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     

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.     

Immunoaffinity purification and characterization of diamine oxidase from Cicer

Antiserum specific for diamine oxidase (DAO;EC 1.4.3.6) from Lens culinaris cross-reacted with DAO from several other members of the Leguminosae when tested by agar double diffusion. Antibodies purified by affinity chromatography were used to make an immunoadsorbent for the one-step purification of DAO from various species of the Leguminosae. This technique has made it possible to purify in one step the already characterized DAO from pea and lentil, and the unknown diamine oxidase from Cicer arietinum. This enzyme was partially characterized; it showed a pH optimum of 7.5 with putrescine as substrate and followed typical Michaelis-Menten kinetics with a K_m of 2.4 × 10^?4 M. Copper ligands and carbonyl group-directed reagents inhibited the enzyme.     

NaCl-Induced Changes in Putrescine Content and Diamine Oxidase Activity in Roots of Rice Seedlings

The effects of NaCl on putrescine (Put) content and diamine oxidase (DAO) activity in roots of rice seedlings were examined. NaCl treatment lowered the content of Put and increased the activity of DAO in roots. Our current results indicate that Cl^– is not required for NaCl-induced decline in Put content and increase in DAO activity in roots. Put content in roots of rice seedlings exposed to NaCl is possibly regulated by DAO activity.     

Helianthus tuberosus and polyamine research: Past and recent applications of a classical growth model

The earliest studies concerning polyamines (PAs) in plants were performed by using in vitro cultured explants of Helianthus tuberosus dormant tuber. This parenchyma tissue was particularly useful due to its susceptibility to several growth substances, including PAs. During tuber dormancy, PA levels are too low to sustain cell division; thus Helianthus represents a natural PA-deficient model. When cultivated in vitro in the presence of auxins, Helianthus tuber dormant parenchyma cells at the G₀ stage start to divide synchronously acquiring meristematic characteristics. The requirement for auxins to induce cell division can be substituted by aliphatic PAs such as putrescine, spermidine or spermine. Cylinders or slices of explanted homogeneous tuber parenchyma were cultured in liquid medium for short-term studies on the cell cycle, or on solid agar medium for long-term experiments. Morphological and physiological modifications of synchronously dividing cells were studied during the different phases of the cell cycle in relation to PAs biosynthesis and oxidation. Long-term experiments led to the identification of the PAs as plant growth regulators, as the sole nitrogen source, as tuber storage substances and as essential factors for morphogenetic processes and cell homeostasis. More recently this system was used to study the effects on plant cell proliferation of platinum- or palladium-derived drugs (cisplatin and platinum or palladium bi-substituted spermine) that are used in human cancer cell lines as antiproliferative and cytotoxic agents. Cisplatin was the most active both in cell proliferation inhibition and on PA metabolism. Similar experiments were performed using three agmatine analogous. Different effects of these compounds were observed on cell proliferation, free PA levels and enzyme activities, leading to a hypothesis of a correlation between their chemical structure and the agmatine metabolism in plants.     

Characterization of gene expression during potato tuber development in individuals and populations using the luciferase reporter system

Analysis of gene expression and enzyme activity in pooled tuber samples has previously indicated different developmental events occurring in a fixed sequential order during tuber development, starting with the up-regulation of starch synthesis then induction of protein storage followed by cell division and cell enlargement. In this report we analysed in vivo promoter activity of genes related to cell division and storage of reserves during tuber development in individual in vitro tubers, using the non invasive firefly luciferase reporter system. The average activity of the storage related promoters (AGPaseS and Pat21) was up-regulated prior to visible swelling, while the average activity of both cell cycle genes (cycB1;1 and CDC2a) showed an up-regulation after the onset of swelling. However, this novel system allowed expression analysis in individual tubers, which showed a variable up-regulation of both storage genes in relation to the moment of swelling, from 4 days before to 10 days after the onset of swelling. We conclude that during the first stages of tuber development, the moment of storage gene induction is independent from swelling. These results indicate that the developmental program of potato tubers does not consist of a fixed sequential order of events, but consists of independent developmental programs (storage and swelling), together resulting in the formation of a potato tuber. It is concluded that analysis of developmental programs by studying individuals may result in new insights, possibly obscured when using pooled samples.     

Transglutaminase-like activity in Chrysanthemum leaf explants cultivated in vitro in relation to cell growth and hormone treatment

In Chrysanthemum leaf explants cultivated in vitro the capacity to covalently link polyamines to protein substances exists. This plant enzyme activity shows some similarities with mammalian transglutaminases. In foliar explants cultured on a medium promoting bud or root formation increasing levels of transglutaminase-like activity occurred during the first days of culture when cell multiplication was rapid then the levels declined as the rate of cell division decreased and differentiation occurred. Undifferentiated callus exhibited low transglutaminase-like activity. Transglutaminase-like activity also increased in rapidly proliferating and growing organs (roots and buds initiated from the foliar explants) and decreased during maturity. The relationship among transglutaminases-like activity, cell division, bud and root formation is discussed.Abbreviations TGase transglutaminase - BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - Put putrescine - Spd spermidine     

Polyamine catabolism in dormant embryos of the spindle tree (Euonymus europaeus L.) and in dormancy break obtained after treatment with gibberellic acid

Previously we showed that dormancy break of spindle tree embryos after gibberellic acid (GA₃) treatment was followed by an increase in arginine decarboxylase (ADC) activity (Béranger-Novat N. et al., Plant Sc. 102: 139–145, 1994). These results indicated that arginine decarboxylase pathway mediate hormone-induced growth responses in spindle tree embryos. In the present investigation we show that in GA₃-treated embryos diamine oxidase (DAO) increases immediately after putrescine content and the increase in DAO activity paralleles the accumulation of putrescine at the beginning of the culture (before the visible appearance of the radicle). In this system polyamine oxidase (PAO) increases immediately after DAO activity and follows closely the increase in spermidine content. These results demonstrate a direct correlation between the biosynthesis and oxidation of putrescine and spermidine. At every stage of development DAO and putrescine levels are lower than spermidine and PAO levels. Dormant embryos can be distinguished from GA₃-treated embryos by a complete lack of putrescine accumulation. In dormant embryos compared to GA₃-treated embryos DAO changed more or less in parallel and on the whole seemed to follow the same content and distribution, but the kinetics of the activation of DAOs were different in dormant embryos with a delay of 1.5 day for the first and 1 day for the second peak. During the first days of culture at least up to 4 days the distribution of spermidine and PAO in GA₃-treated embryos followed the same pattern observed in dormant embryos, but the levels of spermidine and PAO were greatly reduced in dormant embryos. On the other hand the kinetics of the activation of PAOs were different in dormant embryos with a delay of 1 day. The results suggest that dormant embryos are deficient in their ability to synthesize polyamines efficiently and support the view that spermidine catabolism (via PAO pool) is limiting in untreated embryos during the first days of culture.     

Polyamines and Root Formation in Mung Bean Hypocotyl Cuttings : II. Incorporation of Precursors into Polyamines

The incorporation of [¹⁴C]arginine and [¹⁴C]ornithine into various polyamines was studied in mung bean (Vigna radiata [L.] Wilczek) hypocotyl cuttings with respect to the effect of indole-3-butyric acid on adventitious root formation.

Both [¹⁴C]arginine and [¹⁴C]ornithine are rapidly incorporated into putrescine, spermidine, and spermine, with similar kinetics, during 5- to 24-hour incubation periods. The incorporation of arginine into putrescine is generally higher than that of ornithine. The biosynthesis of putrescine and spermidine from the precursors, in the hypocotyls, is closely related to the pattern of root formation: a first peak at 0 to 24 hours corresponding to the period of root primordia development, and a second peak of putrescine biosynthesis at 48 to 72 hours corresponding to root growth and elongation. Indole-3-butyric acid considerably enhances putrescine biosynthesis in both phases, resulting in an increase of the putrescine/spermidine ratio.

It is concluded that the promotive effect of indole-3-butyric acid on putrescine biosynthesis, from both arginine and ornithine, supports the hypothesis that auxin-induced root formation may require the promotion of polyamine biosynthesis.

     

Effects of testosterone and 17, β– estradiol on the polyamine metabolism in cultivated normal rat kidney epithelial cells

Summary. Ornithine decarboxylase (ODC) and diamine oxidase (DAO) are important enzymes involved in the metabolism of polyamines (putrescine, spermidine and spermine). The influence of testosterone (T) and 17, β– estradiol (E₂) on the activity of ODC and DAO was examined in cultivated normal rat kidney (NRK) epithelial cells. The results showed an increase in enzyme activities 4 hours or 12 hours after hormonal treatment. Both T and E₂ led to a significant increase (1.6-fold) in ODC protein level as compared to the controls. Cellular concentration of spermidine and spermine increased (2.2- and 2.6-fold respectively) 4 hours after T addition. A higher levels in concentrations of putrescine (1.4-fold) and spermine (1.5-fold) 12 hours after E₂ treatment were observed. These results suggest that the biosynthesis and terminal oxidation of the polyamines in NRK epithelial cells are androgen- and estrogen-mediated and depend on the hormonal sensitivity of the cells. Received April 5, 1999, Accepted December 20, 1999     

Polyamines and cytokinins in celery embryogenic cell cultures

The effect of inhibitors of polyamine biosynthesis on the development of embryogenic cell cultures of celery (Apium graveolus L.) was studied. Several developmental stages of somatic embryos were compared for differences in the content and biosynthesis of free polyamines and for cytokinin content. Cyclohexylamine and particularly methylglyoxal bis(guanylhydrazone), inhibited both cell division and the organization of polar embryos from globular embryos. Difluoromethylornithine slightly promoted embryo development, especially cell division.The free putrescine content of globular embryos was 6-fold that of fully differentiated plantlets, and that of spermidine 2-fold. Only a slight increase in the spermine content was found with embryo development. These differences were confirmed by data from polyamine biosynthesis. Incorporation of ¹⁴C-arginine into polyamines was slightly higher than that of ¹⁴C-ornithine. Over 96% of this incorporation was detected in the putrescine fraction. Incorporation of ¹⁴C into putrescine in globular embryos was 3 to 4-fold that in fully-differentiated plantlets. Incorporation into spermidine and spermine was, however, higher in plantlets than in globular embryos.Cytokinin analysis revealed considerable differences in the biological activity between the developmental stages of embryogenesis. This could be due to endogenous cytokinins and/or BA taken up from the maintenance medium. Cytokinin levels decreased with increased embryo development. Most of the detected cytokinin-like activity co-chromatographed with BA and its metabolites. Some as yet unidentified peaks of activity were recorded in the globular embryos.The results are considered with respect to the possible participation of polyamines and cytokinins in the development of embryogenic cell cultures of celery. It is suggested that the onset of embryogenesis is characterized by a high content of putrescine and cytokinins, while a decrease in putrescine synthesis and cytokinin content, and an increase in spermidine and spermine content, accompany further embryo development and plantlet formation.Abbreviation ADC arginine decarboxylase - ODC ornithine decarboxylase - 2,4-D dichlorophenoxyacetic acid - DFMA difluoromethylarginine - DFMO difluoromethylornithine - MGBG methylglyoxal bis(guanylhydrazone) - CHA cyclohexylamine - BA benzyladenine - BAR benzyladenine riboside     

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)     

Contribution of putrescine degradation to proline accumulation in soybean leaves under salinity

Proline accumulation was studied in the leaves of Glycine max (L.) Merr. subjected to salt stress in the presence of aminoguanidine (AG, a specific inhibitor of diamine oxidase, DAO) and exogenous putrescine (Put). Both DAO activity and proline content were increased while endogenous Put content was decreased in soybean leaves under 50 to 150 mM NaCl. There was a negative correlation between proline accumulation and endogenous Put content. The addition of AG during NaCl stress inhibited DAO activity, caused Put accumulation and a 15 to 20 % decrease in proline content. Application of 1 mM Put to NaCl solution markedly increased proline content. The promotive effect of Put application could be alleviated by the treatment with Put plus AG. Moreover an application of AG had no effect on proline accumulation in soybean seedlings grown under normal condition. These results indicate that the quantitative contribution of Put degradation to proline formation is 15 to 20 %.     

Distribution of polyamines and their related catabolic enzyme in etiolated and light-grown leguminosae seedlings

Diamine-oxidase (DAO; EC 1.4.3.6) activity and di-and polyamine levels were estimated along the epicotyl and root of light-grown and etiolated lentil (Lens culinaris Medicus) and pea (Pisum sativum L.) seedlings. The activity of DAO was higher in etiolated epicotyls than in lightgrown ones. In both species there was a positive correlation between DAO activity and the diamine (putrescine and cadaverine) levels along the whole epicotyl and root. Polyamine (spermine and spermidine) distribution seemed to be associated with the meristematic and elongating zone of the epicotyl and root. The physiological function of DAO is discussed in relation to its possible role in providing hydrogen peroxide to peroxidase-dependent reactions occurring in the cell wall.Abbreviations CAD cadaverine - DA diamine - DAO diamine oxidase - PA polyamine - PUT putrescine - SPD spermidine - SPM spermine     

Physiological control of arginine decarboxylase activity in k-deficient oat shoots

The effect of K-deficiency on the putrescine biosynthetic enzyme, arginine decarboxylase (ADC), was investigated by growing oat (Avena sativa L. var Victory) plants on a low-K, but otherwise complete nutrient medium in washed quartz sand for up to 18 days. Enzyme activity rose as the concentration of KCl was dropped to 0.6 millimolar or below. However, growth was not inhibited significantly at 0.6 millimolar KCl. ADC activity increased in the whole shoot of K-deficient oats throughout the period of 6 to 18 days, but remained constant in normal plants. At 18 days, ADC activity in entire K-deficient shoots was 6 times greater than in normal shoots, while in the first (oldest) leaf, ADC specific activity increased to more than 30 times the specific activity in the first leaf of normal plants. This effect was due to a moderate rise in total ADC activity in the first leaf between 6 and 18 days, accompanied by a significant decline in protein content. Replacing K⁺ with Na⁺ or Li⁺ significantly inhibited the increase in ADC activity in K-deficient oats, while Rb⁺ depressed the specific activity to a level below that in normal plants. An alternative putrescine biosynthetic enzyme, ornithine decarboxylase, was also examined. The specific activity of a pelletable form of the enzyme was increased 2-fold in the shoots of K-deficient oats.     

Polyamines and nucleic acids during the first cell cycle of Helianthus tuberosus tissue after the dormancy break

Polyamines (spermine, spermidine, and putrescine) and nucleic acids were studied during the first cell cycle after the break of dormancy of tuber slices of Helianthus tuberosus L., cv. OB1. Immediately after the break of dormancy, a marked decrease in stored arginine and glutamine and a corresponding increase of polyamines were observed. This first synthesis of polyamines were observed. This firs synthesis of polyamines occurred very early during the G₁ phase, concomitant to the synthesis of RNAs. A RNA, probably messenger-like RNA, was synthesized very actively only during the first hours of activation in the culture medium plus 2,4-dichlorophenoxyacetic acid, or in water. At the onset of the S phase, after 12h of activation, an incorporation of [³H] thymidine was also detected. A second putrescine synthesis and polyamine accumulation began during the progression of the S phase. During the progression of mitosis, there was a decrease of polyamine synthesis and accumulation.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - GA₇ Gibberellin A₇ - MAK methylated albumin column     

Transglutaminase-like activity in Chrysanthemum leaf explants cultivated in vitro in relation to cell growth and hormone treatment

In Chrysanthemum leaf explants cultivated in vitro the capacity to covalently link polyamines to protein substances exists. This plant enzyme activity shows some similarities with mammalian transglutaminases. In foliar explants cultured on a medium promoting bud or root formation increasing levels of transglutaminase-like activity occurred during the first days of culture when cell multiplication was rapid then the levels declined as the rate of cell division decreased and differentiation occurred. Undifferentiated callus exhibited low transglutaminase-like activity. Transglutaminase-like activity also increased in rapidly proliferating and growing organs (roots and buds initiated from the foliar explants) and decreased during maturity. The relationship among transglutaminases-like activity, cell division, bud and root formation is discussed.     

Association of diamine oxidase and <Emphasis Type="Italic">S</Emphasis>-adenosylhomocysteine hydrolase in <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> extracts

The oxidative deamination of methylated putrescine by a diamine oxidase activity (DAO) is an important step in the biosynthesis of nicotine in tobacco and tropane alkaloids in several Solanaceous plants. A polyclonal rabbit antiserum was previously developed to a purported purified DAO enzyme from Nicotiana tabacum. The antiserum bound to a single 53 kDa protein and immunoprecipitated 80 of DAO activity from tobacco root extracts. In an effort to obtain DAO cDNAs, this antiserum was used to screen a tobacco cDNA expression library and three distinct immunoreactive cDNA clones were isolated. These cDNAs encoded predicted proteins that were either identical or nearly identical to predicted S-adenosylhomocysteine hydrolase (SAHH) from two Nicotiana species. Thus, the rabbit antiserum was not specific to DAO, even though it immunodepleted the majority of DAO activity from root extracts. Alternative hypotheses to explain the DAO immunodepletion results (such as poisoning of DAO activity or that SAHH is a bifunctional enzyme) were tested and ruled out. Therefore, we hypothesize that SAHH associates with DAO as part of a larger multienzyme complex that may function in planta as a nicotine metabolic channel.     

Potato tuber cytokinin oxidase/dehydrogenase genes: Biochemical properties,activity, and expression during tuber dormancy progression

The enzymatic and biochemical properties of the proteins encoded by five potato cytokinin oxidase/dehydrogenase (CKX)-like genes functionally expressed in yeast and the effects of tuber dormancy progression on StCKX expression and cytokinin metabolism were examined in lateral buds isolated from field-grown tubers. All five putative StCKX genes encoded proteins with in vitro CKX activity. All five enzymes were maximally active at neutral to slightly alkaline pH with 2,6-dichloro-indophenol as the electron acceptor. In silico analyses indicated that four proteins were likely secreted. Substrate dependence of two of the most active enzymes varied; one exhibiting greater activity with isopentenyl-type cytokinins while the other was maximally active with cis-zeatin as a substrate. [³H]-isopentenyl-adenosine was readily metabolized by excised tuber buds to adenine/adenosine demonstrating that CKX was active in planta. There was no change in apparent in planta CKX activity during either natural or chemically forced dormancy progression. Similarly although expression of individual StCKX genes varied modestly during tuber dormancy, there was no clear correlation between StCKX gene expression and tuber dormancy status. Thus although CKX gene expression and enzyme activity are present in potato tuber buds throughout dormancy, they do not appear to play a significant role in the regulation of cytokinin content during tuber dormancy progression.