UV-B radiation induces changes in polyamine metabolism in the red seaweed <Emphasis Type="Italic">Porphyra cinnamomea</Emphasis>

Early investigations on the productivity of intertidal seaweeds found that, unlike some seaweeds, members of the genus Porphyra, a Rhodophyte, could tolerate physical stressors such as ultraviolet-B radiation (UV-B) both during immersion and when exposed to air. Increased stress tolerance was thought to be due to an unknown mechanism that operated at the thylakoid level. As recent research has shown that polyamines (PAs), bound to the thylakoid membranes of chloroplasts, play a critical role in protecting the photosynthetic apparatus from high-light and UV damage in both higher plants and in unicellular algae, we investigated PA metabolism in Porphyra cinnamomea exposed to UV-B. Our results show that PA biosynthesis was significantly upregulated in P. cinnamomea in response to UV-B, with the greatest proportional increases being in bound soluble putrescine (PUT), which increased by over 200%, in bound soluble spermidine (SPD) and spermine (SPM) which both increased by more than 150% and in bound insoluble SPM which increased by more than 120%. As PAs can be synthesised from ornithine via ornithine decarboxylase (ODC) or from arginine via arginine decarboxylase (ADC) we investigated the pathway via which polyamines were synthesised in P. cinnamomea. While exposure to UV-B caused increases in the activities of both ADC and ODC, the increase in ADC activity was 10 fold greater than that of ODC, suggesting that the ADC pathway was the principle route by which PA levels increased in response to UV-B. Mechanisms of PA mediated UV-B protection are discussed.     

INFLUENCE OF POLYAMINES ON THE SPORULATION OF GRATELOUPIA (HALYMENIACEAE,RHODOPHYTA)1

Polyamines (PAs) such as putrescine (PUT), spermidine (SPD), and spermine (SPM) are ubiquitous aliphatic amines involved in widely varying physiological behavior, but particularly they are actively involved in cell growth, division, and differentiation during reproductive events in plants. The contents of PUT, SPD, and SPM in infertile and fertile thalli of the red macroalga Grateloupia sp. were compared, and the results revealed a significant decrease in quantity from infertile to fertile status. At the enzymatic level, l ‐ornithine decarboxylase (ODC) was mainly detected, and l ‐arginine decarboxylase activity was not diminished by the inhibition of ODC. The maximum enzymatic activities, within the range of activities observed, correlated with the lower levels of polyamines in fertile thalli. In culture, SPM promoted the maturation of cystocarps to the eventual liberation of spores from aseptic fertile explants. PAs accumulated in cultivated explants as compared with noncultivated, but exogenous SPM addition further increased the endogenous SPM. The addition of berenyl, cordycepin, cyclohexylamine, dicyclohexylamine, and aurintricarboxylic acid blocked the synthesis in culture at the level of PUT, and partially at SPD and SPM synthesis, but the addition of SPM restored the levels of SPD and SPM as SPM accumulated, and they appeared to interconvert each other. The results obtained suggest that the culture in presence of SPM restored a deficient SPM situation in fertile explants, thus promoting sporulation.     

Regulation of polyamine metabolism in Pyropia cinnamomea (W.A. Nelson), an important mechanism for reducing UV‐B‐induced oxidative damage

It is generally accepted that ultraviolet (UV) radiation can have adverse affects on phototrophic organisms, independent of ozone depletion. The red intertidal seaweed Pyropia cinnamomea W.A. Nelson (previously Porphyra cinnamomea Sutherland et al. 2011), similar to many other intertidal macrophytes, is exposed to high levels of UV radiation on a daily basis due to emersion in the upper littoral zone. It has been shown that seaweeds, like higher plants, respond to an increased activity of antioxidative enzymes when exposed to stress. However, earlier investigations have shown that P. cinnamomea also compensates for stress due to UV radiation by increasing polyamine (PA) levels, especially bound‐soluble and bound‐insoluble PAs. The PA precursor putrescine (PUT) can be synthesized via two enzymatic pathways: arginine decarboxylase (ADC) and ornithine decarboxylase (ODC). Both of these enzymes showed increased activity in P. cinnamomea under UV stress. In higher plants, ADC is the enzyme responsible for increased PA levels during stress exposure, while ODC is correlated with cell division and reproduction. However, there are contrary findings in the literature. Using two irreversible inhibitors, we identified the enzyme most likely responsible for increased PUT synthesis and therefore increased stress tolerance in P. cinnamomea. Our results show that changes in the PA synthesis pathway in P. cinnamomea under UV stress are based on an increased activity of ADC. When either inhibitor was added, lipid hydroperoxide levels increased even under photosynthetically active radiation, suggesting that PAs are involved in protection mechanisms under normal light conditions as well. We also show that under optimum or low‐stress conditions, ODC activity is correlated with PUT synthesis.     

Transglutaminase activity decrease during acclimation to hyposaline conditions in marine seaweed Grateloupia doryphora (Rhodophyta, Halymeniaceae)

Polyamines (PAs), such as diamine putrescine (PUT), triamine spermidine (SPD) and tetraamine spermine (SPM) have been related to environmental stress, including salt stress. A marine red macrophyte alga Grateloupia doryphora (Montagne) Howe was used to investigate the role of PAs during acclimation to moderate hyposaline conditions (incubation 24h in 18 psu seawater as compared to 36 psu of natural seawater). The results obtained showed that a moderate hyposaline shock caused an increase in the free fraction of PUT, SPD and SPM, mainly due to a decrease in TGase activity, together with an apparent increase in the l-arginine dependent PAs synthesis (ODC and arginase decreased, and ADC slightly increased). The photosynthetic rate increased in thalli when exposed to free SPD at 18 psu, but it did not increase at 36 psu.     

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.     

Spermidine,a sensor for antizyme 1 expression regulates intracellular polyamine homeostasis

Although intracellular polyamine levels are highly regulated, it is unclear whether intracellular putrescine (PUT), spermidine (SPD), or spermine (SPM) levels act as a sensor to regulate their synthesis or uptake. Polyamines have been shown to induce AZ1 expression through a unique +1 frameshifting mechanism. However, under physiological conditions which particular polyamine induces AZ1, and thereby ODC activity, is unknown due to their inter-conversion. In this study we demonstrate that SPD regulates AZ1 expression under physiological conditions in IEC-6 cells. PUT and SPD showed potent induction of AZ1 within 4 h in serum-starved confluent cells grown in DMEM (control) medium. Unlike control cells, PUT failed to induce AZ1 in cells grown in DFMO containing medium; however, SPD caused a robust AZ1 induction in these cells. SPM showed very little effect on AZ1 expression in both the control and polyamine-depleted cells. Only SPD induced AZ1 when S-adenosylmethionine decarboxylase (SAMDC) and/or ODC were inhibited. Surprisingly, addition of DENSpm along with DFMO restored AZ1 induction by putrescine in polyamine-depleted cells suggesting that the increased SSAT activity in response to DENSpm converted SPM to SPD, leading to the expression of AZ1. This study shows that intracellular SPD levels controls AZ1 synthesis.     

Polyamine metabolism during the growth cycle of tobacco BY-2 cells.

We studied polyamine (PA) biosynthesis, oxidation and conjugation in asynchronously dividing cells of tobacco BY-2 cell suspension culture (Nicotiana tabacum L.) during 7-day growth cycle. We analyzed the levels of free and conjugated PAs and the activities of biosynthetic and catabolic enzymes during the subculture interval. The contents of free spermidine and spermine started to increase after the inoculation into the fresh medium, positively correlated with the mitotic activity of BY-2 cells and reached their maxima at the beginning of exponential phase on day 3. On the contrary, the endogenous level of free Put showed a transient decline in the lag-phase, and then increased till the end of exponential phase (day 5). The time-course of the content of PCA-soluble conjugates showed a trend similar to that of the free PAs. The inoculation of BY-2 cells into the fresh medium resulted in a sharp increase in the activities of ornithine decarboxylase (ODC, EC 4.1.1.17) and S-adenosylmethionine decarboxylase (SAMDC; EC 4.1.1.50). Arginine decarboxylase (ADC; EC 4.1.1.19) activity remained low during the whole subculture interval. The rise of diamine oxidase (DAO; EC 1.4.3.6) in the first day after subculture coincided with the decrease in free Put level. De novo synthesis of PAs in BY-2 cells after inoculation into the fresh medium and the participation of both PA conjugation with hydroxycinnamic acids and Put oxidative degradation in maintaining of free PA levels during the growth cycle are discussed.     

Changes in Polyamine Biosynthesis Associated with Postfertilization Growth and Development in Tobacco Ovary Tissues

Polyamine (PA) titers and the activities of arginine decarboxylase (ADC, EC 4.1.1.19) and ornithine decarboxylase (ODC, EC 4.1.1.17), enzymes which catalyze rate-limiting steps in PA biosynthesis, were monitored during tobacco ovary maturation. In the period between anthesis and fertilization, the protein content of ovary tissues rapidly increased by about 40% and was accompanied by approximately a 3-fold increase in ODC activity, while ADC activity remained nearly constant. PA titers also remained relatively unchanged until fertilization, at which time they increased dramatically and the DNA content of ovary tissues doubled. This increase in PA biosynthesis was correlated with a further 3-fold increase in ODC activity, reaching a maximum 3 to 4 days after fertilization. During this time, ADC activity increased only slightly and accounted for approximately 1% of the total decarboxylase activity when ODC activity peaked. The postfertilization burst of biosynthetic activities slightly preceded a period of rapid ovary enlargement, presumably due to new cell division. During later stages of ovary development, DNA levels fell precipitously, while PA titers and decarboxylase activities decreased to preanthesis levels more slowly. In this period, growth producing a 300% increase in ovary fresh weight appears to be the result of cell enlargement.

Synchronous changes in PA titers and in the rates of PA biosynthesis, macromolecular synthesis, and growth in the tobacco ovary suggest that PAs may play a role in the regulation of postfertilization growth and development of this reproductive organ.

     

Effects of the suicide inhibitors of arginine and ornithine decarboxylase activities on organogenesis, growth, free polyamine and hydroxycinnamoyl putrescine levels in leaf explants of Nicotiana xanthi N.C. Cultivated in vitro in a medium producing callus formation

We studied the effects of dl-α-difluoromethylarginine (DFMA) and dl-α-difluoromethylornithine (DFMO), specific, irreversible inhibitors of arginine decarboxylase (ADC) and ornithine decarboxylase (ODC), respectively, on organogenesis growth and titers of free polyamines and conjugated putrescines (hydroxycinnamoyl putrescines) in tobacco (Nicotiana tabacum cv Xanthi n.c.) calli. These results suggest that ADC and ODC regulate putrescine biosynthesis during early and later stages of tobacco callus development, respectively. ADC appears active in biosynthesis of large levels of free amines (agmatine and putrescine) while ODC appears active only in biosynthesis of large levels of putrescine conjugates (hydroxycinnamoyl putrescines). DFMA inhibits the fresh and dry weight increases of tobacco calli, whereas DFMO even promoted the fresh and dry weight increases, thus supporting the view that ADC is important for cell division and callus induction. Inhibition of ODC activity by DFMO resulting in an amide deficiency after 4 weeks of culture facilates the expression of differentiated cell functions. Formation of buds is associated with a significant decrease of hydroxycinnamoyl putrescines.     

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 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.     

The effect of leaf galls of Cynipidae on accumulation and biosynthesis of plant amines in oak trees

Polyamines (PAs) are involved in plant response to abiotic and biotic stresses, however, their role in biochemical insect-plant interactions is not clear. Therefore, we compared the involvement of polyamines and key enzymes of their biosynthesis in gall formation process. The present study had used galls on oak leaves caused by asexual generation (♀♀) of three Cynipidae species, namely Cynips quercusfolii L., Neuroterus numismalis (Fourc.) and N. quercusbaccarum L., as a model. The obtained results indicate that gall formation on oak leaves affected amine content, but intensity of the changes in their levels were strongly dependent on the insect species. Nevertheless the downward trend was dominant among those changes. Changes in the activity of lysine decarboxylase (LDC), tyrosine decarboxylase (TyDC) and ornithine decarboxylase (ODC) usually corresponded with the direction of changes in polyamine contents. Several cases of divergence between changes in amine levels and the rate of their biosynthesis may suggest the involvement of other regulation mechanisms such as: arginine decarboxylase (ADC) and S-adenosylmethionine decarboxylase (SAMDC) as well as amine oxidases involved in its catabolic pathways. Thus, the future studies on biochemical mechanism of regulation of PAs accumulation during galls formation should be focused on importance of these enzymes.     

In vivo inhibition of polyamine biosynthesis and growth in tobacco ovary tissues

Post fertilization growth of tobacco ovary tissues treated with inhibitors of polyamine (PA) biosynthesis was examined in relation to endogenous PA titers and the activities of arginine decarboxylase (ADC, EC 4.1.1.19) and ornithine decarboxylase (ODC, EC 4.1.1.17). DL-alpha-Difluoromethylornithine (DFMO) and DL-alpha-difluoromethylarginine (DFMA), specific, irreversible ("suicide") inhibitors of ODC and ADC in vitro, were used to modulate PA biosynthesis in excised flowers. ODC represented >99% of the total decarboxylase activity in tobacco ovaries. In vivo inhibition of ODC with DFMO resulted in a significant decrease in PA titers, ovary fresh weight and protein content. Simultaneous inhibition of both decarboxylases by DFMO and DFMA produced only a marginally greater depression in growth and PA titers, indicating that ODC activity is rate-limiting for PA biosynthesis in these tissues. Paradoxically, DFMA alone inhibited PA biosynthesis, not as a result of a specific inhibition of ADC, but primarily through the inactivation of ODC. In vivo inhibition of ODC by DFMA appears to result from arginase-mediated hydrolysis of this inhibitor to urea and DFMO, the suicide substrate for ODC. Putrescine conjugates in tobacco appear to function as a storage form of this amine which, upon hydrolysis, may contribute to Put homeostasis during growth.     

Regulation of human ornithine decarboxylase expression following prolonged quiescence: Role for the c-Myc/Max protein complex

WI-38 cells can remain quiescent for long periods of time and still be induced to reenter the cell cycle by the addition of fresh serum. However, the longer these cells remain growth arrested, the more time they require to enter S phase. This prolongation of the prereplicative phase has been localized to a point early in G₁, after the induction of “immediate early” G₁ genes such as c-fos and c-jun but before maximal expression of “early” G₁ genes such as ornithine decarboxylase (ODC). Understanding the molecular basis for ODC mRNA induction can therefore provide information about the molecular events which regulate the progression of cells out of long-term quiescence into G₁ and subsequently into DNA synthesis. Studies utilizing electrophoretic mobility shift assays (EMSA) of nuclear extracts from short- and long-term quiescent WI-38 cells identified a region of the human ODC promoter at ?491 bp to ?474 bp which exhibited a protein binding pattern that correlated with the temporal pattern of ODC mRNA expression. The presence of a CACGTG element within this fragment, studies with antibodies against c-Myc and Max, the use of purified recombinant c-Myc protein in the mobility shift assay, and antisense studies suggest that these proteins can specifically bind this portion of the human ODC promoter in a manner consistent with growth-associated modulation of the expression of ODC and other early G₁ genes following prolonged quiescence. These studies suggest a role for the c-Myc/Max protein complex in regulating events involved in the progression of cells out of long-term quiescence into G₁ and subsequently into S. © 1995 Wiley-Liss, Inc.     

Participation of ornithine decarboxylase in early stages of tomato fruit development

The apparent association of ornithine decarboxylase (ODC) with rapid cell proliferation in developing tomato (Lycopersicon esculentum Mill. cv. Pearson ms-35) fruits has been previously described. Further evidence is provided by the use of two ODC inhibitors, α-difluoromethylornithine (α-DFMO) and α-methylornithine (α-MO). Fruit development was inhibited by these inhibitors if applied during the period of intensive cell division. When applied in vitro, the two inhibitors were shown to inhibit the activity of ODC but not that of arginine decarboxylase (ADC). When applied in vivo, α-DFMO, a catalytic irreversible inhibitor, caused 97.1% reduction of ODC activity in the dialyzed extract from the treated ovaries, while it had no effect on ADC. On the other hand, α-MO, a reversible inhibitor, did not reduce the activity of these two enzymes in the dialyzed extracts when applied in vivo. The dialysis procedure probably removed α-MO from the enzyme fraction. Putrescine, the product of both ODC and ADC, alleviated the inhibition of fruit development but did not restore ODC activity to the control level. These results suggest that in the young developing tomato fruit, ODC is the enzyme responsible for the synthesis of putrescine, which is essential for the early stages of fruit development. The reduced activity of ODC elicited by putrescine suggests a mechanism of feedback regulation by enzyme repression or release of an ODC anti-enzyme.     

Polyamine- and insulin-like growth factor-I-mediated proliferation of porcine uterine endometrial cells: a potential role for spermidine/spermine N(1)-acetyltransferase during peri-implantation

Insulin-like growth factor-I (IGF-I) and the polyamine catabolic enzyme spermidine/spermine N(1)-acetyltransferase (SSAT) are progesterone-regulated genes with maximal expression at peri-implantation in the porcine uterine endometrium. However, while IGF-I stimulates cell proliferation, SSAT, by acetylating the naturally occurring polyamines (PA) spermine (SPM) and spermidine (SPD), typically functions as a cell growth inhibitor. The present study examined the functional relationships of IGF-I, SSAT, and PA in the control of endometrial cell proliferation. Northern blot analysis indicated that SSAT mRNA levels change with distinct pregnancy stages, in contrast to those for the PA biosynthetic enzyme ornithine decarboxylase (ODC). Primary cultures of luminal and glandular epithelial (LE, GE) and stromal (ST) cells isolated from Day 12 pregnant pig endometrium had IGF-I mRNA levels for ST > LE > GE cells. The mRNA levels for SSAT and ODC were transiently diminished by IGF-I treatment, but only in GE cells. By contrast, SPM and SPD increased SSAT mRNA levels in GE and ST cells, but increased ODC mRNA levels only in GE cells. IGF-I, putrescine (PUT), and SPM individually increased cellular DNA synthesis as measured by tritiated thymidine incorporation in GE and ST cells, while SPD had an effect only in ST cells. IGF-I enhanced the proliferative effect of each PA in GE cells, but only of SPD in ST cells. The mitogen-activated protein kinase inhibitor, PD98059, inhibited the induction by SPM of GE cell DNA synthesis but not that of IGF-I. Wortmannin, a phosphatidylinositol-3-kinase inhibitor had no effect on either IGF-I or SPM induction of GE cell DNA synthesis. The relative concentrations of SPM, SPD, and PUT in uterine luminal fluids differed, with the levels for each PA higher at pregnancy Day 12 than at 11.5. These results suggest that IGF-I and PA act through distinct signaling pathways to mediate cell-type-specific growth of early pregnancy pig uterine endometrium. Further, SSAT, through its control of intracellular PA levels, likely plays a modulatory role in the establishment of an optimal uterine environment for successful embryo attachment.