The influence of NaCl on Δ1-pyrroline-5-carboxylate reductase in proline-accumulating cell suspension cultures of Mesembryanthemum nodiflorum and other halophytes

Pyrroline-5-carboxylate (P-5-C) reductase (EC 1.5.1.2) was extracted from cell suspension cultures, which proved to be very suitable for investigation of proline accumulation. Proline accumulation in cell suspensions of M. nodiflorum L., as well as P-5-C reductase activity and substrate affinity, increased with progressive adaptation to NaCl stress. In vitro NaCl treatment inhibited enzyme activity and decreased substrate affinity, independent of pretreatment of the cells. NaCl concentrations below 100 m M did not inhibit enzyme activity of adapted cells. High substrate concentrations counteracted in vitro NaCl inhibition (up to 200 m M ). Cycloheximide inhibited the increase of P-5-C reductase activity, as well as proline accumulation, after NaCl treatment, indicating stress-induced de novo synthesis of the enzyme. The different reactions of P-5-C reductase upon salt treatment are discussed with respect to its possible role in the regulation of proline accumulation.     

The effect of the embryo axis and exogenous sucrose on lipolysis and glyoxysomal enzyme development in Ricinus communis (castor bean) endosperm and cotyledons

Post-germinative growth in castor bean ( Ricinus communis L. cv. Hale) seedlings was investigated to determine whether lipolytic enzyme synthesis and lipid breakdown was a function of the embryo axis or simply based on a source-sink mechanism connected with sucrose produced during mobilization of storage lipid. Endosperm and cotyledons were excised from the embryo axis at 24 h intervals and were then incubated in Petri dishes containing water or 0.1 M sucrose for 24 h. Excised endosperm showed similar or higher malate synthase (MS, EC 4.1.3.2) and isocitrate lyase (ICL, EC 4.1.3.1) activities and increased lipolysis when compared with endosperm obtained from similarly intact seedlings of the same age. In contrast, cotyledonary ICL and MS activity was up to 50% lower and lipolysis was only slightly affected in excised material when compared with cotyledons obtained from intact seedlings. Incubating endosperm in sucrose had no effect on the development of the above enzyme activities or lipid content, when compared with material incubated in water only. In contrast, cotyledonary MS and ICL activities were up to 70% lower in sucrose and lipolysis substantially inhibited. Lipid breakdown and the development of lipolytic enzyme activity in cotyledons seem to be dependent on the presence of the endosperm. It is concluded that enzyme regulation in castor bean seedlings cannot entirely be explained by axis control or source-sink relationships.     

Effects of NaCl Salinity on Nitrogenous Compounds and Proteases during Germination of Vigna sinensis Seeds

Serido bean (Vigna sinensis (L.) Savi) seeds were sown in water and in NaCl solution of —4.3 bars water potential. Total, insoluble and soluble proteins as well as soluble amino nitrogen and proteolytic activity of cotyledons were studied after 0, 1, 3, 5, 7, and 9 days of germination. Protein breakdown and turnover was delayed by the NaCl treatment as compared to the control. This was not due to the total amount of proteolytic activity, which was unchanged by salinity; even though the specific activity decreased due to the delayed breakdown of proteins under salt stress. The inhibitory effects of salinity on seed protein reserve mobilization may be due more to inhibition of translocation of hydrolysis products than to inhibition of protease activity.     

Mechanism of the Increase in Ribonuclease Activity in Potato Tuber Slices

Upon wounding of potato tubers (Solanum tuberosum L. cv. Spunta)RNase activity increases, peaks in about 16 hours, then declines.To see if the increase of the activity is due to de novo synthesisof the enzyme protein, the extracts were compared for theirability to react with a rabbit antibody prepared against thewound activated RNase. The enzyme was purified by polyacrylamidegel electrophoresis of a RNase preparation, which had been partiallypurified from aged potato slices by ammonium sulfate precipitation,carboxymethyl-Sephadex column chromatography and gel filtrationthrough Sephadex G-100. Using rocket immunoelectrophoresis RNase-proteinimmunoprecipitated by the antibody increased in wounded tissue.This observation implies that the activity increase involvesenzyme synthesis. The increase was inhibited by actinomycinD and cordycepin, but not by 5-fluorouracil, suggesting a requirementfor mRNA synthesis. (Received April 9, 1985; Accepted December 16, 1985)     

Cowpea ribonuclease: properties and effect of NaCl-salinity on its activation during seed germination and seedling establishment

Pitiúba cowpea [Vigna unguiculata (L.) Walp] seeds were germinated in distilled water (control treatment) or in 100 mM NaCl solution (salt treatment), and RNase was purified from different parts of the seedlings. Seedling growth was reduced by the NaCl treatment. RNase activity was low in cotyledons of quiescent seeds, but the enzyme was activated during germination and seedling establishment. Salinity reduced cotyledon RNase activity, and this effect appeared to be due to a delay in its activation. The RNases from roots, stems, and leaves were immunologically identical to that found in cotyledons. Partially purified RNase fractions from the different parts of the seedling showed some activity with DNA as substrate. However, this DNA hydrolyzing activity was much lower than that of RNA hydrolyzing activity. The DNA hydrolyzing activity was strongly inhibited by Cu²⁺, Hg²⁺, and Zn²⁺ ions, stimulated by MgCl₂, and slowly inhibited by EDTA. This activity from the most purified fraction was inhibited by increasing concentrations of RNA in the reaction medium. It is suggested that the major biological role of this cotyledon RNase would be to hydrolyze seed storage RNA during germination and seedling establishment, and it was discussed that it might have a protective role against abiotic stress during later part of seedling establishment.     

Salinity-stress-induced proteins in two nitrogen-fixing Anabaena strains differentially tolerant to salt.

Salinity altered the protein synthesis patterns in two cyanobacterial strains: Anabaena torulosa, a salt-tolerant brackish water strain, and Anabaena sp. strain L-31, a salt-sensitive freshwater strain. The cyanobacterial response to salinity was very rapid, varied with time, and was found to be correlated with the external salt (NaCl) concentration during stress. Salinity induced three prominent types of modification. First, the synthesis of several proteins was inhibited, especially in the salt-sensitive strain; second, the synthesis of certain proteins was significantly enhanced; and third, synthesis of a specific set of proteins was induced de novo by salinity stress. Proteins which were selectively synthesized or induced de novo during salt stress, tentatively called the salt-stress proteins, were confined to an isoelectric pI range of 5.8 to 7.5 and were distributed in a molecular mass range of 12 to 155 kilodaltons. These salt-stress proteins were unique to each Anabaena strain, and their expression was apparently regulated coordinately during exposure to salt stress. In Anabaena sp. strain L-31, most of the salt-stress-induced proteins were transient in nature and were located mainly in the cytoplasm. In A. torulosa, salt-stress-induced proteins were evenly distributed in the membrane and cytoplasmic fractions and were persistent, being synthesized at high rates throughout the period of salinity stress. These initial studies reveal that salinity-induced modification of protein synthesis, as has been demonstrated in higher plant species, also occurs in cyanobacteria and that at least some of the proteins preferentially synthesized during salt stress may be important to cyanobacterial osmotic adaptation.     

In vitro replication of tobacco mosaic virus RNA in tobacco callus cultures: solubilization of membrane-bound replicase and partial purification.

A fraction containing membrane-bound tobacco mosaic virus RNA replicase was isolated form tobacco mosaic virus-infected tobacco callus cultures. The replicase activity reached a maximum 60 h after inoculation and then declined. The enzyme activity was insensitive to actinomycin D and DNase. The corresponding fraction from healthy callus contained essentially no activity. The viral RNA synthesis in vitro proceeded linearly for 30 min and required the four nucleotide triphosphates and Mg2+ ions. Mn2+ was a poor substitute for Mg2+. During RNA synthesis the product was at least 70% resistant to RNase in 2X SSC (0.15 M NaCl plus 0.015 M sodium citrate), but completely digested by RNase in 0.1X SSC. Analysis of the product by polns) that appeared to be replicative form and a partially RNase-resistant structure similar to replicative intermediate form. Washing the membrane-bound replicase with Mg2+-deficient buffer solubilized enzyme. The solubulized enzyme was further purified by DEAE-Sephadex column chromatography. The DEAE-purified enzyme was nearly completely dependent upon tobacco mosaic virus RNA for activity. Analysis of the product on a sucrose gradient revealed a double-stranded RNA with sedimentation of 16S and smaller heterogeneous RNase-sensitive products.     

Induction of phenylalanine ammonia-lyase in hypocotyls of sunflower seedlings by light, excision and sucrose

Light, excision and sucrose increased extractable phenylalanine ammonia-lyase (PAL) activity from hypocotyl tissue of sunflower ( Helianthus annuus L. cv. Peredovik) to 2–6 times the basal level. Intact sunflower seedlings or whole hypocotyls incubated in water or 0.1 M sucrose exhibited, in continuous light, a pattern in which PAL peaked 4 and 28 h after the beginning of the illumination. When 0.5 cm long hypocotyl segments were incubated in water or 0.1 M sucrose, they exhibited, both in continuous light and in the dark, a pattern in which PAL rose during an initial period of 10 h (assay in sucrose and light) to 48 h (assay in water and dark) and then remained nearly constant at a high value for at least the next 10 h. When whole hypocotyls were incubated in 0.1 M sucrose, a third pattern in PAL activity was found in which PAL peaked after 28 h and subsequently declined. In all the above systems the increase in PAL activity was significantly reduced by cycloheximide. Furthermore, the subsequent decay of PAL activity following illumination was prevented by delayed transfer to cycloheximide. It is suggested that the results can be explained on the basis of a turnover mechanism involving continued de novo enzyme synthesis and subsequent synthesis of a PAL-inactivating system.     

Induction of mitochondrial phosphoenolpyruvate carboxykinase in cultured human fibroblasts.

Mitochondria of cultured normal human fibroblast cells were found to contain the enzyme phosphoenolpyruvate carboxykinase. The activity of this enzyme in these cells is increased 2- to 3-fold by addition of 5 . 10(-4) M dibutyryl cyclic AMP, or 1.5- to 2-fold by the addition of dexamethasone (2 . 10(-7) M) or hydrocortisone (1.38 . 10(-6) M). These increases in enzyme activity were inhibited cycloheximide and actinomycin D, suggesting they are dependent upon de novo protein synthesis. Cultured human fibroblasts may thus provide a useful system for studying the regulation of mitochondrial phosphoenolpyruvate carboxykinase.     

Untersuchungen über rnasen in kotyledonen von nachgereiften und dormantenagrostemma githago-samen

Activities of RNasea were studied in cotyledons of dormant and afterripenedAgrostemma githago seeds. Activity of RNase increases during imbibition and germination. This increase in activity cannot be observed in variants which are not able to germinate (dormant seeds and seeds blocked by higher temperature). The development of RNase activities during germination cannot be inhibited by concentrations of cycloheximide or actinomycine D completely preventing phosphatase synthesis. These results may be indicative for the assumption that the increase of RNase during germination is caused by enzyme activation and not by enzyme synthesis. Cytokinins and a combination of cycloheximide and gibberellic acid stimulate the activity of RNase in dormant cotyledons, whereas neither cycloheximide nor gibberellic acid, applicated by themselves, show any effect. Cytokinins and gibberellic acid do not influence the activity of RNase of afterripened cotyledons, abscisic acid inhibits the increase of enzyme activity. There are characteristic changes in the pattern of RNases during germination revealed by polyacrylamide gel electrophoresis. The increase in RNase activity of dormant cotyledons caused by cytokinins is accompanied by obvious changes in the RNase pattern on polyacrylamide gel. Treating dormant cotyledons with cytokinins dormancy is partially overcome. In consequence of the application of cytokinins the differences in the electrophoretic RNase pattern between dormant and afterripened cotyledons can be nearly balanced.     

Identification of luteinizing hormone releasing hormone-like immunoactivity in ovine cotyledons

Luteinizing hormone releasing hormone (LHRH)-like immunoactivity has been identified in cotyledonary extracts prepared from pregnant ewes. This activity displayed similar physico-chemical properties as synthetic LHRH, as determined by reverse-phase HPLC and size-exclusion HPLC. Under reverse-phase conditions, cotyledonary LHRH-like immunoactivity displayed a retention time (10.5 +/- 0.1 min) which was not significantly different from that of synthetic LHRH. When subjected to size-exclusion HPLC, cotyledonary LHRH-like immunoactivity eluted in fractions which corresponded to a molecular weight range of 1100-1200 Da, which was not significantly different from the elution profile observed for synthetic LHRH. The cotyledonary tissue content of LHRH-like immunoactivity averaged 94 +/- 24 pg/mg (n = 6). The results of this study demonstrate the presence of LHRH-like immunoactivity in ovine cotyledons. Although placental synthesis of LHRH-like immunoactive material has been demonstrated in other species, it remains to be established whether this activity, demonstrated in ovine placenta, is the consequence of de novo placental synthesis or represents uptake from the maternal (and/or fetal) circulation.     

Effect of temperature, light and salinity on seed germination and radicle growth of the geographically widespread halophyte shrub Halocnemum strobilaceum

BACKGROUND AND AIMS: The small leafy succulent shrub Halocnemum strobilaceum occurs in saline habitats from northern Africa and Mediterranean Europe to western Asia, and it is a dominant species in salt deserts such as those of north-west China. The effects of temperature, light/darkness and NaCl salinity were tested on seed germination, and the effects of salinity were tested on seed germination recovery, radicle growth and radicle elongation recovery, using seeds from north-west China; the results were compared with those previously reported on this species from 'salt steppes' in the Mediterranean region of Spain. METHODS: Seed germination was tested over a range of temperatures in light and in darkness and over a range of salinities at 25 degrees C in the light. Seeds that did not germinate in the NaCl solutions were tested for germination in deionized water. Seeds from which radicles had barely emerged in deionized water were transferred to NaCl solutions for 10 d and then back to deionized water for 10 d to test for radicle growth and recovery. KEY RESULTS: Seeds germinated to higher percentages in light than in darkness and at high than at low temperatures. Germination percentages decreased with an increase in salinity from 0.1 to 0.75 M NaCl. Seeds that did not germinate in NaCl solutions did so after transfer to deionized water. Radicle elongation was increased by low salinity, and then it decreased with an increase in salinity, being completely inhibited by > or = 2.0 M NaCl. Elongation of radicles from salt solutions < 3.0 M resumed after seedlings were transferred to deionized water. CONCLUSIONS: The seed and early seedling growth stages of the life cycle of H. strobilaceum are very salt tolerant, and their physiological responses differ somewhat between the Mediterranean 'salt steppe' of Spain and the inland cold salt desert of north-west China.     

Effect of NaCl salinity in vivo and in vitro on ribonuclease activity in the halophyte Suaeda maritima

Ribonuclease (EC 2.7.7.17) activity in the obligate halophyte Suaeda maritima (L.) Dum. var. macrocarpa Moq. was studied in relation to salinity (increasing concentrations of NaCl) of incubation and growth media. In vitro, the addition of 50 to 400 m M NaCl did not affect ribonuclease activity. This result, which was also found for Phaseolus vulgaris , indicates that the hydrolase is insensitive to high saline concentrations. The subcellular distribution of RNase activity did not change significantly with the salinity of the medium or with the age of the plant. The microsomal ribonuclease activity expressed on a fresh weight basis represented in every case less than 6% of the total activity. After 23 days of culture, the absence of salt stimulated the activity of soluble ribonuclease in aerial parts of Suaeda ; inversely, the capacity of the enzyme was lower under optimal saline conditions (130 m M NaCl). This was also evidenced by transfer of whole plants from a non-saline to a saline medium. Such a saline shock caused a decrease followed by a stabilization of the capacity of ribonuclease from Suaeda . The influx of NaCl in the tissues lowered the activity of the hydrolase.     

Development of RNase activity in embryonic axes of germinating pea seeds

RNase activity in embryonic pea axes increased in parallel withthe rise of RNA synthesis as germination proceeded. The developmentof this enzymatic activity was modified antagonistically byapplication of GA₃ and ABA and inhibited severely by treatmentwith CH. Sedimentation analysis of ³H-adenosine-labeled RNAindicated that the synthesis of all types of RNA species isuniformly stimulated by GA₃ and inhibited by ABA. However, 5-FUtreatments, which severely inhibited the synthesis of rRNA,with a slight effect on that of mRNA, had no appreciable effecton the development of RNase activity in the axes. These resultsindicate that active RNA synthesis during germination is independentof the development of RNase activity and that the de novo synthesisof RNases may be controlled by the synthesis of their specificmRNAs. Among the three types of RNase (RNase I, II and III) detectedin the embryonic axes, RNase III showed a sharp increase inactivity with embryo growth and the activity of this enzymewas mainly associated with the endoplasmic reticulum. (Received June 5, 1978; )     

Comparative changes in the lipogenic enzyme activities and in the in vivo fatty acid synthesis in liver and adipose tissues during the post-weaning growth of male rats

Changes in the specific activities of acetyl-CoA-carboxylase (ACX), malic enzyme (ME) and glucose-6-phosphate dehydrogenase (G-6-PD) were compared to changes in de novo lipogenesis measured by in vivo incorporation of [3H] of tritiated water into fatty acids of liver and of perirenal and dorsal subcutaneous adipose tissues. In the adipose tissues, the specific activities of the three enzymes rather closely followed fluctuations in the rate of fatty acid synthesis. In the liver, ACX and especially ME activities were satisfactory indicators of de novo lipogenesis; G-6-PD activity did not depend on de novo lipogenesis.     

La glutamine synthètase du Suaeda maritima. Action in vivo et in vitro du NaCl

The glutamine synthetase of Suaeda maritima. In vivo and in vitro action of NaCl Glutamine synthetase (GS; EC 6.3.1.2) was isolated and characterized from roots and aerial parts of the halophyte Suaeda maritima (L.) Dum. var. macrocarpa Moq. Km values of GS were identical in both types of organ and unchanged by the salinity in the medium. Addition of NaCl in the culturing solution increased the specific activity of the enzyme especially in the aerial parts, where GS is more abundant. This increase was all the more pronounced if the plant-salt contact period was extended (between 21 and 45 days). In vitro the addition of 0 to 500 m M of salt did not affect the activity of GS at satured substrate concentrations. At low glutamate concentrations in combination with 300 m M NaCl or more, a slight competitive inhibition was observed, never over 18%. – The remarkable insensibility of GS to salinity in vitro and the stimulating effect of NaCl in vivo on the synthesis of the leaf enzyme indicates that GS plays a fundamental part in the assimilation of NH4⁺ in the halophyte Suaeda maritima var. macrocarpa.     

Nuclear location of ribonuclease H and increased level of magnesium-dependent ribonuclease H in rat liver on thioacetamide treatment

Rat liver nuclei were isolated in aqueous solutions of low ionic strength or anhydrous glycerol. The presence of ribonuclease H (RNase H) [EC 3.1.4.34] activity in the cytoplasm is due to extraction of the nuclear enzyme by buffer and inorganic salts. Two forms of RNase H were separated from rat liver nuclei by affinity chromatography using a DNA-cellulose column. When the RNase H in the wash solution of nuclei with 0.3 M sucrose and in nuclear solution extracted with 0.15 M NaCl were fractionated on a single-stranded DNA-cellulose column, two peaks corresponding to Mn2+- and Mg2+-dependent RNases H were eluted at 0.1 M and 0.2 M NaCl, respectively, and a peak having both RNase H activities was recovered in the wash-through fraction from the column. Among the enzymes from these two fractions in the nuclei, the activity of the Mg2+-dependent RNase H which binds to DNA-cellulose increased several-fold within 24 h of a single injection of thioacetamide. The activities of Mg2+-dependent RNase H extracted with higher-salt solution from the nuclei and recovered in the flow-through fraction from the DNA-cellulose column and the Mn2+-dependent RNase H activities were relatively unaffected by an injection of thioacetamide.     

alpha-D-Mannosidase of Saccharomyces cerevisiae. Characterization and modulation of activity.

A unique and interesting alpha-D-mannosidase (alpha-D-mannoside mannohydrolase EC 3.2.1.24) activity has been isolated from Saccharomyces cerevisiae. The enzyme was localized in a crude particulate fraction of the cell extract and was not solubilized by treatment with detergents or high ionic strength NaCl. The enzyme had a pH optimum of 6.3, Km 50 micron with p-nitrophenyl-alpha-D-mannopyranoside, and was competitively inhibited by D-mannose (Ki 20 mM). The enzyme is not affected by ethylenediaminetetraacetic acid, a number of different cations, or sulfhydryl reagents. It was inhibited by p-chloromercuriphenyl sulfonic acid and this inhibition is prevented by the addition of substrate. The cellular concentration of alpha-D-mannosidase is inversely proportional to growth rate, suggesting that the enzyme is under catabolite repression. The level of enzyme was found to increase approx. 8-fold during sporulation. This is apparently due to de novo synthesis, since inhibition of protein synthesis by cycloheximide prevents the increase in enzyme activity.     

The biosynthesis of ribonuclease and its accumulation in protein bodies in the cotyledons of mung bean seedlings

Germination and seedling growth of mung bean are accompanied by a 7- to 10-fold increase in the ribonuclease content of the cotyledons. The increase occurs during the first 4 days of seedling growth and precedes the senescence of the cotyledons. Separation of the RNases in the cotyledons by polyacrylamide gel electrophoresis indicates the presence of several minor bands in seeds imbibed for 24 hr. On the second day of seedling growth a new major band with an R_f of 0.76 is present. In 4- to 5-day old seedlings this major band accounts for nearly all the RNase activity in the tissue. The characteristics of this RNase show that it is a plant ribonuclease I (pH optimum of 5.0; MW 16,000; activity preferentially inhibited by purine nucleotides; no activity toward DNA; no phosphodiesterase activity). When the seedlings are grown in 66% D₂O the RNase activity undergoes a density shift of 0.61% indicating that the increase in enzyme activity is due to the de novo synthesis of the enzyme molecules. A method is described for the isolation of protein bodies from protoplasts of storage parenchyma cells. Fractionation of protoplast lysates on Ficoll gradients results in the recovery of a high proportion (75%) of intact protein bodies. On these gradients RNase activity comigrates with α-mannosidase, a protein body marker enzyme indicating that the newly synthesized RNase accumulates in the protein bodies. We suggest that the synthesis of RNase in the cotyledons and its accumulation in the protein bodies indicates that protein bodies may function in the degradation of cellular macromolecules other than the reserves stored within them.