Polyamines in marine macroalgae: Levels of putrescine, spermidine and spermine in the thalli and changes in their concentration during glycerol-induced cell growth in vitro

The levels of putrescine (Put), spermidine (Spd) and spermine (Spm) were analyzed in naturally collected samples of the marine macroalgae Dyctiota dichotoma, Gelidium canariensis and Grateloupia doryphora . Polyamines (PAs) appeared in free (35–134 μg g⁻¹ fresh weight) and bound TCA-insoluble form (1 667–2 624 μg g⁻¹ fresh weight). Axenic in vitro cultures of sporelings from G. doryphora were established in the medium containing glycerol. This medium promoted growth and morphogenesis and also increased the free and bound PA levels in the sporelings. Tracer experiments using 70 kBq [U-¹⁴C]-glycerol showed significant quantities of radioactivity in Put, Spd and Spm after 20 h of incubation. The effects of glycerol on growth were inhibited by the ornithine decarboxylase (EC 4.1.1.17) inhibitor α -difluoromethylornithine (DFMO). The presence of DFMO in the incubation medium with [U-¹⁴C]-glycerol also reduced the radioactivity in PAs.     

节瓜茎尖多胺含量和比值与花性别分化的关系

以4个不同基因型的节瓜为材料,通过两个发育时期（10、19片叶展平）茎尖取样,研究了多胺（PA）含量和比值与植株花性别分化的关系。结果表明,节瓜茎尖4种多胺含量差异显著,两个取样时期都是亚精胺（Spd）〉腐胺（Put）〉尸胺（Cad）〉精胺（Spm）。10片叶展平时期多胺含量与节瓜花性别分化之间没有明确的相关性;19片叶展平时期,节瓜茎尖Put、Spd和多胺总量与植株雌花分化比例呈极显著的正相关,而Cad则与雌花分化比例呈极显著的负相关。在两个取样时期,复合指标Spd/PA都与植株雌花分化比例呈显著的正相关,而（Put＋Cad）/（Spd＋Spm）均与之呈显著的负相关,可以较好地预测节瓜的花性别分化状况。     

Polyamine levels in buds and twigs of Tilia cordata from dormancy onset to bud break

The fluctuations of free and bound polyamines (PAs) were studied in vegetative buds and underlying twigs of linden (Tilia cordata L.) from August to May, to assess the connection between PA levels and seasonal cycles of growth and dormancy. Outer and inner bud scales and shoot tips (short shoot tips with leaf initials in contiguous short internodes) were analyzed separately, as were phloem with cortex and xylem with pith tissue from twigs. Seasonal variations in PA levels were present in buds and twigs during the research period. The most abundant PA in buds and twigs in free and bound forms was spermidine followed by putrescine. PA amounts were low in buds and twigs in autumn. In twig tissues, free PAs were predominant whereas in bud scales, bound PAs accumulated over free PAs in autumn, first in inner scales and later in outer scales as well. PA levels did not increase dramatically during the onset of dormancy in autumn but lower temperatures and probable cold hardening correlated positively with bound PAs in bud scales. In shoot tips with leaf initials, and contiguous short internodes, free putrescine and spermidine levels rose simultaneously with bud burst and new growth, while bound PAs diminished quite radically from temporary bud scales and from growing shoot tips.     

Variations in endogenous polyamine content of maize calli obtained from zygotic and androgenetic embryos

A comparative study of polyamine (putrescine, spermidine and spermine) levels was conducted with maize calli originating from a) immature embryos and b) pollen embryos capable of plant regeneration. The differences observed in the studied parameters of the two kinds of calluses are related to their cellular origin and to their regeneration capacity. Moreover, only the calluses proceeding from immature embryos differentiated into preembryogenic structures, which eventually developed into plants. Although total polyamine levels in pollenderived calluses were significantly higher than those from immature embryos, spermidine and spermine were the predominant polyamines in both culture types. Furthermore, polyamine fractions of these calluses also showed differences. All these phenomena may be related with the differences observed in the callus embryogenic response. These findings may be useful in understanding the implication of polyaminesin embryogenetic processes.Abbreviations IEC immature-embryo calluses - PAs polyamines - PEC pollen-embryo calluses - PH insoluble conjugated PA fraction - Put putrescine - S free PA fraction - SH soluble conjugated PA fraction - Spd spermidine - Spm spermine 2,4d-2,4 dichlorophenoxyacetic acid     

Apical dominance in Pssu-ipt-transformed tobacco.

In Pssu-ipt-transformed tobacco, apical dominance was released by defoliation of the upper nodes, while the apex remained intact. After defoliation, the concentration of cytokinins (CKs) increased whereas IAA remained constant, evoking an increase in the CK/IAA ratio in the buds. Moreover, defoliation resulted in a tremendous increase in the concentrations of aromatic amines (AAs): tyramine (TYR), phenethylamine (PEA) and an as yet unidentified compound. Although the total aliphatic monoamine and polyamine (PA) concentration remained constant, putrescine (PUT) and spermidine (SPD) concentrations in the axillary buds decreased, whereas the concentration of spermine (SPM) increased. Similar changes in PAs and AAs could be observed in the buds of untransformed SR1 plants after decapitation, whereas defoliation without removal of the apex had no effect. This is the first report on the possible involvement of PAs and AAs in apical dominance.     

Role of polyamines on de novo shoot morphogenesis from cotyledons of Brassica campestris ssp. pekinensis (Lour) Olsson in vitro

Summary The promotive effect of ethylene inhibitors (Els), i.e. AgNO₃ and aminoethoxyvinylglycine (AVG) on de novo shoot regeneration from cultured cotyledonary explants of Brassica campestris ssp. pekinensis cv. Shantung in relation to polyamines (PAs) was investigated. The endogenous levels of free putrescine and spermidine in the explant decreased sharply after 1–3 days of culture, whereas endogenous spermine increased, irrespective of the absence or presence of Els. AgNO₃ at 30 M did not affect endogenous PAs during two weeks of culture. In contrast, explants grown on medium containing 5 M AVG produced higher levels of free putrescine and spermine which increased rapidly after three days and reached a peak at 10 days. An exogenous application of 5 mM putrescine also resulted in a similar surge of endogenous free spermine of the explant. More strikingly, shoot regeneration from explants grown in the presence of 1–20 mM putrescine, 0.1–2.5 mM spermidine, or 0.1–1 mM spermine was enhanced after three weeks of culture. However, exogenous PAs generally did not affect ethylene production, and endogenous levels of 1-aminocyclopropane-1-carboxylate (ACC) synthase activity and ACC of the explant. This study shows the PA requirement for shoot regeneration from cotyledons of B. campestris ssp. pekinensis in vitro, and also indicates that the promotive effect of PAs on regeneration may not be due to an inhibition of ethylene biosynthesis.Abbreviations PAs polyamines - AVG aminoethoxyvinylglycine - SAM S-adenosylmethionine - ACC 1-aminocyclopropane-1-carboxylate - Els ethylene inhibitors     

Putrescine promotes changes in the endogenous polyamine levels and proteomic profiles to regulate organogenesis in <Emphasis Type="Italic">Cedrela fissilis</Emphasis> Vellozo (Meliaceae)

Improvements to in vitro organogenesis are essential for optimizing shoot development and understanding basic physiological processes. The addition of polyamines (PAs) to the culture medium has been used to modulate organogenesis in plants, and this work evaluated the effects of exogenous PAs on direct organogenesis from apical and cotyledonary nodal Cedrela fissilis explants as well as the effects of putrescine (Put) on endogenous PA levels and variations in protein abundance. The effects of exogenous Put, spermidine, and spermine at 0, 0.5, 1, 2.5, or 5 mM on shoot development were tested. The comparison of the tested PAs to the control treatment revealed that 2.5 mM Put significantly increased the length of shoots from cotyledonary nodal explants, which are more sensitive than apical nodal explants, and treatment with 2.5 mM Put significantly increased the endogenous total free-PA and free-Put levels in shoots compared with the control (no Put). A comparative proteomic analysis of shoots indicated that 2.5 mM Put significantly changed the abundance of proteins, primarily metabolic and cellular proteins associated with stress and energy processes such as cell division. These results show that Put functions in endogenous PA metabolism and alters protein abundance, thereby contributing to shoot development in C. fissilis.     

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。     

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.     

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.     

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.     

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

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

Effects of abscisic acid on the contents of polyamines and proline in common bean plants under salt stress

The effects of ABA treatment on the contents of polyamines (PAs) and proline (Pro) in the glycophyte Phaseolus vulgaris L. during plant adaptation to salt stress were studied. Two-week-old common bean seedlings grown in the phytotron chamber on the Jonson nutrient medium were subjected to salinity for 6 days by one-time NaCl addition to medium up to final concentrations of 50 and 100 mM. During first three days of salinity, the root system was daily treated with ABA (1, 5, 10, or 50 μM) for 30 min. Salt stress (100 mM NaCl) elevated the level of endogenous ABA, increased the content of Pro 14-fold, reduced sharply the content of free PAs (putrescine, spermidine, spermine, and cadaverine), and the accumulation of 1,3-diaminopropan, a product of oxidation of high-molecular PAs. Common bean plant treatment with 1 μM ABA weakened the adverse effects of salt stress (100 mM NaCl), which was manifested in the maintenance of plant growth, stimulation of chlorophyll (a and b) and carotenoid accumulation, a stabilization of water and Na⁺ balance. Seedling treatment with ABA suppressed NaCl-induced Pro and intracellular ABA accumulation and restored the levels of putrescine and spermidine. The content of spermine in the leaves of plants subjected to salt stress and treated with ABA was approximately threefold higher than in control plants, whereas the content of cadaverine increased under similar conditions more than fivefold. Simultaneously, the contents of 1,3-diaminopropan and malondialdehyde as well as activity of superoxide dismutase were reduced, which indicates a weakening of oxidative stress, one of the possible causes of defensive ABA effects against salt stress. In addition, the suppression by exogenous ABA of Pro accumulation and stimulation of PA content under salt stress confirm indirectly our hypothesis that ABA is involved in the coordinated regulation of two biosynthetic pathways, Pro and PA formation, which use a common precursor, glutamate, and play an important protective role during stress in plants.     

Suppression of ethylene levels promotes morphogenesis in pepper (Capsicum annuum L.)

Ethylene and polyamines (PAs) are two phytohormones that play important roles during in vitro morphogenesis of several plant species. The interaction between ethylene and PAs has been of interest because both have S-adenosylmethionine as a precursor. To study the influence of ethylene and PAs on in vitro morphogenesis of an ornamental pepper, we added an ethylene scavenger, PAs, a PA inhibitor, and compounds that affect ethylene biosynthesis and activity to the regeneration medium. Regeneration frequencies increased in response to treatment with ethylene inhibitors (aminoethoxyvinylglycine and silver thiosulfate) and an ethylene scavenger (mercury perchlorate). Treatment with the ethylene precursor 1-aminocyclopropane-1-carboxylic acid reduced the regeneration frequency, increased callus formation, and increased ethylene levels; similar results were obtained in response to treatment with the PA inhibitor methylglyoxal-bis(guanylhydrazone). By contrast, treatment with PAs (particularly spermidine and spermine) decreased ethylene levels, increased the regeneration frequency, and increased shoot bud formation. These results suggest a coordinated regulation of ethylene and polyamines because the suppression of ethylene levels using ethylene inhibitors, polyamines, or mercury perchlorate increased the in vitro regeneration frequency and morphogenic responses of Capsicum annuum L.     

‘神马’菊花花芽分化与内源多胺的关系

用薄层层析-荧光法测定菊花'神马'花芽分化期间顶芽和叶片中多胺的动态变化,分析了菊花花芽分化与多胺的关系.结果表明,花芽分化起始期顶芽中的腐胺(Put)含量急剧下降,此后在低水平上波动;叶片内Put则于总苞鳞片分化初期大幅上升,其后各阶段处于较低的水平.顶芽中精胺(Spm)与亚精胺(Spd)含量呈平行波动上升趋势,顶芽中Spin在小花原基分化初期直到花冠分化中期处于优势地位,而顶芽中Spd并无明显变化.顶芽、叶片中的Spin变化趋势相反,顶芽中Spm、Spd的含量变化趋势十分相似,但叶片中却呈交替性变化.结果显示,菊花花芽分化过程中,Put含量的降低有利于启动菊花花芽分化,后期Spm的增加有利于小花的分化,叶片可能向顶芽提供Spm,顶芽和叶片中的Spd与小花原基分化有密切关系.     

Specific polyamine ratios as indicators of Pinus caribaea microshoot rooting phases

Abstract

This study analysed the changes in the endogenous levels of total and individual polyamines (PAs) during the different rooting phases in Pinus caribaea microshoots: induction, induction-initiation and functional root system emergence. The analysis, performed using high-resolution liquid chromatography, demonstrated that during the inductive phase there is a drop in total PA content in all the fractions, with an increase in the degradation product diaminopropane. In the induction-initiation phase PAs are present in all the fractions. The microshoots with root nodules always presented higher Putrescine (Put) levels, whereas those that did not have them had similar spermidine and Put levels, while maintaining elevated spermine levels. The comparative analysis of microshoots with and without a rooting response indicates that high PA levels are maintained when root manifestation does not occur, coinciding with basal callus formation.     

Polyamines affect the cellular growth and structure of pro‐embryogenic masses in Araucaria angustifolia embryogenic cultures through the modulation of proton pump activities and endogenous levels of polyamines

Polyamines (PAs) are abundant polycationic compounds involved in many physiological processes in plants, including somatic embryogenesis. This study investigates the role of PAs on cellular growth and structure of pro‐embryogenic masses (PEMs), endogenous PA and proton pump activities in embryogenic suspension cultures of Araucaria angustifolia. The embryogenic suspension cultures were incubated with putrescine (Put), spermidine (Spd), spermine (Spm) and the inhibitor methylglyoxal‐bis(guanylhydrazone) (MGBG), respectively (1 mM). After 24 h and 21 days, the cellular growth and structure of PEMs, endogenous PA contents and proton pump activities were analyzed. The addition of Spm reduced the cellular growth and promoted the development of PEMs in embryogenic cultures, which could be associated with a reduction in the activities of proton pumps, such as H⁺‐ATPase P‐ and V‐types and H⁺‐PPases, and alterations in the endogenous PA contents. Spm significantly affected the physiology of the A. angustifolia somatic embryogenesis suspension, as it potentially affects cellular growth and structure of PEMs through the modulation of proton pump activities. This work demonstrates the involvement of exogenous PAs in the modulation of cellular growth and structure of PEMs, endogenous PA levels and proton pump activities during somatic embryogenesis. To our knowledge, this study is the first to report a relationship between PAs and proton pump activities in these processes. The results obtained in this study offer new perspectives for studies addressing the role of PAs and proton pump on somatic embryogenesis in this species.