A Thinopyrum ponticum and Triticum aestivum hybrid. Electrophoretic patterns of their endogenous phosphorylation, Diphosphonucleoside kinases, DNases and RNases

Endogenous protein phosphorylation, DNase and RNase electrophoretic patterns, and the detection of NDP-kinases by TLC (Thin Layer Chromatography) were performed in Thinopyrum ponticum (2n=10x=70), Triticum aestivum (2n=6x=42), and their hybrid seedlings in order to accomplish intergeneric hybridization. Octoploid intergeneric hybrids (2n=8x=56) were synthesized in less than 50% of the hybrids. The F₁ hybrid plants resembled Th. ponticum with regard to morphological features and were sterile. Hybrid seedlings revealed very low endogenous phosphorylation and very low NDP-kinase activity in comparison to their parents. In addition hybrid seedlings expressed a new nuclease. Received: 29 June 2000 / Accepted: 28 July 2000     

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

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 Mg²⁺ 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) Mg²⁺ could substituted by Mn²⁺ 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.     

Effects of melatonin and plane of nutrition on mammary development in prepubertal Boutsiko mountain breed ewe lambs

The effects of melatonin (implants, M or no implants, C) and plane of nutrition (high, H or low, L) on mammary development and growth hormone (GH) concentrations were investigated in prepubertal Boutsiko mountain breed ewe lambs. Eighty female lambs were assigned to each of 4 treatments: ad libitum feeding control (HC), HM, LC and LM. The rearing treatments started and ended at mean ages of 63 and 160 d, respectively. Feed restriction resulted in a mean daily gain of 70.6% of the ad libitum-fed lambs during the experimental period. Melatonin (18 mg Regulin) was administered at 68 d of age (January 10) and replaced on March 1. Blood samples were collected from 10 lambs in each treatment group at the end of the experiment for GH measurements. At a mean age of 160 d, seven lambs from each treatment group were slaughtered and the udder was removed. One udder half was trimmed and the parenchyma and fat pad portions were kept for determination of deoxyribonucleic acid (DNA) content. Melatonin did not influence mammary development parameters, while the mass of parenchyma tended to be greater in lambs on low than high nutrition planes (P<0.10). Mean mammary parenchymal weight and DNA content were 25.1 and 29.2 g and 52.5 and 58.2 mg in high and low nutrition lambs, respectively. Mean plasma GH concentrations were not affected by melatonin treatment and were higher in low than high nutrition lambs (P<0.01). There were no correlations between mean plasma GH concentrations and parenchymal DNA content, or between mean daily weight gain and parenchyma (g), in contrast to those found in a previous experiment with lambs of the same breed but greater age at slaughter. The results suggest that a period of accelerated mammary development occurs later than 140 d of age in Boutsiko mountain breed ewe lambs.     

Paraquat and roundup effects on nucleases activities of alfalfa seedlings and alfalfa nucleases activities on paraquat-treated and roundup-treated nucleic acids

The specific activities of acid (pH 5.5) and neutral (pH 7) DNases and RNases were determined in alfalfa (Medicago sativa L.) seedlings grown in the dark in the presence of 3.7 mM paraquat (PQ) or 1 mM roundup (RD). Seedlings were taken at 0, 1, 3, and 5 days. Plant growth parameters (plant height and fresh weight) were dramatically reduced under these conditions of growth comparing to the control (grown in water). The DNase and RNase specific activities of herbicide-treated seedlings were reduced. The reduction of activities ranged by about 50–90 and 15–70% in PQ- and RD-treated seedlings, respectively. In vitro, PQ- and RD-treated nucleic acids [single-stranded DNA (ssDNA), RNA, and plasmid DNA (pl-DNA)] were incubated with acid and neutral nucleases. Both enzymes were isolated and purified from alfalfa seedlings. Electrophoretic analysis on agarose gel of the above incubated mixtures revealed the following: (a) neutral nuclease (pH 7) was capable of hydrolyzing PQ-treated ssDNA while acid nuclease (pH 5.5) was incapable. This could be due to the fact that acid and neutral nucleases displayed different base linkage specificity toward ssDNA; (b) RD formed strong complexes with ssDNA that were unable to be hydrolyzed by both nucleases; (c) in contrast, both enzymes were capable of hydrolyzing PQ- or RD-treated RNA; (d) neutral nuclease was capable of nicking and linearizing both PQ- and RD-treated pl-DNA while acid nuclease had the same activity only toward the PQ-treated pl-DNA; (e) the enzyme activities were not inhibited in the presence of both herbicides. The data suggest that the complexes of PQ or RD with DNA should not be functional substrates of nucleases, and consequently cell processes (e.g., metabolism of nucleic acids, gene expression, replication), in which DNA and nucleases are involved, could be disturbed.     

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

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 Ni²⁺ or Mn²⁺ (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 Ni²⁺ or Mn²⁺ concentrations showed a negative correlation between the Ni²⁺ or Mn²⁺ concentrations in the nutrient solution, and the chlorophyll concentration, shoot and especially root length. The accumulation of Ni²⁺ or Mn²⁺ in the plant showed a positive correlation with increasing Ni²⁺ or Mn²⁺ concentrations in the nutrient solution. Application of 0.5 mmol/L Ni²⁺ or Mn²⁺ 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 Ni²⁺ or Mn²⁺; they have similar DNase and RNase electrophoretic patterns and show a new DNase form under Ni²⁺ or Mn²⁺ stress. This is the first report on the response of nucleolytic enzymes under metallic elements hyperaccumulation.     

Protein, leucine aminopeptidase, esterase, acid phosphatase and photosynthetic responses of oleander (Nerium oleander L.) during cold acclimation and freezing treatments

Six-month-old oleander (Nerium oleander L.) pot plants, derived from vegetative propagation by cuttings, were tested for their ability to cold hardening. Damage of the non-acclimated (NA) plants was visible when treated by low freezing temperatures (below -2 degrees C). The responses of total proteins, leucine aminopeptidase (LAP), esterase (EST) and acid phosphatase (ACP) isoforms of NA and cold-acclimated (CA; 4 degrees C for 14 days) plants were compared using polyacrylamide gel electrophoresis. These molecular markers were also compared in NA and CA plants which received for 2h temperatures of 0, -2, -4, -6 and -8 degrees C. A new 38-kDa polypeptide appeared from day 7 to 14 during the acclimation treatment in the bark extracts and on day 14 in the leaf extracts. The above-mentioned polypeptide band (38 kDa) strongly appeared in all freezing treatments (0, -2, -4, -6 and -8 degrees C) in both bark and leaf extracts of the CA plants. Alterations in the number and the intensity of LAP and EST isoforms as well as in the intensity of ACP isoforms were observed in both bark and leaf of the CA oleander plants. A newly expressed EST isoform is proposed as biochemical marker for the cold acclimation treatment. CO2 assimilation rates (A) as well as transpiration rates (E) in NA plants were positive in 0 degrees C and negative in all temperatures below zero in the freezing treatments. In contrast, CO2 assimilation rates (A) and transpiration rates (E) were positive in CA plants in all temperatures of freezing treatment. A significant decrease (P<0.05) in chlorophyll (Chl) a, Chl a+b concentration and Chl a/b ratio were noticed in oleander plants during the acclimation treatment (from day 0 to 14), while Chl b concentration was unchanged at the respective time. On the other hand, no significant (P<0.05) differences were observed in the freezing treatments.     

DNase and RNase Responses of Petunia × hybrida During Transition to Flowering as Affected by Light Treatments

The responses of DNase and RNase isoforms and their specific activities following transition to flowering (1 to 6 weeks) were examined in Petunia × hybrida under different light conditions. Petunia × hybrida plants formed flower buds at the 4^th week in the case of high light and at the 6^th week in the far-red light treatment, while no flower bud formation was observed upon red light and control light treatments. The DNase and RNase activities decreased from the 1^st to the 6^th week during transition to flowering. Native-PAGE analysis revealed the appearance of one DNase (D₁) and seven RNase (R₁ - R₇) isoforms in all light treatments. It is assumed that the progress of the flowering could be related to the disappearance or reduction of D₁ DNase band intensity and disappearance of R₁, R₂ and R₇ RNase isoforms. Consequently, these isoforms could be used as potent biochemical markers of flower bud formation under light intensity as well as light quality treatments.     

Purification, properties and specificity of an endonuclease from Agropyron elongatum seedlings.

An endonuclease was isolated from 5 days old Agropyron elongatum 8x = Elytrigia turcica McGuire seedlings. The enzyme was purified by means of ammonium sulfate fractionation, DEAE-cellulose and Heparin Sepharose column. The final preparation, named nuclease A, gave a single band after silver staining had followed SDS-electrophoresis that was identified with nuclease activities. The enzyme also showed a single band after activity staining on gel polymerized in the presence of heat denatured DNA (ssDNA)/RNA. The Mr of native enzyme was 36 and the enzyme's moiety consisted of one polypeptide chain. Nuclease A activity was stimulated in the presence of Zn(2+) and was moderately reduced by NaCl yet strongly by spermine. The enzyme had pH optimum 5.5 and isoelectric point (pI) 4.7. It hydrolyzed the nucleic acids in the order ssDNA > dsDNA > or = RNA; hence it was classified as a plant nuclease type I (EC 3.1.30.2). Synthetic homopolyribonucleotides were hydrolyzed in the order polyU > polyI > or = polyA > polyG > polyC. Nuclease A nicked the supercoiled plasmid DNA while it was incapable of hydrolyzing dinucleoside monophosphates. With regard to nuclease A base linkage specificity towards a synthetic 5'-(32)P labeled deoxydecanucleotide [5'-(32)P]CCTGGCAGTT, the enzyme firstly exhibited a preference to Ap downward arrow G bond and then to Gp downward arrow T, Cp downward arrow A and Gp downward arrow G bonds while it was incapable of hydrolyzing the Cp downward arrow C bond. The substrate's products of nuclease A were oligonucleotides with the monoesterified phosphate at the 3' position. Nuclease A may perform a crucial function in the metabolism of nucleic acids during seedling growth and could be used as a biochemical tool for analysis of nucleic acids structure.     

RNase and DNase activities in the alfalfa and lentil grown in iso-osmotic solutions of NaCl and mannitol

Nucleolytic activities from two plants of Leguminosae family were determined in order to consider if the nucleases of plants which belong to the same family or to the same species responded in similar ways to stress conditions during growth. Growth parameters of both plants were examined in parallel. In detail, seedlings from two plants, alfalfa (Medicago sativa L. cv. Luzerne Euver) and lentil (Lens culinaris cv. Thessalia), showed significant differences in response to iso-osmotic solutions of NaCl (100 mmol · L⁻¹ solution equivalent to conductivity 8.0 dS m⁻¹) and mannitol (190 mmol · kg⁻¹). Plant height and dry weight of mannitol/NaCl-treated seeds in both plants were lower in comparison to controls (water). Mannitol stress reduced height and dry weight in alfalfa seedlings more than did NaCl. By contrast, lentil seedling growth was inhibited more by NaCl stress than mannitol. In addition, DNase and RNase response to mannitol stress differed in each plant compared to the controls. Mannitol stress induced a sharp increase in DNase- and RNase-specific activity during the initial stages of alfalfa seedlings' growth, followed by a decrease during subsequent days; in lentil seedlings, these activities were inhibited throughout the entire growth period. NaCl stress inhibited the above activities in both plants. After native electrophoresis on gels polymerized in the presence of DNA/RNA, the overall band intensities confirmed the above quantitative results of alfalfa RNase and DNase activity. In addition, the active gel analysis revealed that the decrease of nucleolytic activities in mannitol-treated alfalfa seedlings was mainly due to the strong reduction of acid nucleases. This is the first report of different non-ionic osmotic response of type I plant nucleases during seedlings' growth. In vitro, the addition of up to 300 mmol/L mannitol did not affect acid and neutral nuclease activity in enzyme preparations extracted, purified, and separated from control and mannitol-treated alfalfa seedlings.Our results suggest that plant nucleases responded in a different way to osmotic stress and ionic stress conditions during seedlings' growth.     

Purification, properties and specificity of a NDP kinase from Alyssum murale grown under Ni(2+) toxicity

During the growth of Alyssum murale, a nickel accumulator plant, three root peptides chains of 55, 18 and 16kDa undergo phosphorylation. The intensity of the phosphorylated bands decreased in the course of growth in nutrient solution supplied with 0.5mM Ni(2+). In the shoot only two phosphorylated peptide chains with a size of 18 and 16kDa were detected. These two shoot peptides disappeared on the 19th day of growth in Ni(2+)-exposed plants, while the root peptide of 16kDa continued to be present in less intensity. This peptide was identified as the catalytic subunit of nucleoside diphosphate kinase (NDP kinase: E.C. 2.7.4.6) and was named NDPK-B. The enzyme was purified by means of ammonium sulphate precipitation, DEAE-sepharose and hydroxyapatite column chromatography. NDPK-B was thermostable, displayed a molecular mass of 103,000 and was comprised of six catalytic subunits. The autophosphorylated enzyme displayed an isoelectric point (pI) of 6.5. The NDPK-B autophosphorylation activity was metal-dependent. With regard to the transfer reaction, NDPK-B exhibited the following properties: (a) the enzyme had an optimum pH of 7.6; (b) it was capable of using both (gamma-(32)P) ATP and (gamma-(32)P) GTP as phosphate donors and of using all the available NDPs except dCDP as phosphate acceptors; (c) its activity using NDPs as substrates was metal dependent; (d) in the presence of (gamma-(32)P) GTP as the phosphate donor, it phosphorylated exclusively ADP when a mixture of NDPs was added in the reaction mixture; and, (e) ADP had a very low K(m) value towards 8.4nM. This high affinity towards ADP suggests that the enzyme may play a crucial function in the formation of the amount of ATP necessary for Alyssum murale to survive Ni(2+) stress.