Carbon-14 was detected in sucrose in cells which had been enriched for [¹⁴C]glycogen prior to salt stress, inferring that glycogen can act as a carbon source for sucrose synthesis following salt stress. Changes in the specific activity of sucrose are consistent with an initial synthesis of sucrose from glycogen followed by synthesis of sucrose using newly fixed carbon, in response to salt stress.This work was supported by the Agricultural and Food Research Council.     

Peroxidase activity and isoenzymes in the culture medium of NaCl adapted tomato suspension cells

The medium of tomato (Lycopersicon esculentum) cells adapted to grow in the presence of 15 g l^–1 NaCl had a higher peroxidase activity than the medium of an unadapted tomato cell line. When the adapted cells were cultured in a medium without NaCl, the value found for peroxidase activity was intermediate. The increase in peroxidase activity was parallel to an increase of lignin-like compounds in the cell walls, as well as to an increased content or appearance of neutral and basic peroxidase isoenzymes. Apparently, the high values of peroxidase activity in the medium of the salt-adapted cells reflect the changed mechanical properties of the cell wall which, in turn, could be related to the salt adaptation process.Abbreviations LO Control tomato cell line unable to grow in the presence of 15 g 1^–1 of NaCl - L15 tomato cell line adapted to 15 g 1^–1 of NaCl and growing in this salt concentration - L15-0 tomato cell line adapted to 15 g 1^–1 of NaCl and growing in the absence of this salt - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - PBS phosphate buffer saline     

Salt-Adaptation of Tobacco BY2 Cells Induces Change in Glycoform of N-Glycans: Enhancement of Exo- and Endo-Glycosidase Activities by Salt-Adaptation

In this report, we describe that a salt adaptation of plant cells induces glycoform changes in N-glycoproteins. Intracellular and cell-wall glycopeptides were prepared from glycoproteins expressed in wild-type BY2 cells and salt-adapted cells. N-Glycans were liberated from those glycopeptides by hydrazinolysis, and the released oligosaccharides were N-acetylated and pyridylaminated. The structures of pyridylaminated (PA-) N-glycans were analyzed by a combination of two-dimensional sugar-chain mapping, MS analysis, and exoglycosidase digestion. In both wild-type cells and salt-adapted cells, the plant complex type structure was predominant among N-glycans expressed on glycoproteins, but we found that the Man2Xyl1Fuc1GlcNAc2 structure was significantly expressed on intracellular and cell-wall glycoproteins of the salt-adapted cells. Furthermore, enhancement of the specific activities of α-mannosidase and β-N-acetylglucosaminidase was observed in the salt-adapted BY2 cells, suggesting that the glycoform changes are due to changes in glycosidase activities.     

DNA,RNA, and protein synthesis in the cyanobacteriumSynechocystis sp. PCC 6803 adapted to different salt concentrations

A salt shock of 684mm NaCl reduced RNA and DNA synthesis to about 30% of the control level inSynechocystis. DNA synthesis recovered to the initial level within 4 h, while for recovery of RNA synthesis about 8 h were necessary. In cells completely adapted to different salt concentrations (from 171 to 1026mm NaCl), a continuous decrease in the RNA content with increasing salt concentrations up to 684mm NaCl was found, whereas the lowest DNA content was measured around 342mm NaCl, i.e., the salinity at which maximal growth occurred. With the uracil and thymidien incorporation technique, maxima in DNA and RNA synthesis were detected in control cells. Comparing these rates with nucleic acid synthesis rates calculated from the contents of DNA and RNA and the growth rates indicated that adaptation to 1026mm NaCl seemed to lead to an increased RNA turnover inSynechocystis. Analysis of protein synthesis with³⁵S-methionine labeling showed alterations in salt-adapated cells ofSynechocystis. At least three proteins (20.5, 25.8, and 35.8 kDa) were synthesized with highest rates at salinities leading to maximal growth, the synthesis of nine proteins (12.5, 16.9, 19.2, 22.2, 24.7, 28.5, 30.5, 50.3, and 63.5 kDa) increased and that of several other proteins decreased with increasing salinity; but only three proteins (12.5, 22.2, and 30.5 kDa) accumulated under these conditions. The adaptation ofSynechocystis to enhanced salt concentrations led also to increased contents of glucosylglycerol, glycogen, and significant amounts of K⁺ as well as Na⁺ ions.     

Growth and protein profile responses in the halophyte sea aster (Aster tripolium L.) suspension-cultured cells to salinity

We established salt tolerant cell lines, which survived and grew under high salinity conditions with 150 mM (S-150) and 300 mM (S-300) NaCl, to study the effects of salt stress on the proliferation and protein profile of these cells in the halophyte sea aster,Aster tripolium L. These salt-adapted cell lines were produced from leaves and selected by repeated suspension subculture in media containing NaCl every 25 days for five cycles. S-150 cells displayed no inhibition in their growth compared to control cells maintained under non-stressed conditions. S-150 cells exhibited approximately a 15-fold increase in both fresh and dry weight during the 25 days under saline conditions. S-300 cells showed positive growth under severe salt stress, but their dry matter gain was significantly less than that of the S-150 cells, with only a 2.5-fold increase in dry weight. We also detected changes in the protein profile of salt-adapted cells with two specifically induced polypeptides (basic 58.4 and acidic 24.8 kDa) and one enhanced polypeptide (basic 15.1 kDa) in the soluble fraction, and one specifically induced polypeptide (42.0 kDa) in the insoluble fraction.     

Carbon-13 NMR Studies of Salt Shock-Induced Carbohydrate Turnover in the Marine Cyanobacterium Agmenellum quadruplicatum

Carbon turnover in response to abrupt changes in salinity, including the mobilization of glycogen for use in osmoregulation was studied with pulse-chase strategies utilizing nuclear magnetic resonance (NMR)-silent and NMR-detectable ¹²C and ¹³C isotopes, respectively. Growth of Agmenellum quadruplicatum in 30%-enriched¹³C bicarbonate provided sufficient NMR-detectability of intracellular organic osmoregulants for these studies. A comparison of NMR spectra of intact cells and their ethanol extracts showed that the intact cell data were suitable for quantitative work, and, when combined with ESR measurements of cell volumes, yielded intracellular glucosylglycerol concentrations without disrupting the cells. NMR pulse-chase experiments were used to show that ¹³C-enriched glycogen, which had previously been accumulated by the cells under nitrogen-limited growth at low salinities, could be utilized for the synthesis of glucosylglycerol when the cells were abruptly transferred to hypersaline media, but only in the light. It was also shown that the accumulation of glucosylglycerol in the light occurred on a time scale similar to that of cell doubling. Depletion of glucosylglycerol when cells abruptly transferred to lower salinities appeared to be rapid—the intracellular pool of this osmoregulant was decreased 2-fold within 2 hours of hypotonic shock.     

Atrazine degradation in saline wastewater by Pseudomonas sp strain ADP

Wastewater from atrazine manufacturing plants contains large amounts of residual atrazine and atrazine synthesis products, which must be removed before disposal. One of the obstacles to biological treatment of these wastewaters is their high salt content, eg, up to 4% NaCl (w/v). To enable biological treatment, bacteria capable of atrazine mineralization must be adapted to high-salinity conditions. A recently isolated atrazine-degrading bacterium, Pseudomonas sp strain ADP, originally isolated from contaminated soils was adapted to biodegradation of atrazine at salt concentrations relevant to atrazine manufacturing wastewater. The adaptation mechanism was based on the ability of the bacterium to produce trehalose as its main osmolyte. Trehalose accumulation was confirmed by natural-abundance ¹H NMR spectral analysis. The bacterium synthesized trehalose de novo in the cells, but could not utilize trehalose added to the growth medium. Interestingly, the bacterium could not produce glycine betaine (a common compatible solute), but addition of 1 mM of glycine betaine to the medium induced salt tolerance. Osmoregulated Pseudomonas sp strain ADP, feeding on citrate decreased the concentration of atrazine in non-sterile authentic wastewater from 25 ppm to below 1 ppm in less than 2 days. The results of our study suggest that salt-adapted Pseudomonas sp strain ADP can be used for atrazine degradation in salt-containing wastewater. Received 26 August 1997/ Accepted in revised form 06 December 1997     

Enhanced thermotolerance of photosystem II in salt-adapted plants of the halophyte<Emphasis Type="Italic"> Artemisia anethifolia</Emphasis>

Thermotolerance of photosystem II (PSII) in leaves of salt-adapted Artemisia anethifolia L. plants (100–400 mM NaCl) was evaluated after exposure to heat stress (30–45°C) for 30 min. After exposure to 30°C, salt adaptation had no effects on the maximal efficiency of PSII photochemistry (F_v/F_m), the efficiency of excitation capture by open PSII centers (F_v/F_m), or the actual PSII efficiency (_PSII). After pretreatment at 40°C, there was a striking difference in the responses of F_v/F_m, F_v/F_m and _PSII to heat stress in non-salt-adapted and salt-adapted leaves. Leaves from salt-adapted plants maintained significantly higher values of F_v/F_m, F_v/F_m and _PSII than those from non-salt-adapted leaves. The differences in F_v/F_m, F_v/F_m and _PSII between non-salt-adapted and salt-adapted plants persisted for at least 12 h following heat stress. These results clearly show that thermotolerance of PSII was enhanced in salt-adapted plants. This enhanced thermotolerance was associated with an improvement in thermotolerance of the PSII reaction centers, the oxygen-evolving complexes and the light-harvesting complex. In addition, we observed that after exposure to 42.5°C for 30 min, non-salt-adapted plants showed a significant decrease in CO₂ assimilation rate while in salt-adapted plants CO₂ assimilation rate was either maintained or even increased to some extent. Given that photosynthesis is considered to be the physiological process most sensitive to high-temperature damage and that PSII appears to be the most heat-sensitive part of the photosynthetic apparatus, enhanced thermotolerance of PSII may be of significance for A. anethifolia, a halophyte plant, which grows in the high-salinity regions in the north of China, where the air temperature in the summer is often as high as 45°C.     

Simultaneous increases in the levels of compatible solutes by cost-effective cultivation of Synechocystis sp. PCC 6803

Synechocystis sp. PCC 6803, a cyanobacterium widely used for basic research, is often cultivated in a synthetic medium, BG-11, in the presence of 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES) or 2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid buffer. Owing to the high cost of HEPES buffer (96.9% of the total cost of BG-11 medium), the biotechnological application of BG-11 is limited. In this study, we cultured Synechocystis sp. PCC 6803 cells in BG-11 medium without HEPES buffer and examined the effects on the primary metabolism. Synechocystis sp. PCC 6803 cells could grow in BG-11 medium without HEPES buffer after adjusting for nitrogen sources and light intensity; the production rate reached 0.54 g cell dry weight·L⁻¹·day⁻¹, exceeding that of commercial cyanobacteria and Synechocystis sp. PCC 6803 cells cultivated under other conditions. The exclusion of HEPES buffer markedly altered the metabolites in the central carbon metabolism; particularly, the levels of compatible solutes, such as sucrose, glucosylglycerol, and glutamate were increased. Although the accumulation of sucrose and glucosylglycerol under high salt conditions is antagonistic to each other, these metabolites accumulated simultaneously in cells grown in the cost-effective medium. Because these metabolites are used in industrial feedstocks, our results reveal the importance of medium composition for the production of metabolites using cyanobacteria.     

Comparative effects of salt and alkali stresses on growth, osmotic adjustment and ionic balance of an alkali-resistant halophyte Suaeda glauca (Bge.)

Effects of salt and alkali stresses on growth, osmotic adjustment and ionic balance of Suaeda glauca (Bge.), an alkali-resistant succulent halophyte, were compared. The results showed that alkali stress clearly inhibited the growth of S. glauca. Moreover, the concentrations of Na⁺ and K⁺ both increased with increasing salinity under both stresses, suggesting no competitive inhibition between absorptions of Na⁺ and K⁺. The mechanism underlying osmotic adjustment during salt stress was similar to alkali stress in shoots. The shared essential features were that organic acids, betaine and inorganic ions (dominated by Na⁺) mostly accumulated. On the other hand, the mechanisms governing ionic balance under both stresses were different. Under salt stress, S. glauca accumulated organic acids and inorganic anions to maintain the intracellular ionic equilibrium, but the anion contribution of inorganic ions was greater than that of organic acids. However, the concentrations of inorganic anions under alkali stress were significantly lower than those under salt stress of the same intensity, suggesting that alkali stress might inhibit uptake of anions, such as NO₃ ^– and H₂PO₄ ^–. Under alkali stress, organic acids were the dominant factor in maintaining ionic equilibrium. The contribution of organic acids to anions was 74.1%, while that of inorganic anions was only 25.9%. S. glauca enhanced the synthesis of organic acids, dominated by oxalic acid, to compensate for the shortage of inorganic anions.     

Sucrose accumulation in salt-stressed cells of agp gene deletion-mutant in cyanobacterium Synechocystis sp PCC 6803

The agp gene encoding the ADP-glucose pyrophosphorylase is involved in cyanobacterial glycogen synthesis and glucosylglycerol formation. By in vitro DNA recombination technology, a mutant with partial deletion of agp gene in the cyanobacterium Synechocystis sp. PCC 6803 was constructed. This mutant could not synthesize glycogen or the osmoprotective substance glucosylglycerol. In the mutant cells grown in the medium containing 0.9 M NaCl for 96 h, no glucosylglycerol was detected and the total amount of sucrose was 29 times of that of in wild-type cells. Furthermore, the agp deletion mutant could tolerate up to 0.9 M salt concentration. Our results suggest that sucrose might act as a similar potent osmoprotectant as glucosylglycerol in cyanobacterium Synechocystis sp. PCC 6803.     

Energetic basis of development of salt-tolerant transport in a moderately halophilic bacterium,Vibrio costicola

Active transport of -aminoisobutyric acid (AIB) in Vibrio costicola utilizes a system with affinity for glycine, alanine and, to some extent, methionine. AIB transport was more tolerant of high salt concentrations (3–4 M NaCl) in cells grown in the presence of 1.0 M NaCl than in those grown in the presence of 0.5 M NaCl. The former cells could also maintain much higher ATP contents than the latter in high salt concentrations.Transport kinetic studies performed with bacteria grown in 1.0 M NaCl revealed three effects of the Na⁺ ion: the first effect is to increase the apparent affinity (K _t) of the transport system for AIB at Na⁺ concentrations <0.2 M, the second to increase the maximum velocity (V _max) of transport (Na⁺ concentrations between 0.2 and 1.0 M), and the third to decrease the V _max without affectig K _t (Na⁺ concentrations >1.0 M). Cells grown in the presence of 0.5 M or 1.0 M NaCl had similar affinity for AIV. Thus, the differences in salt response of transport in these cells do not seem due to differences in AIB binding. Large, transport-inhibitory concentrations of NaCl resulted in efflux of AIB from cells preloaded in 0.5 M or 1.0 M NaCl, with most dramatic efflux occurring from the cells whose AIB transport was more salt-sensitive. Our results suggest that the degree to which high salt concentrations affect the transmembrane electrochemical energy source used for transport and ATP synthesis is an important determinant of salt tolerance.Abbreviations AIB -aminoisobutyric acid - pmf proton motive force     

The stpA gene form synechocystis sp. strain PCC 6803 encodes the glucosylglycerol-phosphate phosphatase involved in cyanobacterial osmotic response to salt shock.

Mutations in a gene, stpA, had been correlated with the loss of tolerance to high NaCl concentrations in the cyanobacterium Synechocystis sp. strain PCC 6803. Genetic, biochemical, and physiological evidence shows that stpA encodes glucosylglycerol-phosphate phosphatase. stpA mutants are salt sensitive and accumulate glucosylglycerol-phosphate, the precursor of the osmoprotectant glucosylglycerol necessary for salt adaptation of Synechocystis. The consensus motif present in acid phosphatases was found in StpA; however, the homology with other sugar phosphatases is very poor. The amount of stpA mRNA was increased by growth of the cells in the presence of NaCl concentrations above 170 mM. Expression of stpA in Escherichia coli allowed the production of a 46-kDa protein which exhibited glucosylglycerol-phosphate phosphatase activity. The StpA-specific antibody revealed a protein of similar size in extracts of Synechiocystis, and the amount of this protein was increased in salt-adapted cells. The protein produced in E. coli had lost the requirement for activation by NaCl that was observed for the genuine cyanobacterial enzyme.     

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.     

Glyoxysomal malate dehydrogenase of watermelon cotyledons: De novo synthesis on cytoplasmic ribosomes

The development of glyoxysomal malate dehydrogenase (gMDH, EC 1.1.1.37) during early germination of watermelon seedlings (Citrullus vulgaris Schrad.) was determined in the cotyledons by means of radial immunodiffusion. The active isoenzyme was found to be absent in dry seeds. By density labelling with deuterium oxide and incorporation of [¹⁴C] amino acids it was shown that the marked increase of gMDH activity in the cotyledons during the first 4 days of germination was due to de novo synthesis of the isoenzyme. The effects of protein synthesis inhibitors (cycloheximide and chloramphenicol) on the synthesis of gMDH indicated that the glyoxysomal isoenzyme was synthesized on cytoplasmic ribosomes. Possible mechanisms by which the glyoxysomal malate dehydrogenase isoenzyme reaches its final location in the cell are discussed.Abbreviations mMDH mitochondrial malate dehydrogenase - gMDH glyoxysomal malate dehydrogenase - D₂O deuterium oxide - EDTA ethylenediaminetetraacetic acid, disodium salt