Induction and suppression of RNase activity by growth regulators in senescing ragi leaves

Activity of RNase was studied in attached and detached leaves of 7-day-old ragi ( Eleusine coracana Gaertn. cv PR 202) plants during senescence using crude enzyme extracts. The RNase activity was relatively constant in attached leaves. In excised leaves incubated in the dark there was a rapid rise in enzyme activity up to 48 h, followed by a decline. No such decrease was observed in the light. Benzimidazole and gibberellic acid suppressed the activity of RNase up to 48 h in the dark and 96 h in the light. Both the growth regulators also prevented the post-48 h decline in RNase activity of dark incubated excised leaves. Decline in the levels of chlorophyll and RNA in the illuminated excised leaves was not affected by 3-(3,4-dichlorophenyl)-1,1-dimethyIurea, but the inhibitor prevented the photo-induced rise in RNase activity. Cycloheximide and actinomycin D could completely prevent both detachment (increase in enzyme activity after the leaf is excised) and photo-induced rise in RNase activity. Benzimidazole and gibberellic acid prevented the rise in the activity of RNase on one hand and maintained it on the other by their influence on its biosynthesis. Photoinduction of RNase and photo-induced retardation of senescence are concluded to be two different processes.     

Nitrogen-deficiency-induced loss in photosynthesis and modulation of β-galactosidase activity during senescence of <Emphasis Type="Italic">Arabidopsis</Emphasis> leaves

Senescence-induced loss in the content of chlorophyll and the rate of oxygen evolution is remarkably enhanced when the leaves of Arabidopsis thaliana experience nitrogen-deficiency stress. On the other hand, the decline in the level of total soluble sugar during senescence is very slow and nitrogen deficiency does not exhibit any further change. The relative stability in the level of the sugar in the background of severe decline of photosynthesis may suggest the contribution of sugars from other sources to sustain its homeostasis to execute and complete energy-dependent senescence process and stress response. The possible participation of cell wall polysaccharides contributing to sugar homeostasis is predicted. Senescence-induced increase in the activity of β-galactosidase (EC 3.2.1.23) and its further enhancement in senescing leaves experiencing nitrogen stress support the proposition of participation of the enzyme for breakdown of the wall polysaccharides to sugars. The loss of photosynthesis as a possible signal for enhancement in the activity of β-galactosidase has been further examined in the excised leaves incubated in Okada and Shimura (OS) nutrient medium with and without nitrogen. Nitrogen limitation experienced by excised leaves causes rapid loss in photosynthesis with concomitant increase in the activity of the enzyme extracted both from soluble and cell wall fractions. The differential activity of the enzyme from soluble and cell wall fractions during development-dependent leaf senescence and premature senescence in excised leaves induced by nitrogen deficiency appears to be complex and needs to be resolved in the future.     

Purification and characterization of nuclease I associated with rye germ ribosomes

A nuclease has been purified about 100-fold from ammonium chloride wash of rye germ ribosomes. The enzyme was electrophoretically homogeneous. Its M, was 20,000 and pl 4.8. The neclease hydrolyzed endonuclelytically DNA and RNA and was accompanied by 3-nucleotidase activity. The enzyme degraded RNA to oligonucleotides with a phosphomonoester bond at position 5', and both denatured and native DNA to 5'-OH and 3'-phosphate-terminated fragments. Zinc ions and 2-mercaptoethanol stimulated deoxyribonucleolytic activity. EDTA, polyamines and heparin had only little or no effect. The enzyme is a glycoprotein containing 28% of carbohydrate which consists of fucose, mannose and glucosamine. The nuclease isolated is classified as nuclease I.     

Changes in the Level of Peptidase Activities in Pea Ovaries during Senescence and Fruit Set Induced by Gibberellic Acid

The activities and changes in the levels of exopeptidase and endopeptidase activities were characterized in unpollinated ovaries of Pisum sativum L. cv Alaska during senescence and early fruit development induced by gibberellic acid (GA₃). Two aminopeptidases and one iminopeptidase were electrophoretically separated. These peptidases were sensitive to inhibitors of sulfhydryl proteases. Carboxypeptidase activity was inhibited by phenylmethyl sulfonyl fluoride. An azocasein-degrading endopeptidase, sensitive to thiol protease inhibitors, was also found. An increase in the specific activity of aminopeptidase during both fruit development and ovary senescence was observed. In contrast, the specific activity of carboxypeptidase and endopeptidase increased only during senescence of the ovary. Changes in exopeptidase activity in senescing ovaries could be mainly the consequence of a greater stability to proteolysis while the rise in endopeptidase activity appeared to be due to new or increased synthesis of the enzyme. These results suggest that endopeptidase, and not amino or carboxypeptidase, plays a key role in the senescence of pea ovaries and that the changes in unpollinated ovaries leading to ovary senescence or fruit development can be controlled by gibberellins.     

Deoxyribonucleolytic activity of alpha- and beta-momorcharins.

alpha- and beta-Momorcharins were purified by an improved procedure using the affinity Affi-gel Blue gel and the ion exchange Mono-S FPLC column. Both purified alpha- and beta-momorcharins possessed deoxyribonucleolytic activity. Under normal digestion conditions, they cleaved the supercoiled, double-stranded SV-40 DNA to produce nicked circular and linear DNAs. Prolonged incubation did not have any further effects. On the other hand, the linear DNAs, lambda, Ad-2 and T7 were not digested by alpha- nor beta-momorcharin. Thus, it appears that the conformation of the DNA may be the determining factor for the deoxyribonucleolytic activity of these momorcharins.     

Relation between Respiration and Senescence in Oat Leaves

The respiration of excised oat (Avena sativa cv Victory) leaves and their sensitivity to inhibitors was followed during senescence under varied conditions. The respiration rate, which in controls reaches its peak on the third day in darkness, is lowered at the time of fastest loss of chlorophyll (as reported earlier) by seven unrelated reagents that all delay dark senescence. When senescence is delayed by white light or by cytokinins, the respiratory rise is correspondingly delayed. Kinetin and l-serine, which act as antagonists on senescence, also act as antagonists on the respiratory rate. However, an exception to this close correspondence between senescence and the respiratory rise is offered by the lower aliphatic alcohols, which delay dark senescence and yet accelerate the onset of the respiratory rise.     

Characterization of abscisic Acid-induced ethylene production in citrus leaf and tomato fruit tissues

Abscisic acid (ABA) significantly stimulated ethylene production in citrus (Citrus sinensis [L.] Osbeck, cv Shamouti orange) leaf discs. The extent of stimulation was dependent upon the concentration of ABA (0.1-1 milimolar) and the duration of treatment (15-300 minutes). Aging the discs before applying ABA increased ABA-induced ethylene production due to enhancement of both ethylene-forming enzyme activity and the responsiveness of ABA. Discs excised from mature leaves were much more responsive to ABA than discs excised from young or senescing leaves. ABA stimulated ethylene production shortly after application, suggesting that ABA does not enhance ethylene production via the acceleration of senescence. The stimulating effect of ABA on ethylene production resulted mainly from the enhancement of 1-aminocylopropane-1-carboxylic acid synthesis. Stimulation of ethylene production by ABA in intact citrus leaves and tomato (Lycopersicon esculentum Mill., cv Castlemart) fruit was small but could be increased by various forms of wounding.     

Changes in chloroplast peroxidase activities in relation to chlorophyll loss in barley leaf segments

Total peroxidase activity increased during senescence of excised barley ( Hordeum vulgare L. cv. Kashimamugi) leaves. Kinetin treatment furter increased total peroxidase activity but repressed chlorophyll degradation in excised barley leaves. When isoperoxidases were extracted from barley leaf segments. 4 cationic and 4 anionic isozymes were found in polyacrylamide gel electrophorests during leaf senescence. The chloroplasts contained only two cationic isoperoxidase activities. One (designated C4) was repressed by kinetin. and the other (C3) was increased by kinetin. Glucosamine, which also repressed the degradation of chlorophyll, completely repressed C4 activity but did not affect C3 activity. The induction with senescence, and the repression with kinetin and glucosamine, suggest chat chloroplast isoperoxidase C4 may function as a chlorophyll-degrading enzyme during barley leaf senescence.     

N9-substituted derivatives of kinetin: effective anti-senescence agents

The first isolated cytokinin, 6-furfurylaminopurine (kinetin or Kin), was identified almost 55 years ago. Its biological effects on plant cells and tissues include influences on such processes as gene expression, cell cycle, chloroplast development, chlorophyll biosynthesis, stimulation of vascular development, delay of senescence, and mobilization of nutrients. In the present study we prepared a series of eight N9-substituted Kin derivatives, and characterized them with available physicochemical methods such as CI+ mass spectrometry and ¹H NMR spectroscopy. All compounds were tested in three classical cytokinin bioassays: a tobacco callus assay, an Amaranthus assay, and a senescence assay with excised wheat leaves. The ability of the compounds to interact with Arabidopsis cytokinin receptors CRE1/AHK4 and AHK3 was tested in a bacterial receptor assay. Prepared derivatives with certain substitutions of the N9-atom of the purine moiety enhanced the cytokinin activity of the parent compound in the bioassays to a remarkable degree but negatively affected its perception by CRE1/AHK4 and AHK3. The ability of compounds to delay the senescence of excised wheat leaves in both dark and light conditions, was highly correlated with their ability to influence membrane lipid peroxidation, which is a typical symptom of senescence. Our results were corroborated by gene expression profiling of those genes involved in cytokinin metabolism and perception, plant senescence, and the stress response, and suggest that prepared kinetin derivatives might be used as potent anti-senescence agents.     

Ethylene as a regulator of senescence in tobacco leaf discs

The regulatory role of ethylene in leaf senescence was studied with excised tobacco leaf discs which were allowed to senesce in darkness. Exogenous ethylene, applied during the first 24 hours of senescence, enhanced chlorophyll loss without accelerating the climacteric-like pattern of rise in both ethylene and CO₂, which occurred in the advanced stage of leaf senescence. Rates of both ethylene and CO₂ evolution increased in the ethylene-treated leaf discs, especially during the first 3 days of senescence. The rhizobitoxine analog, aminoethoxy vinyl glycine, markedly inhibited ethylene production and reduced respiration and chlorophyll loss. Pretreatment of leaf discs with Ag⁺ or enrichment of the atmosphere with 5 to 10% CO₂ reduced chlorophyll loss, reduced rate of respiration, and delayed the climacteric-like rise in both ethylene and respiration. Ag⁺ was much more effective than CO₂ in retarding leaf senescence. Despite their senescence-retarding effect, Ag⁺ and CO₂, which are known to block ethylene action, stimulated ethylene production by the leaf discs during the first 3 days of the senescing period; Ag⁺ was more effective than CO₂. The results suggest that although ethylene production decreases prior to the climacteric-like rise during the later stages of senescence, endogenous ethylene plays a considerable role throughout the senescence process, presumably by interacting with other hormones participating in leaf senescence.     

Peptide hydrolases in senescing ragi leaves

Two peptide hydrolases — one active at pH 7.0 and the other at pH 3.4 (SH-dependent) were detected in ragi (Eleusine coracana Gaertn. cv. PR 202) leaves. The absolute activities of both acid and neutral peptide hydrolases were move or less stable in attached leaves and exhibited a mild rise in excised leaves, whereas the specific activities exhibited a significant rise. Light and growth regulators were ineffective in changing the course of absolute activity whereas cytokinins stimulated specific activities of both acid and neutral peptide hydrolases. Though the rates were different, protein decline was positively correlated with the changes in activities of peptide hydrolases. Cycloheximide retarded chlorophyll and protein decline and also prevented the rise in peptide hydrolases. Peptide hydrolases thus do not have an active role in senescence.     

Conversion of dNTP to dNMP dependent on DNA synthesis in isolated Yoshida sarcoma nuclei

Nuclei isolated from Yoshida sarcoma cells had activity for conversion of dGTP to dGMP dependent on DNA synthesis. The ratio of nucleotide generation/generation + incorporation was 0.4 ± 0.1, indicating that approx. 40% of the incorporated dGMP was excised. Two lines of evidence indicated the dependence of this activity on DNA synthesis. (1) The activity was observed only in the presence of ATP, which is essential for nuclear DNA synthesis. (2) Inhibitors of DNA synthesis, such as N-ethylmaleimide, aphidicolin, spermine and KCl, also inhibited ATP- or DNA synthesis-dependent dGMP generation. Although nuclei contain nucleoside triphosphatase (N-nucleotidase), this enzyme was not involved appreciably in DNA synthesis-dependent dGMP generation. The reason for this was explained by the following findings. (a) Inhibitors did not decrease dGMP production in the complete absence of DNA synthesis. (b) Inhibitors did not inactivate N-nucleotidase to the same degree as they inhibited DNA synthesis-dependent dGMP generation. (c) Addition of ATP reduced dGTP hydrolysis catalyzed by N-nucleotidase. (d) GDP had no appreciable effect on DNA synthesis-dependent dGMP generation, but had a diluting effect on dGMP production catalyzed by N-nucleotidase. These results show that the pathway of dGMP generation in isolated nuclei was switched on addition of ATP from a N-nucleotidase-catalyzed one to a DNA polymerase-exonuclease-catalyzed one.     

Effects of light and spermine on senescence of hydrilla and spinach leaves

Light treatment markedly accelerated chlorophyll loss in Hydrilla (Hydrilla verticillata [L.f.] Royle) over dark treatment whereas such acceleration could not be observed in spinach (Spinacia oleracea L.) leaf segments. Spermine, a polyamine, retarded the loss of chlorophyll in the dark but markedly accelerated this loss in the light during senescence of Hydrilla leaves. However, such effect of spermine in the dark was not so pronounced in spinach. The loss of protein was slower in the light than in the dark in both the species. Spermine arrested the loss of protein (as in spinach) or even raised the protein level over initial (as in Hydrilla). Loss of both soluble and insoluble protein was slower in light than in darkness. Spermine treatment, either in light or darkness, markedly accelerated the loss of soluble protein but raised the level of insoluble protein over initial in both the species. The pattern of change in α-amino nitrogen in either species could be correlated well with that of protein level. In Hydrilla, light increased the soluble protein fraction over initial and this rise was prevented by cycloheximide and not by chloramphenicol. Also, spermine augmented the protease activity (both acid and neutral) while light retarded the rise in protease activity during senescence of either species. Although spermine treatment reduced the leaching of α-amino nitrogen and electrolytes in Hydrilla, it augmented the same in spinach.     

Delayed leaf senescence by exogenous lyso-phosphatidylethanolamine: Towards a mechanism of action

Exogenous application of the lysophospholipid, lyso-phosphatidylethanolamine (LPE) is purported to delay leaf senescence in plants. However, lyso-phospholipids are well known to possess detergent-like activity and application of LPE to plant tissues might be expected to rather elicit a wound-like response and enhance senescence progression. Since phosphatidic acid (PA) accumulation and leaf cell death are a consequence of wounding, PA- and hormone-induced senescence was studied in leaf discs from Philodendron cordatum (Vell.) Kunth plants in the presence or absence of egg-derived 18:0-LPE and senescence progression quantified by monitoring both lipid peroxidation (as the change in malondialdehyde concentration), and by measuring retention of total chlorophyll (Chl_a+b) and carotenoids (C_c+x). Only abscisic acid (ABA) stimulated lipid peroxidation whereas ABA, 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor to ethylene (ETH), and 16:0–18:2-PA stimulated loss of chloroplast pigments. Results using primary alcohols as attenuators of the endogenous PA signal confirmed a role for PA as an intermediate in both ABA- and ETH-mediated senescence progression. Exogenous 18:0-LPE did not appear to influence senescence progression and was unable to reverse hormone-induced senescence progression. However, when supplied together with 16:0–18:2-PA at 1:1 (mol:mol), activity of phosphatidylglycerol (PG) hydrolase, chlorophyllase (E.C. 3.1.1.14), and progression of leaf senescence were negated. This apparent anti-senescence activity of exogenous 18:0-LPE was associated with induction of the pathogenesis-related protein, extracellular acid invertase (Ac INV, E.C. 3.2.1.26) suggesting that 18:0-LPE like 16:0–18:2-PA functions as an elicitor.     

Poly-beta-hydroxybutyrate Utilization by Soybean (Glycine max Merr.) Nodules and Assessment of Its Role in Maintenance of Nitrogenase Activity

Soybean (Glycine max) nodule bacteroids contain high concentrations of poly-β-hydroxybutyrate and possess a depolymerase system that catalyzes the hydrolysis of the polymer. Changes in poly-β-hydroxybutyrate content and in activities of nitrogenase, β-hydroxybutyrate dehydrogenase, and isocitrate lyase in nodule bacteroids were investigated under conditions in which the supply of carbohydrate from the soybean plants was interrupted. The poly-β-hydroxybutyrate content of bacteroids did not decrease appreciably until the carbohydrate supply from the host plants was limited by incubation of excised nodules, incubation of plants in the dark, or by senescence of the host plant. Isocitrate lyase activity in bacteroids was not detected until poly-β-hydroxybutyrate utilization appeared to begin. The presence of a supply of poly-β-hydroxybutyrate in nodule bacteroids was not sufficient for maintenance of high nitrogenase activity under conditions of limited carbohydrate supply from the host plant. An unusually high activity of β-hydroxybutyrate dehydrogenase was observed in bacteroid extracts but no significant change in the activity of this enzyme was observed as a result of apparent utilization of poly-β-hydroxybutyrate by nodule bacteroids.     

Human Adipose Stromal Cells (ASC) for the Regeneration of Injured Cartilage Display Genetic Stability after In Vitro Culture Expansion

Mesenchymal stromal cells are emerging as an extremely promising therapeutic agent for tissue regeneration due to their multi-potency, immune-modulation and secretome activities, but safety remains one of the main concerns, particularly when in vitro manipulation, such as cell expansion, is performed before clinical application. Indeed, it is well documented that in vitro expansion reduces replicative potential and some multi-potency and promotes cell senescence. Furthermore, during in vitro aging there is a decrease in DNA synthesis and repair efficiency thus leading to DNA damage accumulation and possibly inducing genomic instability. The European Research Project ADIPOA aims at validating an innovative cell-based therapy where autologous adipose stromal cells (ASCs) are injected in the diseased articulation to activate regeneration of the cartilage. The primary objective of this paper was to assess the safety of cultured ASCs. The maintenance of genetic integrity was evaluated during in vitro culture by karyotype and microsatellite instability analysis. In addition, RT-PCR array-based evaluation of the expression of genes related to DNA damage signaling pathways was performed. Finally, the senescence and replicative potential of cultured cells was evaluated by telomere length and telomerase activity assessment, whereas anchorage-independent clone development was tested in vitro by soft agar growth. We found that cultured ASCs do not show genetic alterations and replicative senescence during the period of observation, nor anchorage-independent growth, supporting an argument for the safety of ASCs for clinical use.     

Leaf pretreatment with senescence retardants as a basis for oat protoplast improvement

Protoplasts obtained from oat leaves floated on buffer for 18hr show high nuclease activity, low rates of incorporation ofamino acids and nucleosides into macromolecules, and high ratesof spontaneous lysis. Addition to the leaf flotation mediumof the senescence retardants cycloheximide or kinetin, of thedibasic amino acids L-lysine or L-arginine, or of the diaminesputrescine or cadaverine reduces the rise in nuclease activityand spontaneous lysis of protoplasts, and increases the rateor extent of presumptive protein and nucleic acid synthesis.The diamines, which also retard chlorophyll degradation in theexcised leaves, appear to act both on the membrane and on systemscontrolling macromolecular synthesis and breakdown. By contrast,the senescence promoter L-serine hastens chlorophyll degradationfrom excised leaves and does not improve protoplasts derivedfrom those leaves. (Received July 4, 1977; )     

A DNA polymerase from Ustilago maydis. 2. Properties of the associated deoxyribonuclease activity.

The polymerase and deoxyribonuclease activities of the purified Ustilago maydis DNA polymerase coeluted from a hydroxyapatite column, cosedimented in sucrose gradients in both the absence and presence of salt, possessed similar thermolabilities and reaction requirements. These observations suggest that both activities are associated with the same enzyme and that the deoxyribonuclease activity is not a contaminant. The initial rate of degradation of native 3'-end-group-labelled DNA was similar to that of a heat-denatured substrate, but the final extent was greater for the former. The enzyme exhibits a high specificity for degradation of DNA in a 3' leads to 5' direction. The degradation of a DNA template was inhibited by the presence of the deoxyribonucleoside triphosphates necessary for simultaneous DNA synthesis, but not that of the newly synthesised DNA. About 50%, 29% and 13% of the purine, cytosine and thymine deoxyribonucleotide residues incorporated by the enzyme into DNA respectively, were subsequently excised when monitored by the resulting conversion of the triphosphate substrates to free monophosphate. The majority of the purine deoxyribonucleoside monophosphates appear after the synthetic phase of the reaction has ceased. In many respects, therefore, the deoxyribonuclease activity of the U. maydis DNA polymerase is similar to the bacteriophage T4-induced enzyme.     

Aging of cylinders excised from pulp tissues of the ;golden delicious' apple

Aging cylinders excised from `Golden Delicious' apple (Pyrus malus L.) pulp, like the intact fruit, exhibit some characteristic phenomena such as rise in respiration (climacteric), ethylene synthesis, enzymic changes, and increase in ribosomes and mRNA. Aging of cylinders of pulp tissues may offer a useful physiological tool for the study of maturation and senescence.