Bis(guanylhydrazones) negatively affect in vitro germination of kiwifruit pollen and alter the endogenous polyamine pool

Bis(guanylhydrazones) are a class of compounds known to interfere with the metabolism of polyamines (PAs). Among them, the methylglyoxal derivative (MGBG) has been studied most thoroughly. Because PAs and their biosynthetic enzymes are strongly involved in pollen tube organization, emergence and elongation, a number of these inhibitors have been studied in the present work for their effects on the in vitro performance of kiwifruit (Actinidia deliciosa) pollen. Increasing concentrations of several bis(guanylhydrazones) in the range 0.05-1 mM were checked for their effect on pollen germination. Most of the compounds tested showed a dose-dependent inhibitory effect on tube emergence, which was established very early during incubation. At 0.5 mM, the methylpropylglyoxal derivative (MPGBG) had a stronger inhibitory effect than MGBG. To verify whether the inhibitors reached their metabolic target, PA levels and S-adenosylmethionine decarboxylase (SAMDC) activity were determined in pollen germinated in the presence or absence (controls) of 0.5 mM bis(guanylhydrazones). Spermidine (Spd) content was significantly reduced in the treated pollen, and this effect was more pronounced after treatment with MGBG than with MPGBG. An early and strong reduction in SAMDC activity was observed after exposure to either inhibitor. Inhibition of pollen germination by MGBG or MPGBG could not be reversed by the addition of exogenous Spd, which per se was inhibitory. Taken together, our results suggest that bis(guanylhydrazones) alter PA metabolism and negatively affect kiwifruit pollen germination, even though a strict cause-effect relationship could not be established, and other mechanisms, unrelated to PA activity, must be involved.     

Involvement of polyamines in the post-anthesis development of inferior and superior spikelets in rice

Early-flowered superior spikelets usually exhibit a faster grain filling rate and heavier grain weight than late-flowered inferior spikelets in rice (Oryza sativa L.). But the intrinsic factors responsible for the variations between the two types of spikelets are unclear. This study investigated whether and how polyamines (PAs) are involved in regulating post-anthesis development of rice spikelets. Six rice genotypes differing in grain filling rate were field grown, and PA levels and activities of the enzymes involved in PA biosynthesis were measured in both superior and inferior spikelets. The results showed that superior spikelets exhibited higher levels of free spermidine (Spd) and free spermine (Spm) and higher activities of arginine decarboxylase (ADC, EC 4.1.1.19), S-adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.50) and Spd synthase (EC 2.5.1.16) than inferior spikelets at the early endosperm cell division and grain filling stage. The maximum concentrations of free Spd and free Spm and the maximum activities of ADC, SAMDC and Spd synthase were significantly correlated with the maximum cell division and grain filling rates, maximum cell number and grain weight. Application of Spd and Spm to panicles resulted in significantly higher rates of endosperm cell division and grain filling in inferior spikelets along with the activities of sucrose synthase (EC 2.4.1.13), ADP glucose pyrophosphorylase (EC 2.7.7.27) and soluble starch synthase (EC 2.4.1.21), suggesting that these PAs are involved in the sucrose-starch metabolic pathway. The results indicate that the poor development of inferior spikelets is attributed, at least partly, to the low PA level and its low biosynthetic activity.     

Effect of exogenous spermidine on polyamine metabolism in water hyacinth leaves under mercury stress

The influence of exogenous spermidine (Spd) on arginine decarboxylase (ADC), ornithine decarboxylase (ODC), polyamine oxidase (PAO) activities and polyamines (PAs), proline contents in water hyacinth leaves under Mercury (Hg) stress was investigated after 6 days treatment. The results showed that free putrescine (Put) content increased, the contents of free spermidine (Spd) and spermine (Spm) and the (Spd + Spm)/Put ratio in water hyacinth leaves decreased significantly with the increase of the Hg concentrations. Hg stress also disturbed the activities of ADC, ODC and PAO and caused changes on proline content. Compared to the Hg-treatment only, exogenous Spd (0.1 mM) significantly reduced the accumulation of free Put, increased the contents of free Spd and Spm and the ratio of (Spd + Spm)/Put in water hyacinth leaves. Furthermore, exogenous Spd enhanced the activities of ADC, ODC and PAO and significantly increased proline content. The PS-conjugated PAs and PIS-bound PAs changed in the same trend as free PAs. These results suggest that exogenous Spd can alleviate the metabolic disturbance of polyamines caused by Hg in water hyacinth leaves.     

Loss of viability of tomato pollen during long-term dry storage is associated with reduced capacity for translating polyamine biosynthetic enzyme genes after rehydration

The possibility that a loss of pollen viability during dry storage in a freezer is caused by the reduced pollen capacity to enhance polyamine biosynthetic enzyme activity after rehydration was investigated using pollen grains of tomato (Solanum lycopersicum=Lycopersicon esculentum) stored at -30 degrees C under dry conditions for up to 42 months. Pollen grains showed normal germinability for at least 12 months in storage, but those stored for longer than 24 months exhibited a significant reduction in germinability and fruit-setting ability. This age-dependent reduction in pollen viability coincided with the extent to which the pollen lost the capacity to increase arginine decarboxylase (ADC) and S-adenosylmethionine decarboxylase (SAMDC) activities and polyamine contents upon rehydration. Immunoblot analysis indicated that the capacity of pollen to translate ADC and SAMDC mRNAs was impaired in accordance with the loss of viability. Also, the capacity to synthesize proteins in general decreased with the increase in storage duration. The addition of 1 mM putrescine, spermidine, or spermine to incubation medium promoted germination, impregnation of pollen grains with 1 mM spermidine restored fertilization ability, and the addition of 1 mM spermidine to incubation medium promoted protein synthesis exclusively in pollen grains which had been stored for a long time. These results indicate that the reduction in viability of tomato pollen during long-term dry storage in a freezer involves a decline in the capacity to enhance gene translation for polyamine biosynthetic enzymes upon rehydration.     

Involvement of polyamines in the drought resistance of rice

This study investigated whether and how polyamines (PAs) in rice (Oryza sativa L.) plants are involved in drought resistance. Six rice cultivars differing in drought resistance were used and subjected to well-watered and water-stressed treatments during their reproductive period. The activities of arginine decarboxylase, S-adenosyl-L-methionine decarboxylase, and spermidine (Spd) synthase in the leaves were significantly enhanced by water stress, in good agreement with the increase in putrescine (Put), Spd, and spermine (Spm) contents there. The increased contents of free Spd, free Spm, and insoluble-conjugated Put under water stress were significantly correlated with the yield maintenance ratio (the ratio of grain yield under water-stressed conditions to grain yield under well-watered conditions) of the cultivars. Free Put at an early stage of water stress positively, whereas at a later stage negatively, correlated with the yield maintenance ratio. No significant differences were observed in soluble-conjugated PAs and insoluble-conjugated Spd and Spm among the cultivars. Free PAs showed significant accumulation when leaf water potentials reached -0.51 MPa to -0.62 MPa for the drought-resistant cultivars and -0.70 MPa to -0.84 MPa for the drought-susceptible ones. The results suggest that rice has a large capacity to enhance PA biosynthesis in leaves in response to water stress. The role of PAs in plant defence to water stress varies with PA forms and stress stages. In adapting to drought it would be good for rice to have the physiological traits of higher levels of free Spd/free Spm and insoluble-conjugated Put, as well as early accumulation of free PAs, under water stress.     

Polyamine metabolism of potato seed-tubers during long-term storage and early sprout development

Growth potential of potato (Solanum tuberosum L.) plants is influenced by seed-tuber age. After 24 days of growth, single-eye seedcores from 7-month-old seed-tubers produced 64% more foliar dry matter than those from 19-month-old seed-tubers, reflecting a higher growth rate. This study was initiated to determine if differences in polyamine (PA) metabolism are associated with aging and age-reduced vigor of potato seed-tubers. As tubers aged in storage, putrescine (Put) increased 2.2-fold, while spermidine (Spd) and spermine (Spm) decreased 33% and 38%, respectively. Ethylene content of the tuber tissue also increased with advancing age, suggesting that during the aging process S-adenosylmethionine was directed toward ethylene biosynthesis at the expense of the PAs. Single-eye cores from 7- and 19-month-old tubers were sown and PA levels in core and shoot tissues were monitored during plant development. Put titer of younger cores increased 8.8-fold by 12 days. In contrast, the increase in Put over the initial titer in older cores was 2.9-fold. The reduced ability of older cores to synthesize Put during plant establishment is probably due to a 45% decline in ornithine decarboxylase activity between 12 and 16 days after planting. Lack of available Put substrate limited the biosynthesis of Spd and Spm, and thus their concentrations remained lower in older cores than in younger cores. Lower PA titer in older cores during plant establishment is thus coincident with reduced growth potential. Concentrations of Put and Spd were higher in shoots developing from older cores throughout the study, but there was no age-related difference in Spm content. In contrast, activities of arginine and S-adenosylmethionine decarboxylases were higher in shoots from younger cores during establishment. The results indicate that aging affects PA metabolism in both tuber and developing plant tissues, and this may relate to loss of growth potential with advancing seed-tuber age.     

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.     

Effect of polyamine biosynthetic inhibitors on alkaloids and organogenesis in tobacco callus cultures

We studied the effects of inhibitors of ornithine decarboxylase (ODC), arginine decarboxylase (ADC) and spermidine synthase (Spd synthase) on organogenesis and the titers of polyamines (PA) and alkaloids in tobacco calli. DL--difluoromethylarginine (DFMA) and D-arginine (D-Arg), both inhibitors of ADC activity, were more effective than DL--difluoromethylornithine (DFMO), an inhibitor of ODC, in reducing titers of PA and the putrescine (Put)-derived alkaloids (nornicotine and nicotine). Dicyclohexylammonium sulfate (DCHA), an inhibitor of Spd synthase, was also more efficient than DFMO in reducing PA and alkaloid levels. Root organogenesis is inversely related to the titers of Put and alkaloids. Thus, DFMA and D-Arg, which strongly inhibit Put and alkaloid biosynthesis, markedly promote root organogenesis, while control callus with high Put and alkaloid content showed poor root organization. These results suggest that morphological differentiation is not required for activation of secondary metabolic pathways and support the view that ADC has a major role in the generation of Put going to the pyrrolidine ring of tobacco alkaloids.     

Spermidine biosynthesis as affected by osmotic stress in oat leaves

A new assay for the evaluation of spermidine (Spd) synthase activity was developed. It involves a coupled reaction and avoids the use of decarboxylated S-adenosylmethionine, which is unstable and not easily available. This assay was applied to assess changes in enzyme activity in oat leaves subjected to osmotic stress in the dark. The results indicate that osmotically-induced putrescine (Put) accumulation in cereals results not only from the activation of the arginine decarboxylase pathway, but also from the inhibition of the activity of Spd synthase, the enzyme which catalyzes the transformation of Put to Spd. Other possibilities which could contribute to the decline of Spd and spermine levels under osmotic stress are also discussed.Abbreviations ADC arginine decarboxylase - Dap diaminopropane - DFMA -difluoromethylarginine - MGBG methylglyoxal-bis-guanylhydrazone - MTA 5-deoxy-5-methylthioadenosine - ODC ornithine decarboxylase - PA polyamines - PAO polyamine oxidase - PCA perchloric acid - PLP pyridoxal phosphate - Put putrescine - SAM S-adenosylmethionine - dSAM decarboxylated S-adenosylmethionine - SAMDC S-adenosylmethionine decarboxylase - Spd spermidine - Spm spermine     

水稻细胞质雄性不育系及其保持系幼穗发育过程中的多胺代谢

在幼穗发育过程中,不育系和保持系幼穗多胺含量先剧降后稳定或略回升,精氨酸脱羧酶活性快速下降,而二胶和多胺氧化酶活性缓慢下降。从雌雄蕊形成期到花粉母细胞形成期,不育系的多胺含量和精氨酸脱羧酶活性明显低于保持系;不过,两系二胺氧化酶和多胺氧化酶活性却差别不大。外施D-Arg抑制两系Put合成,也抑制以Put为前体的Spd的合成;外施MGBG抑制Spd和Spm的合成;同时,D-Arg或MGBG对不育系花粉育性影响不大,但明显降低保持系花粉育性,D-Arg＋MGBG对花粉育性的降低效应更强;Put和pd＋Spm可抵消（或部分抵消）D-Arg和MGBG的降低效应。且Put＋Spd＋Spm能使不育系花粉的育性得以轻度恢复。     

Effect of end-of-day irradiations on polyamine accumulation in petal cultures of Araujia sericifera

We have studied photoperiodic control and the effect of phytochrome photoconversion at the end-of-day (EOD) on polyamine (PA) accumulation in petal explants of Araujia sericifera . Petals from immature flowers were cultured under long (LD) and short (SD) days. Light was provided by Gro-lux fluorescent lamps (90–100 µmol m⁻² s⁻¹). Red (R), far red (FR), red followed by far-red (R-FR) and far-red followed by red (FR-R) light treatments were applied daily at the end of the photoperiod. The free and bound putrescine (Put), spermidine (Spd) and spermine (Spm) fractions in petal explants were determined 40 days after the beginning of the culture. We also aimed to clarify the involvement of PA changes by using two inhibitors of PA biosynthesis: D- l -α-difluoromethylarginine (DFMA) and methylglyoxal bis (guanylhydrazone) (MGBG). We found PA accumulation to be under photoperiodic control, and the inhibitory effect of DFMA on this accumulation suggests that arginine decarboxylase (ADC) is the major pathway for Put biosynthesis. Polyamine levels were higher under LD, mainly as a result of the accumulation of free and bound Put. FR-EOD treatment, which dramatically reduced the R : FR ratio after LD, increased the accumulation of PA, mainly as free Put and free and bound Spd. Sequential R-FR and FR-R-EOD treatments strongly increased bound Spd. The concentration of MGBG used increased total PA accumulation, mainly as Put. However, all EOD light treatments dramatically reduced Put accumulation in the presence of MGBG. This may be due to a dual role of FR light in PA accumulation: (1) FR per se stimulates PA production, probably via ADC, and (2) in the presence of MGBG, FR inhibits Put accumulation, probably via ethylene production.     

Polyamines as biomarkers for plant regeneration capacity: improvement of regeneration by modulation of polyamine metabolism in different genotypes of indica rice

The importance of cellular polyamine (PA) levels and the ratio of putrescine (Put) to spermidine (Spd) for plant regeneration ability via somatic embryogenesis in several commercially grown indica rice varieties is reported here. The genotypes namely NDR-624, IR-20, IR-36, BJ-1 (having Put:Spd ratio2.3) showed superior plant regeneration while KL, PB-1 and TN-1 (having Put:Spd ratio3.8) showed moderate plant regeneration ability. The genotypes namely HS, Bindli, DV-85, ACB-72, IR-64 and IR-72 (having Put:Spd ratio5.0) showed poor plant regeneration ability. In contrast KH-7 (Put:Spd ratio10.0) showed no response at all. Favorable modification of cellular PA titers and their Put:Spd ratio by the addition of exogenous PAs (Put, Spd) or their biosynthesis inhibitor, difluoromethylarginine (DFMA) led to the induction/promotion of plant regeneration in poorly responding genotypes. These results showed a close relationship between cellular PA levels and their Put:Spd ratio with in vitro morphogenetic capacity in indica rice and suggest that the cellular PAs and Put:Spd ratios are important determinants (biomarkers) of plant regeneration ability in indica rice, and the improvement/induction of plant regeneration in morphogenetically poor and recalcitrant species could be achieved by modulating PA metabolism.     

Effect of salt and osmotic stress on changes in polyamine content and arginine decarboxylase activity in Lupinus luteus seedlings

The effects of NaCl (260 mM) and sorbitol (360 mM) isoosmotic stresses on polyamine titers in lupin (Lupinus luteus L. var. Ventus) in relation to organ-specific responses were investigated. Analysis showed that during the first few hours (4 h) of salt and osmotic stress higher amounts of putrescine (Put) and spermidine (Spd) were accumulated in the roots and leaves of lupin seedlings. After exposing the plants to a longer duration (24 h) of exposure to NaCl, the level of free Put decreased in roots and cotyledons by about 48% and 54%, respectively, and increased in hypocotyls and leaves by about 27% and 73%, respectively. The Level of free Spd also decreased in roots by about 50%, in contrast to the increase of Spd observed in hypocotyls and leaves by about 50% and 70%, respectively. The effect of non-ionic stress on the level of Put and Spd in studied organs of lupin was similar to that of NaCl. Free spermine was at an undetectable level in examined organs. However, in the roots of lupin growing for 24 h in the presence of NaCl and/or sorbitol, the activity of arginine decarboxylase (ADC) (EC 4.1.1.19) increased by about 66% and 80%, respectively. ADC activity in leaves was similar to that observed in the control. Additionally, in the roots and leaves of lupin growing under the stress condition (NaCl or sorbitol), a higher level of polyamines (PAs) bound to microsomal membranes was observed. It is probable that PAs bound to microsomal membranes prevent stress-induced damage. We conclude that both stresses induce biosynthesis of Put and other PAs in the roots, as well as Put accumulation in the leaves, and this may indicate translocation of Put from the roots to the shoot. The possible role of PAs in adaptive mechanisms to stress is discussed.     

Modulation of SAMDC expression in Arabidopsis thaliana alters in vitro shoot organogenesis

S -Adenosylmethionine decarboxylase (SAMDC, EC 4.1.4.50) is a rate-limiting enzyme in the biosynthesis of polyamines (PAs) from putrescine. To gain more insight into the role of PAs in shoot organogenesis, a reverse genetic approach has been used to study in vitro shoot organogenesis by manipulating SAMDC expression in Arabidopsis . Up- and downregulation of SAMDC expression was achieved by transferring sense, antisense and double-stranded Arabidopsis SAMDC complementary DNA constructs back into Arabidopsis via Agrobacterium tumefaciens . Results show that the biosynthesis of PAs and ethylene is mutually antagonistic by manipulation of SAMDC expression. Further results demonstrate that increased shoot organogenesis seems to be directly related to PA accumulation. This effect of PA may be further enhanced with reduced ethylene. It also suggests that spermidine is involved in the process of acquiring organogenesis competence through downregulation of ethylene production and shoot organogenesis, which might result from the concerted action of PAs and plant hormones such as auxin, cytokinins and ethylene.     

Involvement of polyamines in the control of fruitlet physiological abscission in grapevine (Vitis vinifera)

The potential contribution of polyamines (PAs) in the regulation of physiologically induced fruitlet abscission was investigated in cuttings from two cultivars of Vitis vinifera L., Pinot noir (PN) and Merlot (MRT). Abscission was higher in MRT than in PN and was preceded by a decrease in free PA levels. This decline was more pronounced in inflorescences than in leaves of the sensitive cultivar. Soluble conjugated PA showed an opposite trend in both cultivars. This suggests a cause-effect relationship between free and/or conjugated PA levels in floral organs and susceptibility to abscission. Spermidine (Spd), but not putrescine (Put) or diaminopropane, supplied at 0.5–1 m M to the nutritive medium prior to the anthesis, increased free and conjugated PA levels in the inflorescences and markedly inhibited abscission. α -Difluoromethylarginine, an inhibitor of arginine decarboxylase, but not α -difluoromethylornithine, an inhibitor of ornithine decarboxylase, lowered PA levels and increased abscission. Treatment with cyclohexylamine or β -hydroxyethylhydrazine as potent inhibitors of Spd synthase and PA oxidases, respectively, reduced the Spd and/or spermine levels and enhanced free Put in the inflorescences, inducing an increased abscission of floral organs shortly after anthesis. These data suggest that PAs, particularly Spd, could be involved in the regulation of grapevine fruitlet physiological abscission.     

Biosynthesis profile and endogenous titers of polyamines differ in totipotent and recalcitrant plant protoplasts

The expression of totipotency in plant protoplasts is a complex developmental phenomenon and is affected by genetic and physiological factors. Polyamines (PAs) are known to be involved in a variety of growth and developmental processes in higher plants, as well as in adaptation to stresses. In this study, we present the homeostatic characteristics of the endogenous PA putrescine (Put), spermidine (Spd), and spermine (Spm) in totipotent (T) and non-totipotent (NT) tobacco protoplasts and in recalcitrant (R) grapevine protoplasts. T-tobacco protoplasts, with high division rates, have the highest level of endogenous PAs. In these protoplasts, the soluble-hydrolyzed fraction predominates and increases, and the insoluble-hydrolyzed fraction also increases, whereas soluble (S) PAs decrease rapidly during culture. The isolation process contributes to the increased Put levels, which are higher in freshly isolated NT-tobacco protoplasts than in T-protoplasts. During culture, total Put predominates over Spd and Spm, and the highest accumulation is found in T-protoplasts. Ornithine decarboxylase and arginase activities both increase in T-protoplasts, whereas arginine decarboxylase activity causes Put accumulation in NT-tobacco protoplasts. R-grapevine protoplasts show a different PA profile, mostly due to the lower PA content, the higher S-fraction, and the higher ratio of Spm to total PAs. The data suggest that the levels and metabolism of the intracellular PAs could be related to the expression of totipotency of plant protoplasts.