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.     

Inhibition of polyamine biosynthesis and growth in plant pathogenic fungi in vitro

Polyamine (PA) biosynthesis inhibitors, difluoromethylornithine (DFMO), difluoromethylarginine (DFMA), methylglyoxal bis-(guanylhydrazone) (MGBG) and bis-(cyclohexylammonium) sulphate (BCHA) have been tested for their effects on colony diameters at different intervals after inoculation of four plant pathogenic fungi (Helminthosporium oryzae, Curvularia lunata, Pythium aphanidermatum and Colletotrichum capsici). All these inhibitors, except DFMA had strongly retarded the growth of four fungi in a dose- and species-dependent fashion, and H. oryzae and C. lunata were found to be most sensitive to the effects of PA inhibitors. P. aphanidermatum and C. capsici were relatively insensitive and required rather high concentrations of inhibitors to get greater inhibition of mycelial growth, except DFMA which had stimulatory effect on the growth of these two fungi. However DFMA had greatly suppressed the growth of H. oryzae and C. lunata. The effect was generally more pronounced with MGBG than with DFMO and BCHA, and 1 mM Put completely prevented the inhibitory effects of 1 and 5 mM DFMO. Analysis of free and conjugated PAs in two sensitive fungi (H. oryzae and C. lunata) revealed that Put was present in highest concentrations followed by Spd and Spm and their levels were greatly reduced by DFMO application, and such inhibitions were totally reversed by exogenously supplied Put; in fact, PA titers were considerably increased by 1 mM Put alone and in combination with 1 mM DFMO. These results suggest that PA inhibitors, particularly DFMO and MGBG may be useful as target-specific fungicides in plants.     

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     

Effects of polyamines and polyamine biosynthetic inhibitors on mitotic activity of Allium cepa root tips

The genotoxic and cytotoxic effects of exogenous polyamines (PAs), putrescine (Put), spermidine (Spd), spermine (Spm) and PA biosynthetic inhibitors, alpha-difluoromethylornithine (DFMO), cyclohexilamine (CHA), methylglioxal bis-(guanylhydrazone) (MGBG) were investigated in the root meristems of Allium cepa L. The reduction of mitotic index and the induction of chromosomal aberrations such as bridges, stickiness, c-mitotic anaphases, micronuclei, endoredupliction by PAs and PA biosynthetic inhibitors were observed and these were used as evidence of genotoxicity and cytotoxicity.     

Effect of polyamines on stabilization of molecular complexes in thylakoid membranes of osmotically stressed oat leaves

Monocotyledonous leaves subjected to osmotica used for protoplast isolation accumulate a massive amount of putrescine (Put), lose chlorophyll and senesce rapidly. Treatment with spermidine (Spd) or spermine (Spm) prevents the loss of chlorophyll, indicating preservation of the thylakoid membranes at the site of the chlorophyll-protein complexes. Using several recently produced antibody probes, the effects on the stabilization of thylakoid membranes of applying either difluoromethylarginine (DFMA), a specific inhibitor of putrescine synthesis via arginine decarboxylase, or the polyamines Spd, Spm, or diaminopropane (Dap) to osmotically shocked oat leaves (Avena sativa L.) have been investigated. High protein levels were maintained in thylakoid membranes of leaf tissue incubated in the dark in the presence of 0.6 M sorbitol when pretreated with DFMA. After 48 h incubation, the level of the thylakoid protein D1, at the core of photosystem II, was higher in the DFMA-pretreated leaves as was the stromal protein ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco; as indicated by the level of large subunits). Applications of Spd, Spm or Dap were effective in retarding the loss of D1, D2 and cytochrome f from the thylakoid membranes as well as Rubisco large subunits and chlorophyll from the leaf tissue. The effects of polyamine applications may be mediated through Dap since most of the added Spd or Spm was converted to Dap within 6 h. The possible mechanisms of action of polyamine applications and DFMA-pretreatment on stabilizing the composition of the thylakoid membrane are also discussed.Abbreviations Cyt cytochrome - Dap diaminopropane - DFMA DL--difluoromethylarginine - LSU large subunit (of Rubisco) - Put putrescine - Rubisco ribulose-1,5-bisphosphate carboxylase-oxygenase - Spd spermidine - Spm spermine - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis This research was supported by the Agricultural and Food Research Council and by the British-Spanish joint research programme Acción Integrade HB-079 (R.T.B. and A.F.T.), British Council SPN/BAR/991 (R.T.B.) and Comision Interministerial de Cienica y Tecnologia 90-130 (A.F.T.). We thank Merrell Dow Research Center (Cincinnati, Ohio) for the gift of DFMA and Teresa Capell and Xavier Figueras (Univ. Barcelona) for help and suggestions.     

Do exogenous polyamines have an impact on the response of a salt-sensitive rice cultivar to NaCl?

In order to analyze the putative impact of polyamines (PAs) on the plant response to salt, seedlings from the salt-sensitive rice cultivar I Kong Pao (IKP) were exposed for 5, 12 and 19 days to 0, 50 or 100 mM NaCl in the absence, or in the presence of exogenous PAs (putrescine (Put), spermidine (Spd) or spermine (Spm) 1mM) or inhibitors of PA synthesis (methylglyoxalbis-guanyl hydrazone (MGBG) 1mM, cyclohexylammonium (CHA) 5mM and D-arginine (D-Arg) 5mM). The addition of PAs in nutritive solution reduced plant growth in the absence of NaCl and did not afford protection in the presence of salt. PA-treated plants exhibited a higher K+/Na+ ratio in the shoots, suggesting an improved discrimination among monovalent cations at the root level, especially at the sites of xylem loading. The diamine Put induced a decrease in the shoot water content in the presence of NaCl, while Spd and Spm had no effects on the plant water status. In contrast to Spd, Spm was efficiently translocated to the shoots. Both PAs (Spd and Spm) induced a decrease in cell membrane stability as suggested by a strong increase in malondialdehyde content of PA-treated plants exposed to NaCl. These results are discussed in relation to the putative functions of PAs in stressed plant metabolism.     

渗透胁迫对小麦胚芽鞘内多胺的种类、形态和含量的影响

用高压液相色谱法研究了豫麦18（抗旱性较强）和扬麦9号（抗旱性较弱）小麦胚芽鞘中三种不同形态的多胺（polyamine,PA）：游离态多胺（PA）、高氯酸可溶性结合态多胺（ps结合态PA）和高氯酸不溶性结合态多胺（PIS结合态PA）与渗透胁迫的关系。结果发现：渗透胁迫2d,豫麦18胚芽鞘中的游离态Spd和游离态Spm的含量明显上升,而扬麦9号的游离态Put的上升明显。S-腺苷蛋氨酸脱羧酶（S—AMDC）的抑制剂——甲基乙二醛-双（鸟嘌呤腙）（MGBG）处理豫麦18,明显抑制了渗透胁迫诱导的游离态Spd和游离态Spm的增加,并且加重了渗透胁迫伤害,外源Spd处理扬麦9号明显促进了渗透胁迫诱导的游离态Spd和游离态Spm的增加,并且减缓了渗透胁迫的伤害。渗透胁迫下,豫麦18胚芽鞘中的PS结合态PA和PIS结合态PA的上升幅度都明显大于扬麦9号。菲咯啉（o—Phen）抑制渗透胁迫下PIS结合态PA的合成并加重了渗透胁迫对胚芽鞘的伤害。这些结果表明：小麦胚芽鞘中的游离态Spd、游离态Spm、PS结合态PA和PIS结合态PA的升高有利于增强渗透胁迫抗性。     

Effects of spermidine and spermine levels on salt tolerance associated with tonoplast H+-ATPase and H+-PPase activities in barley roots

The effects of polyamines (Putrescine— Put; Spermidine—Spd; and Spermine—Spm) on␣salt tolerance of seedlings of two barley (Hordeum vulgare L.) cultivars (J4, salt-tolerant; KP7, salt-sensitive) were investigated. The results showed that, the salt-tolerant cultivar J4 seedlings accumulated much higher levels of Spd and Spm and lower Put than the salt-sensitive cultivar KP7␣under salt stress. At the same time, the dry weight of KP7 decreased significantly than that of␣J4. After methylglyoxal bis(guanylhydrazone) [MGBG, an inhibitor of S-adenosylmethionine decarboxylase (SAMDC)] treatment, Spd and Spm levels together with the dry weight of both cultivars were reduced, but the salt-caused dry weight reduction in two cultivars could be reversed by the concomitant treatment with Spd. MGBG decreased the activities of tonoplast H⁺-ATPase and H⁺-PPase too, but the experiments in vitro indicated that MGBG was not able to affect the above two enzyme activities. However, the polyamines, especially Spd, promoted their activities obviously. These results suggested that the conversion of Put to Spd and Spm and maintenance of higher levels of Spd and Spm were necessary for plant salt tolerance.     

水稻细胞质雄性不育系幼穗发育过程中多胺与乙烯的关系初探(英文 )

利用ＨＰＬＣ和ＧＣ分别测定了水稻细胞质雄性不育系及其保持系幼穗多胺（ 腐胺,亚精胺和精胺） 含量和乙烯释放速率,并研究了外施多胺合成抑制剂ＭＧＢＧ 和乙烯前体ＡＣＣ生成抑制剂ＡＶＧ 对两系幼穗多胺含量和乙烯释放速率以及花粉育性的影响。结果表明, 不育系幼穗乙烯释放速率显著高于其保持系幼穗, 外施ＡＶＧ 引起两系幼穗乙烯释放速率下降,并使不育系花粉育性得以部分恢复; 不育系幼穗多胺含量显著低于保持系幼穗, 外施ＭＧＢＧ 使两系幼穗Ｓｐｄ 和Ｓｐｍ 含量下降, 并使保持系花粉育性降低。外施ＡＶＧ 抑制乙烯释放,促进多胺合成;而外施ＭＧＢＧ 抑制Ｓｐｄ和Ｓｐｍ 合成, 却促进乙烯的释放; 而且,乙烯释放速率与多胺（精胺和亚精胺） 含量呈显著负相关。提示在水稻ＣＭＳ 系及其保持系幼穗发育过程中乙烯与多胺（ 精胺和亚精胺） 的生物合成竞争ＳＡＭ。     

水分胁迫下山黧豆多胺代谢与β_N_草酰_L_α,β_二氨基丙酸积累相关性的研究

Seedlings of grass pea (Lathyrus sativus L.) were treated with polyethylene glycol (PEG), in addition with putrescine (Put) and α-difluoromethylarginine (DFMA) in order to study the relation between polyamine metabolism and accumulation of β-N-oxalyl-L-α, β-diaminopropionic acid (ODAP) under water stress. The results indicated that Put, spermidine (Spd) and spermine (Spm) contents of leaves gradually increased along with the extension of PEG-treatment time. Especially, the increase in Spm contents was significant together with a gradual increase of ODAP contents. Put and Spd contents of leaves markedly increased along with the extension of PEG+Put treatment time. There was little effect on Spm contents and ODAP contents in leaves with PEG+Put treatment. The accumulation of Put, Spd, Spm and ODAP in leaves was inhibited by PEG+DFMA treatment. The inhibition of DFMA for Put and Spd biosynthesis was partially overcome by adding exogenous Put, but little effect for the inhibition of DFMA on Spm biosynthesis by adding exogenous Put. The accumulation of ODAP was inhibited with PEG+DFMA+Put treatment in leaves. This indicated that there was a relationship between the increase of Spm contents and the accumulation of ODAP in leaves under water stress.     

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.     

Effects of exogenous polyamines and difluoromethylornithine on seed germination and root growth of Arabidopsis thaliana

Effects of exogenous polyamines and difluoromethylornithine (DFMO) on seed germination and seedling root growth of Arabidopsis thaliana were investigated. Root growth was stimulated by low concentrations of putrescine but was increasingly inhibited by high concentrations of putrscine. DFMO inhibited root growth and this inhibition was reversed by applying putrescine. In contrast, both spermidine and spermine had no effect on root growth but inhibited seed germination. The results suggest a possible requirement of endogeneous putrescine for normal root growth of Arabidopsis seedlings.Abbreviations DFMO difluoromethylornithine - DFMA difluoromethylarginine - ODC ornithine decarboxylase - Put Putrescine - Spd Spermidine - Spm Spermine     

Polyamine metabolism during seedling development in rice

The main free amines identified during growth and development of rice seedlings were agmatine, putrescine, spermidine, diaminopropane and tyramine. Amine composition differed according to tissue and stages of development. Conjugated amines were only found in roots. We present evidence that arginine decarboxylase (ADC) regulates putrescine during the development of rice seedlings. When ADC action was blocked by DFMA (-DL-difluoromethylarginine, a specific irreversible inhibitor of ADC), polyamine titers and seedling development were diminished; when agmatine or putrescine was added, normal polyamine titers and growth were restored. The effects of DFMA were concentration dependent. DFMO (-DL-difluoromethylornithine, a specific irreversible inhibitor of ornithine decarboxylase or ODC) promoted growth and development at concentrations below 2 mM. This effect was probably related to its unexplained, but consistently observed slight enhancement of rice ADC. When the increase in the concentration of spermidine was prevented by CHA (cyclohexylammonium sulfate), the number of roots increased and the increase in length of leaves and roots was strongly inhibited. The addition of exogenous spermidine at the time of treatment with CHA reversed the inhibition by CHA.Abbreviations ADC arginine decarboxylase - ODC ornithine decarboxylase - DFMA -DL-difluoromethylarginine - DFMO -DL-difluoromethylornithine - CHA cyclohexylammonium sulfate     

Polyamines and related enzymes in rice seeds differing in germination potential

In ungerminated rice seeds, (Japonica rice variety, CV Tapei 309), the content of free amines (putrescine, spermidine, spermine, tyramine) was higher in seed lots having a low germination frequency compared to those with high germination potential. Conversely, amine conjugates (di-feruloylputrescine, di-feruloylspermidine, diferuloyldiaminopropane and feruloyltyramine) decreased with loss of viability. Thus, these compounds appeared to constitute biochemical markers of seed viability. In seeds with high germination potential, conjugates decreased drastically during germination, with an early and rapid increase in free amines (putrescine, spermidine, tyramine). Arginine decarboxylase (ADC) activity was highest during the germination of high germination potential seeds, its activity gradually declining with loss of viability and being closely correlated with agmatine content. The polyamine biosynthetic inhibitors (-DL-difluoromethylarginine, DFMA, a specific and irreversible inhibitor of ADC; -DL-difluoromethylornithine, DFMO, a specific irreversible inhibitor of ornithine decarboxylase (ODC); cyclohexylammonium sulfate, CHA, inhibitor of spermidine synthase) neither depleted putrescine and spermidine levels nor inhibited germination in high germination potential seeds. In low germination potential seeds, the germination process was inhibited by DFMA or CHA. Application of agmatine resulted in a reversal of inhibition. DFMA inhibited ADC activity in both categories of seeds. In low germination potential seeds treated with CHA no ADC activity was found. These results suggest that amines are involved in the germination process of rice seeds. It appears that amine conjugates may serve as a storage form of amines which, upon enzymatic hydrolysis, could supply the cell with an additional amine reserve and influence cell division and/or cell elongation.Abbreviations ADC arginine decarboxylase - ODC ornithine decarboxylase - DFMA -DL-difluoromethylarginine - DFMO -DL-difluoromethylornithine - CHA cyclohexylammonium sulfate     

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.     

Amelioration of salinity stress by exogenously applied spermidine or spermine in three varieties of indica rice differing in their level of salt tolerance

We present here the comparative protective potentiality of exogenously applied polyamines (PAs), namely spermidine (Spd) and spermine (Spm), in mitigating NaCl toxicity and inducing short-term salinity tolerance in three indica rice varieties, namely M-1-48 (salt-sensitive), Nonabokra (salt-tolerant) and Gobindobhog (highly sensitive). The retardation in root length or shoot length and toxic Na⁺ accumulation or K⁺ loss, the considerable increment in malondialdehyde/H₂O₂ accumulation or lipoxygenase activity, all of which were particularly noteworthy in M-1-48 and Gobindobhog during salinity stress, was appreciably reduced by co-treatment with Spd or Spm. Both the PAs also inhibited the extent of salt-induced protein carbonylation in all the varieties and enhanced protease activity, especially in Gobindobhog. The prevention of chlorophyll degradation was better with Spd in Nonabokra and Gobindobhog. While the salt-induced increase in anthocyanin or reducing sugar level was further prompted by Spd or Spm in all the varieties, the proline content was elevated by Spd particularly in Gobindobhog. During salinity stress, both the PAs were effective in lowering the putrescine accumulation in M-1-48 and Gobindobhog, and strikingly increasing the Spm level in all the varieties, the highest being in Gobindobhog. In addition, they enhanced the activity of peroxidases and compensated for the decreased catalase activity in all the varieties. Thus the two PAs could recuperate all the three varieties from salt-induced damages to different degrees. The salt injuries, encountered in M-1-48 and Gobindobhog, both of which showed greater susceptibility to salinity stress, were more pronouncedly alleviated and counteracted by the PAs, than the salt-tolerant Nonabokra. The reversal of inhibitory effect of salinity stress was conferred by preventing growth inhibition or various forms of cellular damages, maintaining proper K⁺/Na⁺ balance or triggering the level of osmolytes and activity of antioxidant enzymes. Our communication offers a referenced evidence for an understanding of the mechanism by which higher PAs relieve the damages particularly in salt-sensitive rice varieties.     

Cytological events induced by the inhibition of polyamine biosynthesis in thin cell layers of tobacco

Summary The proliferative growth of thin cell layers ofNicotiana tabacum cultured on a rhizogenic medium was markedly disturbed when polyamine biosynthesis was inhibited. Treatments with polyamine inhibitors led to cell expansion, accompanied by thinning of the cell wall and inhibition of cell division, and frequent cases of nucleolar extrusion, mainly in the parenchymal layer in contact with the medium. Nucleolar extrusion was not correlated with cell expansion. The highest incidence of nucleolar extrusion occurred when the pathways of putrescine biosynthesis were inhibited and when spermidine synthesis, via S-adenosylmethionine decarboxylase, was blocked. The duration of the growth phase with nuclear amitotic divisions was prolonged in the presence of the inhibitors and root meristem formation delayed. When polyamines were added with the inhibitors, all reactions proceeded as in the controls.Abbreviations CHA cyclohexylamine - DFMA DL--difluoromethyl-arginine - DFMO DL--difluoromethylornithine - LS longitudinal section - MGBG methylglyoxal-bis(guanylhydrazone) - PA polyamine - Pu putrescine - RLS radial longitudinal section - S.E. standard error - Spd spermidine     

多胺及其合成抑制剂MGBC对稀脉萍成花反应的影响

腐胺、亚精胺和精胺对稀脉萍的成花均有一定的抑制作用,这种作用随多肢的浓度增加而增强。多胺合成抑制剂MGBG强烈抑制稀脉萍群体的增殖速率,并使稀脉萍群体在非诱导光周期下开花。这种由MGBG引起的增殖速率的降低及成花诱导作用均可被多胺逆转。稀脉萍成花诱导过程中,内源腐胺含量显著升高,亚精胺则下降。     

Treatment with alpha-difluoromethylornithine plus a spermidine analog leads to spermine depletion and growth inhibition in cultured L1210 leukemia cells

Of the three biological polyamines, putrescine (Put), spermidine (Spd), and spermine (Spm), the relevance of Spm to cell proliferation has yet to be defined because of our general inability to deplete it selectively in intact cells. In the present study, Spm depletion was accomplished by treating cultured L1210 cells for 96 hr with alpha-difluoromethylornithine (DFMO) and an analog of Spd such as aminopropylcadaverine, N4-methylSpd, N4-ethylSpd, or homoSpd. DFMO, a specific inhibitor of ornithine decarboxylase, halts continued polyamine biosynthesis and the Spd analog serves as a functional substitute for Spd. Thus, while the Spd analog fulfills the role(s) of Spd in cell proliferation, Spm becomes steadily depleted. In cells treated with DFMO plus the analog, aminopropylcadaverine, Spm pools decline steadily and growth inhibition occus after 48 hr (when Spm pools decline to 60% of control). By 96 hr, Spm is approximately 15% of control and growth is less than 30%. Prevention studies with exogenous polyamines confirm a causal relationship between Spm depletion and growth inhibition. The critical levels of polyamines for cell proliferation to take place were found to be 30% of control for Spd and 60% for Spm. The use of DFMO plus a Spd analog is proposed as a system for studying the cellular consequences of Spm depletion. Spd depletion can be achieved for comparison purposes by treating cells with DFMO alone.     

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.