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     

Polyamine titer and biosynthetic enzymes during tuber formation ofHelianthus tuberosus

During the formation ofHelianthus tuberosus tubers the activities of arginine decarboxylase (ADC) and S-adenosylmethionine decarboxylase (SAMDC), examined in medullary parenchyma cells, increase with the increase in weight of the tuber. The ornithine decarboxylase (ODC) activity is about 100-fold less with respect to ADC activity, and it was detected only during the deceleration phase of the growth curve. Spermidine and spermine content are strictly related to the SAMDC activity and tuber growth. The increase of ADC and SAMDC activity is directly related to cell extension and increase in weight. The limited area of cell division in parenchyma tissue found during the first stage of tuber formation could justify the low ODC activity. The data suggest that ADC affects mainly growth processes, while ODC seems to be preferentially related to cell division.     

Effects of ethylene and auxin on polyamine levels in suspension-cultured tobacco cells

The effects of ethylene and auxin on polyamine levels were studied in suspension-cultured cells of tobacco ( Nicotiana tabacum . L). Treatment of 4-day-cultured cells with ethylene increased the levels of spermidine and spermine. The activities of arginine decarboxylase (ADC; EC 4.1.1.19), ornithine decarboxylase (ODC: EC 4.1.1.17), and S -adenosylmethionine decarboxylase (SAMDC: EC 4.1.1.50) rapidly increased between 3 and 12 h. An auxin, indole-3-acetic acid (IAA), increased polyamine levels and activities of ADC, ODC and SAMDC. The spermine level continued to increase significantly during a 24-h incubation with IAA. The increases in polyamine accumulation induced by ethylene were partially offset by an inhibitor of ethylene action, 2,5-norbornadiene. It is suggested that the auxin-induced polyamine accumulation occurred directly, without metabolic competition between ethylene and polyamine biosynthesis, and indirectly, through auxin-induced ethylene formation.     

Regulation of DNA synthesis and cell division by polyamines in Catharanthus roseus suspension cultures

Summary Various inhibitors of polyamine biosynthesis were used to study the role of polyamines in DNA synthesis and cell division in suspension cultures of Catharanthus roseus (L.) G. Don. Arginine decarboxylase (ADC; EC 4.1.1.19) was the major enzyme responsible for putrescine production. DL -difluoromethylarginine inhibited ADC activity, cellular putrescine content, DNA synthesis, and cell division. The effect was reversible by exogenous putrescine. Ornithine decarboxylase (ODC; EC 4.1.1.17) activity was always less than 10% of the ADC activity. Addition of DL -difluoromethylornithine had no effect on ODC activity, cellular polyamine levels, DNA synthesis, and cell division within the first 24 h but by 48 to 72 h it did inhibit these activities. Methylglyoxal bis(guanyl-hydrazone) inhibited S-adenosylmethionine decarboxylase (EC 4.1.1.50) activity without affecting DNA synthesis and cell division.Abbreviations ADC arginine decarboxylase - ODC ornithine decarboxylase - SAMDC S-adenosylmethionine decarboxylase - DFMA DL -difluoro-methylarginine - DFMO DL -difluoromethylornithine - MGBG methylglyoxal bis(guanylhydrazone)     

Polyamine Biosynthesis and Effect of Dicyclohexylamine during the Cell Cycle of Helianthus tuberosus Tuber

Polyamine content and the activities of their main biosynthetic enzymes, ornithine decarboxylase (ODC, EC 4.1.1.17), arginine decarboxylase (ADC, EC 4.1.1.19), S-adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.50), and arginase (EC 3.5.3.1.), were examined in crude extracts of Helianthus tuberosus tuber slices during the first synchronous cell cycle, induced by synthetic auxin, with or without the addition of 1 or 5 millimolar dicyclohexylamine (DCHA), an inhibitor of spermidine synthase. In the DCHA-treated slices a peak of accumulation of the drug was observed at 12 hours. Bound DCHA was also found. Free polyamine content generally increased, reaching a maximum at 12 to 18 hours in the S phase of the cycle; while spermidine content was decreased slightly with DCHA after 12 hours, putrescine almost doubled at 18 hours. Bound polyamines were also present. ODC and ADC showed a maximum activity at 15 and 18 to 21 hours, respectively, i.e. in the S phase; both activities increased slightly in the presence of 5 millimolar DCHA at or near the time of maximum activity. Arginase was initially very high and then rapidly decreased although a small peak of activity occurred at 15 hours. SAMDC, which had two peaks of activity, was initially inhibited by DCHA, and then stimulated, especially at 12 hours and in coincidence with the main peak, at 21 hours. Thus ODC, ADC, and SAMDC activities as well as polyamine titer increased before and during the S phase of the cell cycle and all declined during cell division. The slight inhibitory effect of DCHA was possibly due to its degradation in the tissue and to the fact that putrescine could substitute for the function(s) of spermidine.     

Polyamine metabolism and in vitro cell multiplication and differentiation in leaf explants of Chrysanthemum morifolium Ramat

Arginine decarboxylase (ADC), ornithine decarboxylase (ODC), diamine oxydase (DAO) free amine and conjugated amine titers were estimated in leaf explants of Chrysanthemum morifolium Ramat. var. Spinder cultivated in vitro in relation to hormone treatment. Addition of benzyladenine (BA) to a basal medium caused the formation of buds on the explants. BA plus 2,4 dichlorophenoxyacetic acid (2,4 D) caused callus formation and proliferation. Formation of roots was obtained by addition of indolylacetic acid (IAA). Arginine decarboxylase (ADC) ornithine decarboxylase (ODC) and diamine oxidase (DAO) activities increased during the first days of culture when cell multiplication was rapid, followed by a sharp decline as the rate of cell division decreased and differentiation took place. DAO activities increased rapidly in proliferating and growing organs and decreased during maturity. This increase was concomitant with ADC and ODC activities and polyamine content (free and conjugated polyamines). The biosynthesis and oxidation of polyamines which occurred simultaneously in physiological states of intense metabolism such as cell division or organ formation were directly correlated. In callus cultures DAO activity was blocked throughout development and regulated neither the cellular levels of polyamines nor polyamine conjugates. Levels of polyamine conjugates were high in callus cultures throughout development. In foliar explants cultivated on a medium promoting callus, inhibition of ODC activity by DFMO (-DL-difluoromethylornithine, a specific enzyme-activated ODC inhibitor) resulting in an amide deficiency facilated the expression of differentiated cell function; substantial activation of DAO was observed until the emergence of the buds. On a medium promoting bud formation, -OH ethylhydrazine (DAO inhibitor) promoted callus formation without differentiation. In this system DAO activity was blocked and there were high levels of polyamines, especially polyamine conjugates, throughout the culture period. The relationship among free and conjugated polyamines related biosynthetic enzyme activities, DAO activities, cell division and organ formation is discussed.Abbreviations ADC = arginine decarboxylase - ODC = ornithine decarboxylase - DOA = diamine oxidase - DFMA = -DL-difluoromethylarginine - DFMO = -DL-difluoromethylornithine - Put = putrescine     

Changes in cellular polyamine contents and activities of their biosynthetic enzymes at each phase of the cell cycle in by-2 cells

We analyzed changes in polyamine contents and the activities of biosynthetic enzymes during each phase of the cell cycle for a synchronized population of BY-2 cells. Based on our analysis of H³-thymidine incorporation flow cytometry, and the mitotic index, the M and G₂ phases seemed to occur at 8 h and from 2.5 to 8 h, respectively, after the release of aphidicolin. The respective activities of arginine decarboxylase (ADC), Ornithine decarboxylase (ODC), and S-adenosyl methionine decarboxylase (SAMDC) at the beginning (7.4, 11.2, and 5.5 nmol mg^-1 protein h^-1) were increased to 22.6, 22.1, and 15.1 nmol mg^-1 protein h^-1. However, those increases do not coincide with the general change in polyamines reported from animal cells. In addition, the bi-phasic activation of polyamine biosynthetic enzymes, such as those found in the general animal model, was observed with ADC and ODC but not with SAMDC. These results suggest that the general animal model for explaining polyamine changes and SAMDC activation in the cell cycle cannot be applied to BY-2 cells. Further, our flow-cytometric analysis of cell populations may be a useful tool for evaluating the effects of polyamines on cell cycle progression in BY-2 cells.     

Biosynthesis of spermidine, a direct precursor of pyrrolizidine alkaloids in root cultures of Senecio vulgaris L.

 The polyamine spermidine is an essential biosynthetic precursor of pyrrolizidine alkaloids. It provides its aminobutyl group which is transferred to putrescine yielding homospermidine, the specific building block of the necine base moiety of pyrrolizidine alkaloids. The enzymatic formation of spermidine was studied in relation to the unique role of this polyamine as an alkaloid precursor. S-adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.50) and spermidine synthase (SPDS, EC 2.5.1.16) from root cultures of Senecio vulgaris were partially purified and characterized. The SAMDC-catalyzed reaction showed a pH optimum of 7.5, that of SPDS an optimum of 7.7. The K _m value of SAMDC for its substrate S-adenosylmethionine (SAM) was 15 μM, while the apparent K _m values of SPDS for its substrates decarboxylated SAM (dSAM) and putrescine were 4 μM and 21 μM, respectively. The relative molecular masses of the two enzymes, determined by gel filtration, were 29 000 (SAMDC) and 37 000 (SPDS). Studies with various potential inhibitors revealed, for most inhibitors, profiles that were similar to those established with the respective enzymes from other plant sources. However, putrescine which is not known to be an inhibitor of plant SAMDC, strongly inhibited the enzyme from S. vulgaris roots. Spermidine synthase was sensitive to inhibition by its product spermidine. In the presence of the stationary tissue concentrations of the two polyamines (ca. 0.1 mM each) the activities of SAMDC and SPDS would be inhibited by >80%. The results are discussed in relation to the role of spermidine in primary and secondary metabolism of alkaloid-producing S. vulgaris root cultures. Received: 15 September 1999 / Accepted 10 December 1999     

Polyamine metabolism and physiological responses of <Emphasis Type="Italic">Potamogeton crispus</Emphasis> leaves under lead stress

Aquatic macrophytes were found to be the potential scavengers of heavy metals from aquatic environment. In this study, influences of ladder concentration of lead (Pb) on the leaves of Potamogeton crispus Linn were studied after 7 days of treatment. The accumulation of Pb, nutrient element contents, the generation rate of superoxide radical (O₂^·−), MDA, proline, and polyamine (PAs) contents, as well as the activities of diamine oxidases (DAO), polyamine oxidases (PAO), arginine decarboxylase (ADC), and ornithine decarboxylase (ODC) in P. crispus leaves were investigated. The result indicated that Pb treatment decreased the activity of DAO, whereas the proline content, MDA content, the generation rate of O₂^·− and the activity of ODC increased in different degrees. Meantime, Pb treatment significantly increased the free putrescine (Put) level and made other PAs levels dynamic changes. The activities of PAO and ADC were declined firstly and then enhanced with the increase in the Pb concentration.