Traianos Yupsanis

Activity and isoforms of peroxidases, lignin and anatomy, during adventitious rooting in cuttings of Ebenus cretica L.

Adventitious rooting of Ebenus cretica cuttings was studied in order to examine a) the rooting ability of different genotypes in relation to electrophoretic patterns of peroxidases. b) the activity and electrophoretic patterns of soluble and wall ionically bound peroxidases, the lignin content and anatomical changes in the control and IBA treated cuttings of and genotypes in the course of adventitious root formation. In addition, a fraction of soluble cationic peroxidases was separated by gel filtration chromatography from the total soluble peroxidases of a genotype. No rooting occurred in cuttings without IBA-treatment. In both genotypes, electrophoretic patterns of soluble anionic peroxidases revealed two common peroxidase isoforms, while a fast-migrating anionic peroxidase isoform (A3) appeared only in genotypes. Both genotypes showed similar patterns of soluble, as well as wall ionically bound cationic peroxidase isoforms. The number of isoforms was unchanged during the rooting process (induction, initiation and expression phase) but an increase in peroxidase activity (initiation phase) followed by decrease has been found in IBA-treated cuttings. During initiation phase the lignin content was almost similar to that on day 0 in genotype while it was reduced at by about 50% in genotype at the respective time. Microscopic observations revealed anatomical differences between genotypes. According to this study, the and genotypes display differences in anatomy, lignin content, activity of soluble peroxidases and the electrophoretic patterns of soluble anionic peroxidase isoforms. The A3-anionic peroxidase isoform could be used as biochemical marker to distinguish and genotypes of E. cretica and seems to be correlated to lignin synthesis in rooting process.

Protein Phosphorylation-Dephosphorylation in Alfalfa Seeds Germinating under Salt Stress

Summary When seeds of alfalfa (Medicago sativa L. cv. Luzerne Euver) were germinated under iso-osmotic solutions of NaCl (0.10M solution equivalent to conductivity 8.0 dSm-1) and mannitol (0.19m) or an increased level of NaCl (0.15M solution equivalent to conductivity 11.6dSm-1) seedling growth indicated a continuous decrease in plant height and dry weight in subsequent days of germination. In contrast, the extractable proteins of NaCl and mannitol stressed seedlings were more comparable to controls. Protein kinase (EC 2.7.1.37) and protein phosphatase (EC 3.1.3.16) specific activities were determined in crude extracts of alfalfa seedlings. At both concentrations of NaCl protein kinase specific activities towards endogenous substrates were higher than the control or mannitol except at day 7 under 11.6 dSm-1 NaCl. Unlike protein kinase specific activities, the protein phosphatase specific activities against 32P-labeled bovine casein were inhibited in the presence of NaCl at all days of germination, except at day 5, when 8.0 dSm-1 NaCl enhanced them. The presence of mannitol (0.19 m) also inhibited protein phosphatase specific activites. These observations suggest that some specific ionic effect of salt was responsible for the behaviour of the enzymes. SDS-electrophoresis followed by autoradiography revealed three major groups of in vitro phosphorylated proteins (in the presence of Ca2+ or Mn2+) in ungerminated seeds: a low molecular group (Mrs ca. 10–20,000), a medium molecular group (Mrs ca. 35–50,000) and a high molecular group (Mrs ca. 50-86,000). The latter was absent from the seedlings in all treatments. An increase in salinity (11.6dS m-1 NaCl) retarded the disappearance of medium molecular group protein bands in contrast to control or mannitol where these almost disappeared from the first day of germination. A condition of normal alfalfa seedling growth may be regulated by dephosphorylation of medium molecular group protein bands.

Aluminum toxicity effects on durum wheat cultivars

Abstract The effects of aluminum on biomass, nutrients and kinases were studied in two durum wheat cultivare (Triticum durum Desf. cvs Sapfo, Capeiti 82) grown in nutrient solutions (pH 4.5) at seven Al levels (0, 9.3, 18.5, 37.1, 74.1, 148, and 297 μM). The most evident Al toxicity symptom was a reduction in root growth. Sapfo showed greater Al tolerance than Capeiti 82. However, both cultivare must be characterized as Al‐sensitives. Aluminum in the nutrient solution above 74.1 μM significantly (P<0.05) reduced root growth of both durum wheat cultivare. The concentration of 74.1 μM Al can be suggested for screening of durum wheats on the basis of tolerance to Al. The concentrations of the nutrients Ca, Mg, K, and Fe in the plant tissues of both cultivare decreased even at low Al levels. The decrease of Ca+2 content in leaves and roots of the two durum wheat cultivare was almost the same while the less sensitive cultivar Sapfo retained larger amounts of Mg+2 in roots and leaves compared with the more sens...

Nucleolytic activities and appearance of a new DNase in relation to nickel and manganese accumulation inAlyssum murale

Summary Serpentine and non-serpentine plants of Alyssum murale, a nickel (Ni) accumulator plant, from North Greece, were studied in order to examine: (1) The ability of natural plants to accumulate metals; (2) the ability of their seedlings to tolerate increasing concentrations of Ni 2+ or Mn 2+ (0, 0.16, 0.32, 0.5 and 1 mmol/L), when grown in nutrient solution; (3) the activities and electrophoretic patterns of root and shoot DNases and RNases under the above conditions. Measurements of metal concentrations in serpentine and non-serpentine natural plants and the respective soils revealed: (1) Very low calcium (Ca)/magnesium (Mg) (∼0.16) ratio and high concentration of Ni in serpentine soil; (2) very high Ca/Mg (∼17) ratio and high concentration of manganese (Mn) in non-serpentine soil; (3) the ability of serpentine natural plants to accumulate Ni and the inability of plants of both serpentine and non-serpentine populations to accumulate Mn. A. murale plants grown in nutrient solution with increasing Ni 2+ or Mn 2+ concentrations showed a negative correlation between the Ni 2+ or Mn 2+ concentrations in the nutrient solution, and the chlorophyll concentration, shoot and especially root length. The accumulation of Ni 2+ or Mn 2+ in the plant showed a positive correlation with increasing Ni 2+ or Mn 2+ concentrations in the nutrient solution. Application of 0.5 mmol/L Ni 2+ or Mn 2+ resulted in the inhibition of DNase activities and the appearance of a new DNase form, in both root and shoot detected by electrophoresis in active ssDNA polyacrylamide gel. The new gel-extracted DNase showed nicking action against plasmid DNA and has been characterised as an endo-DNase. In contrast, electrophoretic patterns and RNase activities were unaffected. According to our studies on growth, both serpentine and non-serpentine plants of A. murale have a constitutive ability to tolerate and accumulate Ni 2+ or Mn 2+ ; they have similar DNase and RNase electrophoretic patterns and show a new DNase form under Ni 2+ or Mn 2+ stress. This is the first report on the response of nucleolytic enzymes under metallic elements hyperaccumulation.

Fractionation and Electrophoretic Patterns of Storage Proteins of Ebenus cretica. A preliminary Survey as a Tool in Taxonomy

Seed storage proteins of Ebenus cretica were fractionated to albumins, globulins, prolamins and glutelins according to their solubility in water, 0.5 M NaCl solution, 55 % propanol-2 and 0.125 M sodium borate (pH 9.0) containing 0.5 % SDS (sodium dodecyl sulfate) solution, respectively. Glutelins consist of the major (about 81 %) fraction of the total extracted proteins. Analysis by SDS-PAGE revealed that the total extracted protein patterns from different racemes of the same plant were similar, while those from seeds of different plants were different. In addition, distinct differences were observed within protein patterns of alkaline extractable glutelin fractions and salt soluble globulin fractions. In E. cretica four ecotypes (A – D) were distinguished by SDS-PAGE of total extracted seed proteins. The last method was more simple and rapid than others and was suggested for screening analysis.

Peroxidase, acid phosphatase, RNase and DNase activity and isoform patterns during in vitro rooting of Petunia × hybrida microshoots

Specific activities and isoform patterns of peroxidases, acid phosphatases, DNases and RNases were studied in relation to in vitro rooting of Petunia × hybrida microshoots in the presence of 4 µM indole-3-butyric acid (IBA). Specific activities of the above enzymes increased in the course of rooting. Rhizogenesis could be related with an increased specific activity of peroxidases during the initiation phase, in parallel with increased lignin content. Twelve peroxidases, six anionic (A1–A6) and six cationic (C1–C6), seven acid phosphatases (ACP1–ACP7), seven RNases (R1–R7) and four DNases (D1–D4) isoforms were detected following native PAGE. Variation in the number of the above isoforms and their quantity was observed during different stages of rooting. Particularly, A2, A3, C3, C4, C5, ACP2, R1, R2, R3, and D4 isoforms appeared after the induction phase and could be related to emergence of root primordia. Additionally, R3 and D4 could be associated with cell division and differentiation, since these are only expressed in rooted microshoots. Moreover, the higher number of roots in IBA-treated microshoots could be related to the higher expression of RNase and DNase isoforms during initiation and expression phases.

Similarities and differences in the properties of multiple NDP-kinase isoforms of Alyssum murale, Ni2+-accumulator species.

Two isoforms of NDPKs (diphosphonucleoside kinases: E.C. 2.7.4.6.) named S-NDPK-A and S-NDPK-B were separated and purified from shoots of Alyssum murale (19th day of growth), a nickel accumulator plant, by a four-step procedure involving ammonium sulphate precipitation and DEAE-sepharose and hydroxyapatite column chromatography. Shoot NDPKs underwent autophosphorylation, proved thermostable, displayed similar molecular mass of 105,000, and consisted of six catalytic subunits. The size of subunits of S-NDPK-A and S-NDPK-B were 18 and 16kDa, respectively. The autophosphorylated S-NDPK-A and S-NDPK-B displayed isoelectric points (pI) of 5.8 and 6.6, respectively. The shoot NDPKs using NDPs (diphosphonucleosides) as substrates were metal dependent, while these underwent autophosphorylation in the absence of metal. The specificity of S-NDPK-A, S-NDPK-B and root NDPK-B (R-NDPK-B); towards mixtures of purino- and pyrimidino-NDPs were tested by TLC (thin layer chromatography). UDP and CDP (pyrimidino-NDPs) and GDP (purino-NDP) were exclusively phosphorylated, using (gamma-(32)P) ATP as phosphate donor, by S-NDPK-A and R-NDPK-B, respectively. Both purino- and pyrimidino-NDPs were phosphorylated by S-NDPK-B. The above isoforms also displayed differences in preference towards a mixture of ADP, GDP, dGDP, TDP, dCDP, CDP and UDP in the presence of Cu(2+), Zn(2+), Mg(2+), Mn(2+), Ni(2+), Ca(2+), Hg(2+) or Co(2+). For example, GDP was mainly phosphorylated by R-NDPK-B independently of the metal used, TDP was mainly phosphorylated by S-NDPK-A in the presence of Mg(2+), Mn(2+), Ca(2+) or Co(2+) and S-NDPK-B was capable of phosphorylating more or less independently of the metal used. The purified and characterized NDPK isoforms may play different biological roles according to their preference towards NDPs.

Similarities and differences in the properties of multiple isoforms of maize endosperm casein kinase I (CK-I)

Summary Two novel type I casein kinases named CK-1B and CK-1C have been purified from maize endosperm (three weeks after anthesis) by a six step procedure involving ammonium sulfate precipitation, DEAE-cellulose, Sephadex G-75, Heparin-sepharose, and ATP-agarose chromatography. The catalytic subunits of both enzymes were identified as a 35-37 kDa polypeptide doublet by in situ phosphorylation after SDS/PAGE in active casein gel. Both enzymes required 5-10 mmol · L −1 Mg 2+ for maximal activity, could utilize only ATP as phosphate donor, were insensitive to heparin, were not autophosphorylated, had a pH optimum at pH 7 to 8.5, and exclusively phosphorylated acidic proteins (casein, phosvitin). Regarding the enzyme differences, their properties were as follows: a) CK-1B could bind on ATP-agarose affinity column, while CK-1C could not; b) the activity of CK-1C was strongly stimulated at low concentrations (1 mmol/L) of spermine, while that of CK-1B was inhibited; c) CK-1B and CK-1C Km values for ATP were 11 μmol · L −1 and 26 μmol · L −1 , respectively; d) Mg 2+ could substituted by Mn 2+ in the CK-1B catalytic activity (by about 80 percnt;); e) CK-1B phosphorylated serine, while CK-1C both serine and threonine on casein. The combination of these results with those from Babatsikos and Yupsanis (2000) brings the number of investigated maize endosperm CK-I isoforms to three (CK-1B, CK-1C, and CK-1E). This is the first biochemical approach demonstrating that multiple isoforms of CK-I casein kinases are present in the same plant tissue.

Usage of DNases and NDP kinases as qualitative markers to discriminate apomictic embryos of Poncirus trifoliata L. and Citrus aurantium L. rootstocks

The aim of the present work was to study the qualitative changes of DNases and NDP kinases of Poncirus trifoliata L. and Citrus aurantium L. rootstocks between apomictic pairs of cotyledons of different developmental stage (large and smaller pair) in mature seeds. In nucleolytic analysis of P. trifoliata, differences were observed between large and small pairs of cotyledons. Specifically, in small pairs two DNase isoforms were formed, while in the large pairs, an additional isoform appeared. In C. aurantium, four DNase isoforms were present. The first isoform appeared in all treatments, and the three other isoforms only in large pair extracts. In both P. trifoliata and C. aurantium SDS-PAGE, large pair of cotyledons showed peptide bands more intense compared to pairs of small cotyledons. When cotyledonary extract was incubated in the presence of different metals, different number and intensity of peptide bands was appeared. From TLC autoradiography, it was found that all phosphorylated bands were catalytic subunits of NDP kinases, but showed different specificity towards substrates (phosphorylated NDPs). DNases and NDP kinase activity depend on the developmental stage of nucellar embryos’ cotyledons of P. trifoliata and C. aurantium and they could be used as an efficient and precise scientific method for indicating it.