Excretion of polyamines in alfalfa and tobacco suspension-cultured cells and its possible role in maintenance of intracellular polyamine contents

Changes in polyamines (PAs) in cells and cultivation media of alfalfa (Medicago sativa L.) and tobacco bright yellow 2 (BY-2) (Nicotiana tabacum L.) cell suspension cultures were studied over their growth cycles. The total content of PAs (both free and conjugated forms) was nearly 10 times higher in alfalfa, with high level of free putrescine (Put) (in exponential growth phase it represented about 65-73% of the intracellular Put pool). In contrast, the high content of soluble Put conjugates was found in tobacco cells (in exponential phase about 70% of the intracellular Put). Marked differences occurred in the amount of PAs excreted into the cultivation medium: alfalfa cells excreted at the first day after inoculation 2117.0, 230.5, 29.0 and 88.0 nmol g(-1) of cell fresh weight (FW) of Put, spermidine (Spd), spermine (Spm) and cadaverine (Cad), respectively, while at the same time tobacco cells excreted only small amount of Put and Spd (12.7 and 2.4 nmol g(-1) FW, respectively). On day 1 the amounts of Put, Spd, Spm and Cad excreted by alfalfa cells represented 21, 38, 12 and 15% of the total pool (intra- plus extra-cellular contents) of Put, Spd, Spm and Cad, respectively. In the course of lag-phase and the beginning of exponential phase the relative contents of extracellular PAs continually decreased (with the exception of Cad). On day 10, the extracellular Put, Spd, Spm and Cad still represented 11.3, 10.9, 2.1 and 27% of their total pools. The extracellular PAs in tobacco cells represented from day 3 only 0.1% from their total pools. The possible role of PA excretion into the cultivation medium in maintenance of intracellular PA contents in the cells of the two cell culture systems, differing markedly in growth rate and PA metabolism is discussed.     

Polyamines and environmental challenges: recent development

In this review, we will try to summarize some recent data concerning the changes in polyamine metabolism (biosynthesis, catabolism and regulation) in higher plants subjected to a wide array of environmental stress conditions and to describe and discuss some of the new advances concerning the different proposed mechanisms of polyamine action implicated in plant response to environmental challenges. All the data support the view that putrescine and derived polyamines (spermidine, spermine, long-chained polyamides) may have several functions during environmental challenges. In several systems (except during hypoxia, and chilling tolerance of wheat and rice) an induction of polyamines (spermidine, spermine) not putrescine accumulation, may confer a stress tolerance. In several cases stress tolerance is associated with the production of conjugated and bound polyamines and stimulation of polyamine oxidation. In several environmental challenges (osmotic-stress, salinity, hypoxia, environmental pollutants) recent results indicate that both arginine decarboxylase and ornithine decarboxylase are required for the synthesis of putrescine and polyamines (spermidine and spermine). Under osmotic and salt-stresses a production of cadaverine is observed in plants. A new study demonstrates that under salt-stress putrescine catabolism (via diamine oxidase) can contribute to proline (a compatible osmolyte) accumulation.     

Ornithine decarboxylase activity in Helianthus tuberosus

Ornithine decarboxylase (ODC, EC 4.1.1.17) was studied in crude extracts of parenchyma slices of dormant tubers activated for 12 h, tuber shoots and shoot apices. It was highest in shoot apices. The enzyme activity was measured by the production of ¹⁴CO₂ from labelled ornithine; V_max was 450 nmol (mg protein)^-1h^-1, K_m for ornithine and pyridoxal phosphate were, respectively, 30 m M and 5μ M . Only when partially purified, the ¹⁴CO₂ production was inhibited by α-difluoromethylornithine, while in crude extracts dithiothreitol was inhibitory. Ornithine and arginine decarboxylase (ADC, EC 4.1.1.19) activities from parenchyma tubers were not greatly altered by exogenously supplemented ornithine, even though its endogenous pool increased. Exogenously supplemented arginine enhanced ornithine decarboxylase activity, whereas putrescine decreased it slightly. The possibility of artifactual activities in the crude extract is also discussed.     

Polyamine metabolism and gene regulation during the transition of autonomous sugar beet cells in suspension culture from quiescence to division

Sugar beet cells grown in batch suspension culture have been used to study the regulation of polyamine levels during the transition from a quiescent to a proliferating state. The quiescent state was achieved by maintenance of the phytohormone autonomous cells in the stationary phase of the batch culture cycle. After subculture into fresh medium there was an increase in DNA synthesis which was accompanied by a dramatic increase in cellular polyamine levels. The levels of both free and bound cellular putrescine and spermidine within the cells reached a peak before the onset of the first synchronous division. The levels of putrescine, spermidine and to some extent spermine in the culture medium also increased dramatically shortly after subculture. The increase in polyamines was preceded by a rapid but transient increase in omithine decarboxylase (EC 4.1.1.17) and S -adenosylmethionine decarboxylase (EC 4.1.1.50). Arginine decarboxylase (EC 4.1.1.19) and S -adenosylmethionine synthetase (EC 2.5.1.6) activity did not show the same pattern of cell division-related variation. Inhibition of S -adenosylmethionine biosynthesis with methylglyoxal bis-(guanylhydra-zone) (MGBG) reduced cell division in the suspension culture. Inhibitors of ornithine decarboxylase and arginine decarboxylase individually had little effect on cell division, but in combination led to a reduction in cell division. Addition of polyamines and their precursors to cells in the stationary phase of a batch culture cycle led to the induction of expression of a mitotic cyclin sequence ( BvcycII ).     

Polyamine metabolism and gene regulation during the transition of autonomous sugar beet cells in suspension culture from quiescence to division

Sugar beet cells grown in batch suspension culture have been used to study the regulation of polyamine levels during the transition from a quiescent to a proliferating state. The quiescent state was achieved by maintenance of the phytohormone autonomous cells in the stationary phase of the batch culture cycle. After subculture into fresh medium there was an increase in DNA synthesis which was accompanied by a dramatic increase in cellular polyamine levels. The levels of both free and bound cellular putrescine and spermidine within the cells reached a peak before the onset of the first synchronous division. The levels of putrescine, spermidine and to some extent spermine in the culture medium also increased dramatically shortly after subculture. The increase in polyamines was preceded by a rapid but transient increase in omithine decarboxylase (EC 4.1.1.17) and S -adenosylmethionine decarboxylase (EC 4.1.1.50). Arginine decarboxylase (EC 4.1.1.19) and S -adenosylmethionine synthetase (EC 2.5.1.6) activity did not show the same pattern of cell division-related variation. Inhibition of S -adenosylmethionine biosynthesis with methylglyoxal bis-(guanylhydra-zone) (MGBG) reduced cell division in the suspension culture. Inhibitors of ornithine decarboxylase and arginine decarboxylase individually had little effect on cell division, but in combination led to a reduction in cell division. Addition of polyamines and their precursors to cells in the stationary phase of a batch culture cycle led to the induction of expression of a mitotic cyclin sequence ( Bvcycll ).     

Polyamine concentrations and arginine decarboxylase activity in wheat exposed to osmotic stress

The activity of L-arginine decarboxylase (ADC: EC 4.1.1.19)and polyamine content were examine in intact wheat plants ( Triticum aestivum L. cv. Sappo) exposed to osmotic stress (0.4 M mannitol) for 5 days. ADC activity was increased in first and second leaves and in roots of mannitol-stressed plants. Concentrations of putrescine, cadaverine and spermine were generally increased in leaves and roots of plants exposed to mannitol, whereas spermidine was reduced in first leaves and roots of these plants. In an attempt to determine the localization of mannitol in stressed wheat. ¹⁴C-mannitol was fed to plants grown in liquid culture. Most of the mannitol was detected in roots (84%), while small amounts were found in first (9%) and second (7%) leves.
Since it seemed possible that some of the effects on polyamine metabolism caused by exposure to mannitol could have been the result of water stress. polyamine metabolism was also studied in plants water stressed by exposure to 2% polyethylene glycol (PEG) 4000. ADC activity was not altered by exposure to PEG. but concentrations of putrescine, spermidine and spermine were generally reduced in leaves and roots of stressed plants. Cadaverine concentrations were not significantly affected by exposure to PEG. Spermidine and spermine concentrations were reduced in first and second leaves but remained unchanged in roots of plants exposed to PEG.     

Characterization of arginine decarboxylase in rat brain and liver: distinction from ornithine decarboxylase

We compared the properties of mammalian arginine decarboxylase (ADC) and ornithine decarboxylase (ODC) in rat liver and brain. Mammalian ADC is thermally unstable and associated with mitochondrial membranes. ADC decarboxylates both arginine (Km = 0.75 mM) and ornithine (Km = 0.25 mM), a reaction not inhibited by the specific ODC inhibitor, difluoromethylomithine. ADC activity is inhibited by Ca2+, Co2+, and polyamines, is present in many organs being highest in aorta and lowest in testis, and is not recognized by a specific monoclonal antibody to ODC. In contrast, ODC is thermally stable, cytosolic, and mitochondrial and is expressed at low levels in most organs except testis. Although ADC and ODC are expressed in cultured rat C6 glioma cells, the patterns of expression during growth and confluence are very different. We conclude that mammalian ADC differs from ADC isoforms expressed in plants, bacteria, or Caenorhabditis elegans and is distinct from ODC. ADC serves to synthesize agmatine in proximity to mitochondria, an organelle also harboring agmatine's degradative enzyme, agmatinase, and a class of imidazoline receptor (I2) to which agmatine binds with high affinity.     

The effect of salt stress on polyamine biosynthesis and content in mung bean plants and in halophytes

The activity of L-arginine decarboxylase (EC 4.1.1.19) and L-ornithine decarboxylase (EC 4.1.1.17), polyamine content, and incorporation of arginine and ornithine into polyamines, were determined in mung bean [Vigna radiata (L.) Wilczek] plants subjected to salt (hypertonic) stress (NaCl at 0.51–2.27 MPa). Changes in enzyme activity in response to hypotonic stress were determined as well in several halophytes [Pulicaria undulata (L.), Kostei, Salsola rosmarinus (Ehr.) Solms-Laub, Mesembryanthemum forskahlei Hochst, and Atriplex halimus L.]. NaCl stress, possibly combined with other types of stress that accompanied the experimental conditions, resulted in organ-specific changes in polyamine biosynthesis and content in mung bean plants. The activity of both enzymes was inhibited in salt-stressed leaves. In roots, however, NaCl induced a 2 to 8-fold increase in ornithine decarboxylase activity. Promotion of ornithine decarboxylase in roots could be detected already 2 h after exposure of excised roots to NaCl, and iso-osmotic concentrations of NaCl and KCl resulted in similar changes in the activity of both enzymes. Putrescine level in shoots of salt-stressed mung bean plants increased considerably, but its level in roots decreased. The effect of NaCl stress on spermidine content was similar, but generally more moderate, resulting in an increased putrescine/spermidine ratio in salt-stressed plants. Exposure of plants to NaCl resulted also in organ-specific changes in the incorporation of both arginine and ornithine into putrescine: incorporation was inhibited in leaf discs but promoted in excised roots of salt-stressed mung bean plants. In contrast to mung bean (and several other glycophytes), ornithine and arginine decarboxylase activity in roots of halophytes increased when plants were exposed to tap water or grown in a pre-washed soil—i.e. a hypotonic stress with respect to their natural habitat. NaCl, when present in the enzymatic assay mixture, inhibited arginine and ornithine decarboxylase in curde extracts of mung bean roots, but did not affect the activity of enzymes extracted from roots of the halophyte Pulicaria. Although no distinct separation between NaCl stress and osmotic stress could be made in the present study, the data suggest that changes in polyamines in response to NaCl stress in mung bean plants are coordinated at the organ level: activation of biosynthetic enzymes concomitant with increased putrescine biosynthesis from its precursors in the root system, and accumulation of putrescine in leaves of salt-stressed plants. In addition, hypertonic stress applied to glycophytes and hypotonic stress applied to halophytes both resulted in an increase in the activity of polyamine biosynthetic enzymes in roots.     

The effect of submergence,ethylene and gibberellin on polyamines and their biosynthetic enzymes in deepwater-rice internodes

Submergence and treatment with ethylene or gibberellic acid (GA₃) stimulates rapid growth in internodes of deepwater rice (Oryza sativa L. cv. Habiganj Aman II). This growth is based on greatly enhanced rate of cell-division activity in the intercalary meristem (IM) and on increased cell elongation. We chose polyamine biosynthesis as a biochemical marker for cell-division activity in the IM of rice stems. Upon submergence of the plant, the activity of S-adenosylmethionine decarboxylase (SAMDC; EC 4.1.1.50) in the IM increased six- to tenfold within 8 h; thereafter, SAMDC activity declined. Arginine decarboxylase (ADC; EC 4.1.1.19) showed a similar but less pronounced increase in activity. The activity of ornithine decarboxylase (ODC; EC 4.1.1.17) in the IM was not affected by submergence. The levels of putrescine and spermidine also rose in the IM of submerged, whole plants while the concentration of spermine remained low. The increase in SAMDC activity was localized in the IM while the activity of ADC rose both in the node and the IM above it. The node also contained low levels of ODC activity which increased slightly following submergence. Increased activities of polyamine-synthesizing enzymes in the nodal region of submerged plants probably resulted from the promotion of adventitious root formation in the node. Treatment of excised rice-stem sections with ethylene or GA₃ enhanced the activities of SAMDC and ADC in the IM and inhibited the decline in the levels of putrescine and spermidine. We conclude that SAMDC and perhaps also ADC may serve as biochemical markers for the enhancement of cell-division activity in the IM of deepwater rice.Abbreviations ADC arginine decarboxylase - GA gibberellin - IM intercalary meristem - ODC ornithine decarboxylase - SAM S-adenosylmethionine - SAMDC SAM decarboxylase     

Inhibition of ornithine decarboxylase and S-adenosylmethionine decarboxylase synthesis by antisense oligodeoxynucleotides

Oligodeoxynucleotides 18 nucleotides in length having sequences complementary to regions spanning the initiation codon regions of ornithine decarboyxlase or S-adenosylmethionine decarboxylase mRNAs were tested for their ability to inhibit translation of these mRNAs. In reticulocyte lysates, a strong and dose dependent reduction of ornithine decarboyxlase synthesis in response to mRNA from D-R L1210 cells was brought about by 5-AAAGCT GCTCATGGTTCT-3 which is complementary to the sequence from - 6 to + 12 of the mRNA sequence but there was no inhibition by 5-TGCAGCTTCCATCACCGT-3. Conversely, the latter oligodeoxynucleotide which is complementary to the sequence from – 6 to + 12 of the mRNA of S-adenosyl methionine decarboxylase was a strong inhibitor of the synthesis of this enzyme in response to rat prostate mRNA and the antisense sequence from ornithine decarboxylase had no effect. The translation of ornithine decarboxylase mRNA in a wheat germ system was inhibited by the antisense oligodeoxynucleotide at much lower concentration than those needed in the reticulocyte lysate suggesting that degradation of the hybrid by ribonuclease H may be an important factor in this inhibition. These results indicate that such oligonucleotides may be useful to regulate cellular polyamine levels and as probes to study control of mRNA translation.Abbreviations ODC ornithine decarboxylase - AdoMetDC S-adenosylmethionine decarboxylase - DFMO difluoromethylornithine     

Purification and characterization of arginine decarboxylase from cucumber (Cucumis sativus) seedlings

A simple, reproducible and rapid protocol for the purification of arginine decarboxylase fromCucumis sativus seedlings has been standardised. The purification steps involved ion-exchange chromatography on diethylaminoethyl-cellulose followed by gel filtration on Sephadex G-l 50. The purified enzyme preparation migrated as a single stainable band on Polyacrylamide gels at both basic and acidic pH, but under denaturing and reducing conditions on sodium dodecyl sulphate-polyacrylamide gels resolved into polypeptides of molecular weight 48,000,44,000 and 15,000. However, in the absence of 2-mercaptoethanol on electrophoresis on sodium dodecyl sulphate-polyacrylamide gels, the enzyme moved as single band with a molecular weight of 150,000. Evidence was obtained to indicate that these three polypeptides were probably derived from a single larger molecular weight enzyme. On storage of the purified protein, the 48,000 species was preferentially degraded to smaller polypeptides. The preliminary data suggested that the 48,000 and 44,000 species shared many common tryptic peptides as revealed by finger printing of the [¹²⁵I ]-labelled protein. The purified enzyme was a glycoprotein and had aK _m of 0.5 mM for arginine. Its activity was stimulated by dithiothrietol and pyridoxal phosphate. EDTA did not inhibit the enzyme activity. Mn²⁺ at 1 mM stimulated arginine decarboxylase activity but was inhibitory at higher concentration     

mRNA结构及其稳定性的关系

mRNA结构与mRNA稳定性关系密切,mRNA稳定性与基因表达调控之间也有着紧密的联系。现从mRNA结构中所含的5'端帽结构、3'端poly(A)尾、5'非翻译区、3'非翻译区、编码区、富AU元件等方面综述了mRNA结构与mRNA稳定性之间的关系,为深入了解基因表达调控的分子机制提供理论基础。     

Polyamines in discrete regions of barley leaves infected with the powdery mildew fungus, Erysiphe graminis

The concentrations of putrescine, spermidine and spermine and the activities of arginine decarboxylase (ADC; EC 4.1.1.19) and ornithine decarboxylase (ODC: EC 4.1.1.17) were determined in discrete regions of barley leaves ( Hordeum vulgare L. cv. Golden Promise) infected with the powdery mildew fungus ( Erysiphe graminis f.sp. hordei Marchal). Polyamine concentrations and the activities of both enzymes were always greatest within the region surrounding the fungal pustule, with the lowest values always being found in the region furthest away from the pustule. Although the concentrations of the three amines and ADC and ODC activities within the fungal pustule were always less than values from the zone surrounding the pustule, these differences were never significant. Polyamine concentrations and ODC activity were not significantly reduced, and ADC activity remained unchanged in mildewed leaves with all surface fungal growth removed. It would appear therefore that not only does most of the increase in amines and ODC activity reside in the leaf itself, but that very little of this increase is due to fungal growth and sporulation. Furthermore, it seems possible that the increase in polyamines in mildewed barley could be involved in 'green-island' formation, where regions around mildew pustules remain green and physiologically active while the rest of the leaf senesces.