Measurement of enzymes related to sucrose metabolism in permeabilized Chlorella vulgaris cells

Sucrose phosphate synthase (UDP-glucose: D-fructose-6-phosphate-2-glucosyl transferase, EC 2.4.1.14), sucrose synthase (UDP-glucose: D-fructose-2-glucosyl transferase, EC 2.4.1.13) and invertase (β-D-fructofuranoside fructohydrolase, EC 3.2.1.26) were measured in toluene permeabilized cells of Chlorella vulgaris Beijerinck. All three activities were detected at all stages of the growth curve; sucrose synthase and sucrose phosphate synthase showed a zone of maximum activity, while invertase increased with time of growth. Sucrose phosphate synthase and sucrose synthase (sucrose synthesis direction) were stimulated by divalent cations and inhibited by UDP. This inhibition could be reversed by Mg²⁺ or Mn²⁺. Sucrose phosphate synthase activity was inhibited by inorganic phosphate and was enhanced by glucose-6-phosphate, but was insensitive to sucrose. Arbutine decreased sucrose synthase activity in both directions. Sucrose cleavage was inhibited by divalent cations and by pyrophosphate. The effects on the enzyme activities of the presence of 2,4-dichlorophenoxyacetic acid (2,4-D), gibberellic acid, abscisic acid and kinetin in the growth medium were investigated. Sucrose synthase activity was practically unaffected by all plant hormones tested, except for the presence of kinetin which stimulated the activity. Sucrose phosphate synthase activity was increased by both kinetin and abscisic acid. The effect of the latter was partially reversed by the presence of gibberellic acid. 2,4-D and kinetin were potent stimulators of invertase activity.     

Cytokinin control in subcellular localization of indoleacetic acid oxidase and peroxidase

IAA oxidase and peroxidase were found in all subcellular fractions of tobacco callus cells. The bound and cytoplasmic fractions differed greatly in IAA oxidase/peroxidase ratio and in isoperoxidase composition. The IAA oxidase/peroxidase ratio was particularly high in the plasma membrane-rich fraction. Kinetin had profound effects on IAA oxidase and peroxidase. The appearance of fast migrating isoperoxidases in response to 0·2 μM kinetin was found only in cytoplasmic, plasma membrane and ribosome-rich fractions; a high concentration of kinetin inhibited their formation. High kinetin concentrations also lowered the specific activity of IAA oxidase and peroxidase in all subcellular fractions, but the effect was much greater on peroxidase than on IAA oxidase, thus resulting in a drastic increase in IAA oxidase/peroxidase ratio. Evidently the activities of IAA oxidase and peroxidase were not equivalent and should be considered separately.     

Peroxidase and Senescence

Kinetin and a, á-dipyridyl prevented the rapid decreaseof chlorophyll content in detached oat leaves senescing in thedark. In the light, detachment caused a 27–40% rise in peroxidaseactivity and kinetin enhanced the enzyme in the segments byabout 80%. Darkness prevented any detachment-induced rise ofthe activity and decreased the stimulating action of kinetinand mechanical injury. The effect of dipyridyl on peroxidaseactivity in the dark was similar to that of kinetin. Kinetin enhanced the same distinctive isoperoxidases under lightand dark conditions. Neither horseradish peroxidase nor that extracted from oat leavesshowed any ability to hydroxylate free proline in vitro. A systemwhich supposedly led to peroxidase-catalysed proline hydroxylationyielded small amounts of hydroxyproline in the absence of theenzyme. Staining with Fast Blue BB salt in the presence of IAA as asubstrate after electrophoresis indicated that all detectedoat isoperoxidases had an IAA oxidase activity visually parallelingtheir peroxidase activity. Crude extracts contained IAA oxidaseinhibitors that could be partially or fully removed by dialysis. The possible significance of the rise in peroxidase activityduring senescence is discussed.     

Role of antioxidant systems in wild plant adaptation to salt stress

Wild plants differing in the strategies of adaptation to salinity were grown for six weeks in the phytotron and then subjected to salt stress (100 mM NaCl, 24 h). The activities of principal antioxidant enzymes and the accumulation of sodium ions and proline were studied. Independently of the level of constitutive salt tolerance, plants of all species tested accumulated sodium ions under salinity conditions but differed in their capability of stress-dependent proline accumulation and superoxide dismutase (SOD) and guaiacol-dependent peroxidase activities. Proline-accumulating species were found among both halophytes (Artemisia lerchiana and Thellungiella halophila) and glycophytes (Plantago major and Mycelis muralis). The high activities of ionically-bound and covalently bound peroxidases were characteristic of Th. halophila plants. High constitutive and stress-induced SOD activities were, as a rule, characteristic of glycophytes with the low constitutive proline level: Geum urbanum and Thalictrum aquilegifolium. Thus, a negative correlation was found between proline content and SOD activity in wild species tested; it was especially bright in the halophyte Th. halophila and glycophyte G. urbanum. An extremely high constitutive and stress-induced levels of proline and peroxidase activity in Th. halophila maybe compensate SOD low activity in this plant, and this contributed substantially into its salt resistance. Thus, monitoring of stress-dependent activities of some antioxidant enzymes and proline accumulation in wild plant species allowed a supposition of reciprocal interrelations between SOD activity and proline accumulation. It was also established that the high SOD activity is not obligatory trait of species salt tolerance. Moreover, plants with the high activity of peroxidase and active proline accumulation could acclimate to salts stress (100 mM NaCl, 24 h) independently of SOD activity.     

Cd induced changes in proline level and peroxidase activity in roots of rice seedlings

The effects of Cd on changes in proline level and peroxidase activity in roots of rice seedlings were investigated. CdCl₂ was effective in inhibiting root growth and in accumulating proline in roots. The inhibition of root growth by Cd is reversible. The reduction of root growth induced by Cd is closely associated with accumulation of proline in roots. External application of proline markedly inhibited root growth of rice seedlings in the absence of Cd. Ionically bound, but not soluble, peroxidase activity in roots was increased by CdCl₂. Proline treatment also resulted in an increase in ionically bound peroxidase activity in roots. The relationship between growth inhibition of roots induced by Cd and changes of proline level and peroxidase activity is discussed.Abbreviations POX peroxidase     

Role of kinetin in alleviation of copper and zinc toxicity in Lupinus termis plants

The effect of exogenous kinetin application on the growth and some physiological processes of Lupinus termis plants growing in metal containing solutions with excess concentrations of Cu and Zn ion were studied. Generally, plants growing in these solutions had a lower chlorophyll (Chl.) content, leaf relative water content (RWC) and produced less biomass than the control plants. Proline content was higher in metal-treated plants than in untreated controls. Chromatography of cell-free-extracts of roots and shoots indicated three main protein peaks with molecular weights about 170, 75--70 and 5--45 kDa. These peaks were coincident with Cu or Zn maxima. Addition of kinetin reduced the decline in Chl. content in metal-treated plants, improved water status of the plants and enhanced growth of the shoots and roots. The Cu or Zn content expressed on a per mg protein basis was raised when kinetin was applied to the growing shoots. Kinetin (Kin), Cu and Zn, singly and in the presence of kinetin (Cu × Kin and Zn × Kin), significantly affected the parameters tested. Only the effects of Cu × Kin and Zn × Kin interactions on shoot fresh weight and Cu × Kin on root length were statistically insignificant. Based on the calculated coefficient of determination ( ²) the roles of Cu and Zn in affecting Chl. content and growth were dominant in comparison to kinetin. Kinetin effect was dominant for root length and proline content, but the role of the interaction was subdominant. The results of this study indicate that kinetin can alleviate the harmful effects of Cu and Zn on the growth of lupin plants through stimulation of Cu and Zn incorporation into metal-binding proteins.     

Plant enzyme synthesis: Hormonal regulation of invertase and peroxidase synthesis in sugar cane

Summary Using sugar-cane internodal tissue in which RNA synthesis was ratelimiting for invertase of peroxidase synthesis, measurements were made of enzymeforming-capacity after blocking further RNA synthesis with actinomycin D or 6-methylpurine. In this way it was possible to determine whether added auxin (naphthaleneacetic acid) or gibberellic acid (GA₃) affected steps prior or subsequent to synthesis of the RNA fractions specifically required for synthesis of either enzyme. Both auxin and GA₃ increased the enzyme-forming-capacity for invertase but not for peroxidase. The effects of the two hormones are interpreted as causing stabilization of mRNA for invertase.Abscisic acid (ABA) increased the rate of synthesis of invertase but not peroxidase. ABA did not change the rate of loss of invertase when peptide-bond formation was blocked with cycloheximide, but stimulated its synthesis when RNA synthesis was blocked with 6-methyl purine. Hence, the site of action of ABA is subsequent to invertase-mRNA formation and prior to invertase destruction.Kinetin had no short-term effects when RNA synthesis was limiting for invertase production, and does not appear to directly modulate mRNA synthesis or stabilization, or amino-acid-polymerization steps. In treatments longer than 5 hours, kinetin inhibited synthesis of all three enzymes studied, so that its effect on enzyme synthesis in this tissue appears to be unspecific.Abbreviations used throughout text ABA (±)-abscisic acid (abscisin II, dormin) - GA₃ gibberellic acid - NAA -naphthaleneacetic acid     

Effect of water stress on growth and proline metabolism of Phaseolus vulgaris L.

Summary The effect of water stress on growth (fresh weight, dry weight), water relations (water saturation deficit, water potential, osmotic pressure), and proline metabolism in Phaseolus vulgaris were studied.Experimentally, water deficit was produced by reduced watering of the bean plants. This resulted in a decrease in water potential and leaf fresh and dry weight. Increases in the water saturation deficit and the osmotic pressure of the sap were, however, recorded. Water stress was also induced by treatment of the plants with polyethylene glycol, but its effects on the above mentioned parameters were different. In addition, necrosis of the foliage was observed. According to the present results, polyethylene glycol seems to be suitable only for the induction of short-termed water stress conditions.The effects of water stress on growth and on water relations of the plants were accompanied by a marked increase in the free amino acid content, especially that of the free proline content of the plants. The activities of the proline dehydrogenase and glutamate dehydrogenase were stimulated under water stress conditions, indicating that proline accumulation in water stressed plants is not attributed to an inhibited proline breakdown. The accumulated proline was metabolized rapidly once the water deficit of the plant was relieved by watering. The ability of the plant to accumulate proline might be of ecological importance for the plant and might be an adaption mechanism of the plant to overcome short periods of drought.Abbreviations -Kg -ketoglutarate - GDH glutamate dehydrogenase - PDH proline dehydrogenase - P5C -pyrroline-5-carboxylic acid - PEG polyethylene glycol - WSD water saturation deficit     

Seed Germination of Triglochin Maritima as Influenced by Salinity and Dormancy Relieving Compounds

Triglochin maritima had only 40 % germination in distilled water, but germination was substantially enhanced when seeds were exposed to ethephon, fusicoccin, proline, kinetin, and thiourea. Also, the effect of low salinity on germination was alleviated by fusicoccin, kinetin, nitrate and thiourea, whereas, the reduction in germination at high salinity was partially countered by ethephon, kinetin, thiourea and nitrate.     

Effects of ALA on Photosynthesis,Antioxidant Enzyme Activity,and Gene Expression,and Regulation of Proline Accumulation in Tomato Seedlings Under NaCl Stress

This study examined the effects of 5-aminolevulinic acid (ALA) application on photosynthesis, activity and gene expression of key antioxidant enzymes, and on proline accumulation in tomato (Lycopersicon esculentum Mill. ‘Hezuo 903’) seedlings under NaCl stress. NaCl stress significantly decreased the net photosynthetic rates and inhibited the activity of photosystem II, whereas exogenous ALA application significantly restored the net photosynthetic rates, quantum yield of electron transport, and energy conversion efficiency of photosystem II of tomato under NaCl stress. Production of superoxide, hydrogen peroxide, and malondialdehyde strongly increased in response to NaCl stress, and these increases were significantly counteracted by ALA. ALA increased the activity of reactive oxygen species (ROS) scavenging antioxidant enzymes, including superoxide dismutase, catalase, ascorbate peroxidase, and peroxidase, and upregulated the expression of SOD, APX, and POD, genes that encode these enzymes in NaCl-treated plants. ALA simultaneously increased proline accumulation in tomato seedlings under NaCl stress by regulating the expression of genes that encode ALA biosynthetic enzymes and that control proline biosynthesis and metabolism, for example, expression of GluRS and GluTR was downregulated, accompanied by a significant increase in the expression of P5CS and decline in the expression of ProDH. ALA provided protection against NaCl stress by increasing photosynthetic capacity, regulating antioxidant enzyme gene expression and proline accumulation, and decreasing ROS accumulation and lipid peroxidation in tomato.     

Cytokinin-like growth regulators mitigate toxic action of zinc and nickel ions on maize seedlings

Maize (Zea mays L.) seedlings grown in water culture in the presence of zinc and nickel ions were used with an effort to alleviate heavy metal toxicity by treating seeds with thidiazuron and kinetin (synthetic growth regulators with cytokinin-like activity). Heavy metals were shown to decrease germinability of seeds, suppress seedling growth, alter membrane permeability, and inhibit the activity of ascorbate peroxidase. Synthetic cytokinin-like agents alleviated deteriorative effects of heavy metals; the extent of alleviation depended on toxicant species and its concentration. The toxic effect of Zn²⁺ was effectively relieved by kinetin, whereas the Ni²⁺ toxicity was preferentially alleviated by thidiazuron.     

Cyclic AMP Promotion and Abscisic Acid Inhibition of {alpha}-Amylase Activity in the Seeds of Rice (Oryza sativa L.)

Cyclic AMP and GA₃ stimulated both -amylase activity in riceendosperm and the germination of the seed. In combination theyalso induced germination of ABA-treated seeds but cyclic AMPalone failed to neutralize the inhibitory effect of ABA; withadded kinetin, however, it promoted the -amylase activity ofthe dormant seeds. The enzyme activity decreased as the storageperiod of seeds increased. Cyclic AMP and GA     

Synergistic effect of kinetin and spermine on some physiological aspects of seawater stressed Vigna sinensis plants

The effect of kinetin, spermine and their combination on growth vigor, photosynthetic pigments, some metabolites, some enzymes, polyamines and productivity of salt-stressed Vigna sinensis plants was investigated. Salt stress reduced all evaluated growth criteria and yield components of used plants. Chlorophyll (CHL (a, b, carotenoids, carbohydrates, protein, spermidine and spermine level as well as and amylase activity were also decreased in response to salinity. On the other hand, proline, K⁺, Na⁺ and putrescine concentration, and peroxidase activity were increased in the salt-stressed plants. Exogenous application of kinetin and spermine mitigated the deleterious effects of salinity stress on growth and yield of the used plants. Conversely, the combined treatment of kinetin and spermine induced additional reduction in growth and yield of the stressed plants, and the effect appeared to be constitutive. The protective effect of kinetin and spermine on V. sinensis plants appeared mainly due to the enhancement effect of these growth regulators on chlorophylls and protein content and polyamines titer.     

Hormonal control of isoperoxidases in lentil embryonic axis

Detachment of the cotyledons from the lentil (Lens culinaris Med.) embryonic axis causes in the latter an increase in total peroxidase activity which is shown to be due to enhancement of specific cathodic isoperoxidases. Kinetin treatment of attached or detached axes promotes activity of essentially the same cathodic isoperoxidases. In addition kinetin enhances the activity of two anodic peroxidases and represses specifically that of a cathodic one. Abscisic acid inhibits the production of all isoenzymes in the presence or absence of kinetin. Cytokinin and abscisic acid actions are discussed in relation to the nature of a wounding hormone and the role of natural inhibitors in cotyledons during germination. Indoleacetic acid stimulates the activity of certain isoenzymes which are also stimulated by kinetin, whereas in others the effects of the two hormones are different. Specific inverse effects of indoleacetic acid and kinetin are demonstrated on the two most cathodic isoperoxidases. Indoleacetic acid-kinetin interactions on the cathodic isoperoxidases have been found in the literature and are discussed as a possible mechanism for explaining interactions of the two regulators on growth and other physiological processes.     

Effects of exogenous spermine on sweet sorghum during germination under salinity

Seedlings of Sorghum bicolor (L.) Moench were subjected to 180 mM NaCl with or without 0.25 mM spermine (SPM) for 7 d. NaCl treatment resulted in the inhibition of growth and increased the content of free proline, soluble protein and malondialdehyde (MDA). Additionally, it also enhanced the activity of catalase (CAT), peroxidase (POX) in both shoots and roots, while decreased that of glutathione reductase (GR). When exogenous spermine was added to the test solution, the growth of sweet sorghum seedlings was improved, and a smaller increase in the free proline and MDA contents was observed. The addition of spermine also partially increased the activities of POX and GR, but had no effects on soluble protein content or the activity of CAT.     

NaCl induced changes in ionically bound peroxidase activity in roots of rice seedlings

The changes in ionically bound peroxidase activity in roots of NaCl-stressed rice seedlings and their correlation with root growth were investigated. Increasing concentrations of NaCl from 50 to 150 mM progressively decreases root growth. The reduction of root growth by NaCl is closely correlated with the increase in ionically bound peroxidase activity. Since proline and ammonium accumulations are associated with root growth inhibition caused by NaCl, we determined the effects of proline or NH4Cl on root growth and ionically bound peroxidase activity in roots. External application of proline or NH4Cl markedly inhibited root growth and increased ionically bound peroxidase activity in roots of rice seedlings in the absence of NaCl. An increase in ionically bound peroxidase activity in roots preceded inhibition of root growth caused by NaCl, NH4Cl or proline. Mannitol inhibited root growth, but decreased rather than increased ionically bound peroxidase activity at the concentration iso-osmotic with NaCl. The inhibition of root growth and the increase in ionically bound peroxidase activity in roots by NaClis reversible and is associated with ionic rather than osmotic component. This revised version was published online in June 2006 with corrections to the Cover Date.     

The Effect of Salicylic Acid on Peroxidase Activity in Wheat Calli Cocultured with the Bunt Pathogen Tilletia caries

The effect of salicylic acid (SA) on peroxidase activity in wheat (Triticum aestivum L.) calli cocultured with the bunt pathogen Tilletia caries was studied. Fungal infection was shown to activate cytoplasmic peroxidase. SA suppressed total peroxidase activity but did not inhibit the peroxidase with pI 9.8. A novel chitin-specific peroxidase with pI 3.5 appeared after the SA treatment. The infection of SA-treated cells with Tilletia caries activated the isoenzymes with pI 3.5, 4.8, and 7.5 and stimulated their secretion into the culture medium. The ability of SA to control wheat peroxidase activity during pathogenesis is discussed. The important role of this control in plant defense responses to the bunt pathogen is emphasized.     

Alterations in tissue glutathione antioxidant system in streptozotocin-induced diabetic rats

Changes in tissue glutathione antioxidant system in streptozotocin-induced diabetic rats for a period of 15 weeks were examined. Total glutathione level was significantly increased in kidney tissue, but were slightly decreased and increased in liver and heart tissues, respectively. The small changes in total glutathione level in the liver and heart, though not statistically significant, were associated with reciprocal alterations in the activity Of -glutamylcysteine synthetase (GCS). While the GCS activity was not changed in kidney tissue, the activity of -glutathione peroxidase was significantly increased in kidney tissue. Insulin treatment could completely or partly normalize almost all of these changes induced by diabetes. However, the decrease in hepatic glutathione S-transferases activity in diabetic rats was not reversed by the insulin treatment. The ensemble of results suggests that the diabetes-induced alterations in tissue glutathione antioxidant system may possibly reflect an inter-organ antioxidant response to a generalized increase in tissue oxidative stress associated with diabetes.Abbreviations AGES advanced glycosylation end-products - EDTA ethylenediamine tetraacetic acid - GCS -glutamylcysteine synthetase - GlyHb glycated hemoglobin - GPX Se-glutathione peroxidase - GRD glutathione reductase - GSH reduced glutathione - GSSG oxidized glutathione - GST glutathione S-transferases - SSA sulfosalicylic acid - STZ streptozotocin     

Transport and catabolism of proline betaine in salt-stressed Rhizobium meliloti

Exogenous proline betaine (N,N-dimethylproline or stachydrine) highly stimulated the growth rate of Rhizobium meliloti, in media of inhibitory concentration of NaCl whereas proline was ineffective. High levels of proline betaine uptake occurred in cells grown in media of elevated osmotic strength; on the contrary, only low activity was found in cells grown in minimal medium. The apparent K _m was 10 M with a maximal transport rate of 25 nmol min^-1 mg^-1 of protein in 0.3 M NaCl-grown cells. The concentrative transport was totally abolished by KCN (2 mM), 2,4-dinitrophenol (2 mM), and carbonyl cyanide-m-chlorophenyl hydrazone (CCCP 10 M) but was insensitive to arsenate (5 mM). Glycine betaine was a very potent inhibitor of proline betaine uptake while proline was not. Proline betaine transport was not reduced in osmotically shocked cells and no proline betaine binding activity was detected in the crude periplasmic shock fluid. In the absence of salt stress, Rhizobium meliloti actively catabolized proline betaine but this catabolism was blocked by increasing the osmotic strength of the medium. The osmolarity in the growth medium regulates the use of proline betaine either as a carbon and nitrogen source or as an osmoprotectant.Abbreviations LAS lactate-aspartate-salts - MSY mannitol-salts-yeast - CCCP carbonyl cyanide-m-chlorophenyl hydrazone - DCCD dicyclohexylcarbodiimide - KCN potassium cyanide - Hepes 4-(2-hydroxyethyl)-1-piperzine-ethanesulphonic acid