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     

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     

Developmental regulation of polyamine metabolism in growth and differentiation of carrot culture

Polyamine levels and the activities of two polyamine biosynthetic enzymes, arginine decarboxylase (EC 4.1.1.19) and S-adenosylmethionine decarboxylase (EC 4.1.1.50), were determined during somatic embryogenesis of carrot (Daucus carota L.) cell cultures. Embryogenic cultures showed severalfold increases in polyamine levels over nondifferentiating controls. A mutant cell line that failed to form embryos but grew at the same rate as the wild-type line also failed to show increases in polyamine levels, thus providing evidence that this increased polyamine content was in fact associated with the development of embryos. Furthermore, inhibition of these increases in polyamines caused by drugs inhibited embryogenesis and the effect was reversible with spermidine. The activities of arginine decarboxylase and Sadenosylmethionine decarboxylase were found to be suppressed by auxin; however, the specific effects differed between exogenous 2,4-dichlorophenoxyacetic acid and endogenous indole-3-acetic acid. The results indicate that increased polyamine levels are required for cellular differentiation and development occurring during somatic embryogenesis in carrot cell cultures.Abbreviations ADC arginine decarboxylase - 2,4-D 2,4-dichlorophenoxyacetic acid - DFMA difluoromethylarginine - DCHAS dicyclohexylammonium sulfate - SAMDC S-adenosylmethionine decarboxylase     

Methyl jasmonate upregulates biosynthetic gene expression, oxidation and conjugation of polyamines, and inhibits shoot formation in tobacco thin layers

The effect of methyl jasmonate (MJ) on de novo shoot formationand polyamine metabolism was investigated in thin layer explantsof tobacco (Nicotiana tabacum L. cv. Samsun). A relatively lowconcentration of MJ (0.1 µM) enhanced explant fresh weight,but had no effect on the final number of shoots per explantwhile higher concentrations (1 and 10 µM) significantlyinhibited organogenesis. The histological study revealed that,with increasing concentrations of MJ, the formation of meristemoidsand shoot domes declined and the incidence of cell hypertrophyincreased. In explants cultured with 0.1, 1 or 10 µM MJ,the endogenous levels of free putrescine, spermidine and sperminegenerally declined compared with controls, after 7 and 15 d.Perchloric acid-soluble conjugated polyamines accumulated dramaticallyduring culture, but much more so in the presence of MJ thanin controls. Acid-insoluble conjugated spermidine alone increasedin response to the elicitor. Activities of the putrescine biosyntheticenzymes arginine decarboxylase (ADC, EC 4.1.1.19) and ornithinedecarboxylase (ODC, EC 4.1.1.17) in the soluble fraction ofMJ-treated explants displayed up to 3-fold increases relativeto control explants. However, the most relevant increases inthese enzyme activities occurred in the particulate fraction.The activity of S-adenosylmethionine decarboxylase (SAMDC, EC4.1.1.21), an enzyme involved in spermidine and spermine biosynthesis,was also stimulated by exposure to MJ. Northern analyses revealedMJ-induced, generally dose-dependent, increases in the mRNAlevels of all three enzymes. Diamine oxidase (DAO, EC 1.4.3.6)activity was stimulated by MJ mainly in the cell wall fraction.The upregulation of polyamine metabolism is discussed in relationto the morphogenic behaviour of MJ-treated explants. Key words: Nicotiana tabacum, thin layers, shoot formation, methyl jasmonate, polyamine metabolism.     

Polyamines and somatic embryogenesis in two Vitis vinifera cultivars

Polyamine content and activities of enzymes of polyamine biosynthesis were assayed during somatic embryogenesis in Vitis vinifera callus cultures of Chardonnay and Brachetto 'a grappolo lungo' (Brachetto g.l.) cultivars. The analyses were carried out on embryogenic callus samples, embryos at different stages and developing plants. Polyamine content, both in the free and PCA-soluble conjugated form, was higher in Brachetto g.l. than in Chardonnay, and putrescine was present at higher concentrations than the other polyamines. In all samples of both cultivars, ornithine decarboxylase activity (ODC, EC 4.1.1.17) was higher than arginine decarboxylase (ADC, EC 4.1.1.19), with a maximum in developing plant roots. S -Adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.50) activity displayed a similar trend. The activities of all three enzymes were detected both in the supernatant and pellet fractions, indicating for the first time the presence of SAMDC activity in the particulate fraction. Particularly in the Chardonnay cultivar, an increase in the mRNAs expression patterns of ODC and SAMDC during morphogenesis from small embryos to plantlets was detected by northern blot, suggesting a direct correlation with enzymatic activities.     

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.     

Growth and polyamine metabolism inPyrenophora avenae exposed to cyclohexylamine and norspermidine

Summary The effectiveness of inhibitors of polyamine biosynthesis in controlling plant pathogenic fungi is well established. The spermidine synthase inhibitor cyclohexylamine (CHA) and the spermidine analogue norspermidine were evaluated againstin vitro growth of the oat stripe pathogenPyrenophora avenae. Mycelial growth was reduced by 55% upon exposure to 2.0mM CHA while the same concentration of norspermidine reduced growth by 63%. Neither inhibitor had any effect on ODC or AdoMetDC activities, nor the flux of label from ornithine through to the polyamines. Levels of free polyamines in fungal tissue exposed to 0.01 mM norspermidine were unaltered, although 1.0mM CHA did produce a 75% increase in fungal putrescine content. These data suggest that CHA and norspermidine do not reduce fungal growth as a result of a perturbation in polyamine biosynthesis.Abbreviations ODC ornithine decarboxylase - ADC arginine decarboxylase - AdoMetDC S-adenosylmethionine decarboxylase - DFMO adifluoromethylornithine - CHA cyclohexylamine     

Spermine facilitates recovery from drought but does not confer drought tolerance in transgenic rice plants expressing <Emphasis Type="Italic">Datura stramonium</Emphasis><Emphasis Type="Italic">S</Emphasis>-adenosylmethionine decarboxylase

Polyamines are known to play important roles in plant stress tolerance but it has been difficult to determine precise functions for each type of polyamine and their interrelationships. To dissect the roles of putrescine from the higher polyamines spermidine and spermine, we generated transgenic rice plants constitutively expressing a heterologous S-adenosylmethionine decarboxylase (SAMDC) gene from Datura stramonium so that spermidine and spermine levels could be investigated while maintaining a constant putrescine pool. Whereas transgenic plants expressing arginine decarboxylase (ADC) produced higher levels of putrescine, spermidine and spermine, and were protected from drought stress, transgenic plants expressing SAMDC produced normal levels of putrescine and showed drought symptoms typical of wild type plants under stress, but the transgenic plants showed a much more robust recovery on return to normal conditions (90% full recovery compared to 25% partial recovery for wild type plants). At the molecular level, both wild type and transgenic plants showed transient reductions in the levels of endogenous ADC1 and SAMDC mRNA, but only wild type plants showed a spike in putrescine levels under stress. In transgenic plants, there was no spike in putrescine but a smooth increase in spermine levels at the expense of spermidine. These results confirm and extend the threshold model for polyamine activity in drought stress, and attribute individual roles to putrescine, spermidine and spermine.     

多胺对荔枝胚性愈伤组织增殖与体胚发生的影响

为探讨外源多胺(PAs)对荔枝胚性愈伤组织(EC)增殖及体胚发生的影响机制,该研究以“妃子笑”荔枝EC为材料,采用单因素法在增殖培养基中添加腐胺(Put)、亚精胺(Spd)及精胺(Spm),分析了不同PAs处理后EC的形态、结构、内源PAs含量及相关酶指标的变化。结果表明:(1)外源Put、Spd和Spm处理均显著提高了EC增殖率,减少了体胚诱导及萌发数量。经外源PAs处理增殖的EC胚性细胞大小较一致,染色深且均匀,多细胞原胚减少,可见已经分化完全的早期子叶胚。(2)外源PAs处理均显著提高了EC中内源PAs含量,其中Put处理的EC中各类内源PAs及总PAs含量最高; 当在含外源PAs培养基上增殖的EC转入不含外源PAs的培养基上增殖时(恢复培养),EC中的Put含量仍然显著高于对照,内源Spd和Spm则显著降低。(3)外源Put处理显著提高了EC中的鸟氨酸脱羧酶(ODC)、精氨酸脱羧酶(ADC)和二胺氧化酶(DAO)活性,而外源Spd、Spm处理显著降低了EC中的ODC及ADC活性,外源Spd显著提高了多胺氧化酶(PAO)活性; 恢复培养后,EC中ADC和DAO活性比恢复培养前显著降低,ODC和PAO无显著性差异。综上认为,外源PAs可以通过调节PAs代谢相关酶活性影响内源PAs含量,进而影响荔枝EC增殖和体胚诱导。该研究结果为进一步研究PAs调节荔枝体胚发生机制及提高荔枝离体再生效率提供了基础。     

Polyamine homeostasis in transgenic plants overexpressing ornithine decarboxylase includes ornithine limitation

It was reported recently that overexpression of human ornithine decarboxylase (ODC) cDNA in transgenic rice plants resulted in increased steady-state concentration of polyamines, i.e., enough biosynthetic control is invested at this step to enable adjustment of polyamine levels. To investigate critically whether constitutive overexpression of ODC is sufficient to control steady-state polyamine levels, we expressed an ODC cDNA from Datura stramonium in transgenic tobacco plants. Transgenic progeny of self-fertilised primary transformants exhibited increases in ODC activity of 25-fold in leaves and 5-fold in flower buds. However, the increase in putrescine levels was only 1.5- to 2.1-fold in leaves and 1.1- to 1.3-fold in flower buds. Emphatically, no changes to spermidine or spermine steady-state levels or to soluble or insoluble hydroxycinnamic acid-conjugated polyamines were observed. Ornithine feeding to cell suspension cultures derived from the transgenic plants indicated that putrescine accumulation was limited in part by ornithine availability. These results demonstrate that a large increase in the capacity of the tobacco plants to decarboxylate ornithine does not result in a comparable increase in the level of free or conjugated polyamines. Plant polyamine homeostatic mechanisms efficiently accommodate increased ODC activity, suggesting that polyamine biosynthetic control is invested at multiple interdependent steps.     

Over-expression of a cDNA for human ornithine decarboxylase in transgenic rice plants alters the polyamine pool in a tissue-specific manner

We investigated how over-expression of a cDNA for human ornithine decarboxylase (odc) affects the polyamine pools in transgenic rice. We further investigated tissue-specific expression patterns and product accumulation levels of the transgene driven by either constitutive or seed-specific promoters. Our results indicate that: (1) whereas the expression of a heterologous arginine decarboxylase (adc) cDNA in rice resulted in increased putrescine and spermine levels only in seeds, plants engineered to express odc cDNA exhibited significant changes in the levels of all three major polyamines in seeds and also in vegetative tissues (leaves and roots); (2) there was no linear correlation between odc mRNA levels, ODC enzyme activity and polyamine accumulation, suggesting that control of the polyamine pathway in plants is more complex than in mammalian systems; (3) ODC activity and polyamine changes varied in different tissues, indicating that the pathway is regulated in a tissue-specific manner. Our results suggest that ODC rather than ADC is responsible for the regulation of putrescine synthesis in plants.     

Differential induction by methyl jasmonate of genes encoding ornithine decarboxylase and other enzymes involved in nicotine biosynthesis in tobacco cell cultures

A cDNA of tobacco BY-2 cells corresponding to an mRNA species which was rapidly induced by methyl jasmonate (MeJA) in the presence of cycloheximide (CHX) was found to encode ornithine decarboxylase (ODC). Another cDNA from a MeJA-inducible mRNA encoded S-adenosylmethionine synthase (SAMS). Although these enzymes could be involved in the biosynthesis of polyamines, the level of putrescine, a reaction product of ODC, increased slowly and while the levels of spermidine and spermine did not change following treatment of cells with MeJA. However, N-methylputrescine, which is a precursor of pyrrolidine ring of nicotine, started to increase shortly after MeJA-treatment of cells and the production of nicotine occured thereafter. The levels of mRNA for arginine decarboxylase (ADC), an alternative enzyme for putrescine synthesis, and that for S-adenosylmethionine decarboxylase (SAMDC), required for polyamine synthesis, were not affected by MeJA. In addition to mRNAs for ODC and SAMS, mRNA for putrescine N-methyltransferase (PMT) was also induced by MeJA. Unlike the MeJA-induction of ODC mRNA, MeJA-induction of SAMS and PMT mRNAs were blocked by CHX. The level of ODC mRNA declined after 1 to 4 h following MeJA treatment, while the levels of mRNAs for SAMS and PMT continued to increase. Auxin significantly reduced the MeJA-inducible accumulation of mRNAs for ODC, SAMS and PMT. These results indicate that MeJA sequentially induces expression of a series of genes involved in nicotine biosynthesis by multiple regulatory mechanisms.p>     

Short-term polyamine response in TMV-inoculated hypersensitive and susceptible tobacco plants

The short-term polyamine response to inoculation, with tobacco mosaic virus (TMV), of TMV-inoculated NN (hypersensitive) and nn (susceptible) plants of Nicotiana tabacum (L.) cv. Samsun was investigated. Free and conjugated polyamine concentrations, putrescine biosynthesis, evaluated through arginine decarboxylase (ADC) and ornithine decarboxylase (ODC) activities, and putrescine oxidation, via diamine oxidase (DAO) activity, were analysed during the first 24 h from inoculation. Results were compared with those of mock-inoculated control plants. In NN TMV-inoculated plants undergoing the hypersensitive response (HR), free putrescine and spermidine concentrations had increased after 5 h compared with controls; polyamine conjugates also tended to increase compared with controls. In both virus- and mock-inoculated plants, ADC and ODC activities generally increased whereas DAO activity, which was present in controls, was detectable only in traces in inoculated tissues.
In TMV-infected susceptible plants, free putrescine and spermidine concentrations were lower at 5 h relative to controls, as were polyamine conjugates. No differences were revealed in ADC and ODC activities whereas DAO activity was not detectable. These results further support the hypothesis that polyamines are involved in the response of tobacco to TMV and that, only a few hours after inoculation, the response of hypersensitive plants is distinct from that of susceptible ones.     

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.