Excretion of polyamines in alfalfa and tobacco suspension-cultured cells and its possible role in maintenance of intracellular polyamine contents

Changes in polyamines (PAs) in cells and cultivation media of alfalfa (Medicago sativa L.) and tobacco bright yellow 2 (BY-2) (Nicotiana tabacum L.) cell suspension cultures were studied over their growth cycles. The total content of PAs (both free and conjugated forms) was nearly 10 times higher in alfalfa, with high level of free putrescine (Put) (in exponential growth phase it represented about 65-73% of the intracellular Put pool). In contrast, the high content of soluble Put conjugates was found in tobacco cells (in exponential phase about 70% of the intracellular Put). Marked differences occurred in the amount of PAs excreted into the cultivation medium: alfalfa cells excreted at the first day after inoculation 2117.0, 230.5, 29.0 and 88.0 nmol g(-1) of cell fresh weight (FW) of Put, spermidine (Spd), spermine (Spm) and cadaverine (Cad), respectively, while at the same time tobacco cells excreted only small amount of Put and Spd (12.7 and 2.4 nmol g(-1) FW, respectively). On day 1 the amounts of Put, Spd, Spm and Cad excreted by alfalfa cells represented 21, 38, 12 and 15% of the total pool (intra- plus extra-cellular contents) of Put, Spd, Spm and Cad, respectively. In the course of lag-phase and the beginning of exponential phase the relative contents of extracellular PAs continually decreased (with the exception of Cad). On day 10, the extracellular Put, Spd, Spm and Cad still represented 11.3, 10.9, 2.1 and 27% of their total pools. The extracellular PAs in tobacco cells represented from day 3 only 0.1% from their total pools. The possible role of PA excretion into the cultivation medium in maintenance of intracellular PA contents in the cells of the two cell culture systems, differing markedly in growth rate and PA metabolism is discussed.     

黄瓜花性别分化与内源多胺的关系

研究了黄瓜雌、雄花几个主要发育时期和性别逆转过程中内源多胺的变化。结果表明 ,雄花在不同发育时期 ,内源腐胺含量均高于雌花 ,腐胺含量的显著升高伴随着花粉粒的形成 ,高腐胺含量是雄花发育的特征。雌花在大孢子母细胞时期以后直到雌花发育成熟 ,其内源尸胺含量均高于雄花 ,高尸胺含量可能有利于雌花的发育。高水平的内源多胺、精胺和亚精胺可能有利于雌花大孢子母细胞的形成。亚精胺和腐胺含量随着大小孢子四分体形成和大孢子核的连续分裂而分别表现下降和上升。雌性系黄瓜经硝酸银诱导雄花处理后 ,茎尖内源亚精胺含量下降 ,腐胺含量上升 ,从而诱导雄花形成 ;雄性系黄瓜经乙烯利诱导雌花处理后 ,茎尖内源亚精胺含量上升 ,腐胺含量下降 ,从而诱导雌花形成     

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。     

盐适应过程中香根草内源游离态、结合态、束缚态多胺含量的变化

研究了不同浓度NaCl胁迫下,香根草（Vetiteria zizanioides）根、叶中的游离态、结合态、束缚态多胺（PAs）[包括腐胺（Put）,尸胺（Cad）,亚精胺（Sod）和精胺（Spm）]含量的变化。在中度盐胁迫（100,200mmol L^-1NaCl）9天时,香根草基本能够正常生长,但在重度盐胁迫（300mmol L^-1NaCl）下,其生长受到严重抑制。在上述3个不同浓度的NaCl胁迫下,香根草根、叶中游离态Put,Cad,spd,Stma和总的游离态PAs含量明显下降,在高盐浓度下下降幅更大;结合态Put,Cad,Sod,Spm和总的结合态PAs含量显著上升,但在重度盐胁迫下升幅较小或与对照相当;束缚态Put,Cad和总的束缚态PAs含量均减少,而束缚态Spd和Spm含量在叶中是下降的,在根中则增加,且在中度盐胁迫下更明显。对根和叶片而言,除游离态（Spd＋Spm）,Put比值在重度盐胁迫下较对照显著下降外,其它游离态、结合态、束缚态和总的（Spd＋Spm）／Put比值在不同盐胁迫下均上升,在中度盐胁迫下更明显。这表明,维持多胺总量的稳态和较高的（Spd＋Spm）／Put比值是香根草适应中度盐胁迫的一个重要机制。     

Role of some growth regulators on cytogenetic activity of barley under salt stress

The effects of gibberellic acid (GA₃), kinetin (KIN), benzyladenine and ethylene (E) on mitotic activity and chromosomal aberrations in root tips of barley seeds (Hordeum vulgare L. cv. “Bülbül 89”) germinated under salt stress were investigated. It was determined that all of these plant growth regulators (PGRs) decreased mitotic index in root tips of barley seeds germinated at 20 °C and in distilled water. Furthermore, some of the PGRs studied increased significantly the frequency of chromosomal aberrations. The frequency of chromosomal aberrations in seeds treated with E and KIN was considerably higher than in the seeds germinated under nonstress conditions. The inhibitory effect of salt stress on mitotic index increased with increasing salt concentration (0.30, 0.35, 0.40 and 0.45 molal, m). GA₃ and KIN pretreatments showed a successful performance in ameliorating the negative effects of increasing salinity on mitotic activity. The number of chromosomal aberrations also increased with increasing NaCl concentration. However, most of the PGR pretreatments studied alleviated the detrimental effects of increasing salinity on chromosomal aberrations. KIN pretreatment at 0.30 and 0.35 m salinity could not rescued the cytogenetic activity of salt stress on this parameter.     

Chromotoxic effects of exogenous hydrogen peroxide (H<Subscript>2</Subscript>O<Subscript>2</Subscript>) in barley seeds exposed to salt stress

The effect of exogenous hydrogen peroxide (H₂O₂) on mitotic activity and chromosomal aberrations in root tip meristems of barley (Hordeum vulgare L. var. Tokak 157/37) germinated under salinity was analyzed. The inhibitory effect of salinity on mitotic index and the frequency of chromosomal aberrations increased with increasing salt concentration (0.00 control, 0.35, 0.40, 0.45 M, molal NaCl). The frequency of chromosomal aberrations of seeds germinated in medium with 0.40 M NaCl after pretreatment with H₂O₂ (30 μM, micromolal) was significantly higher than the control group. The highest concentration of NaCl (0.45 M) together with H₂O₂ caused total inhibition of germination. In this study, the intention was to determine the performance of H₂O₂ in alleviating detrimental effect of salt stress on mitotic activity and chromosomal aberrations. However, H₂O₂ did not reduce the detrimental effect of NaCl on these parameters. Also, it caused higher chromotoxic effect compared to those of control groups.     

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.     

Are polyamines directly involved in silymarin production in the milk thistle [<Emphasis Type="Italic">Silybum marianum</Emphasis> (L.) Gaernt]?

The possible role of polyamines (PAs) in the regulation of silymarin (Sm) production in milk thistle [Silybum marianum (L.) Gaernt] cell suspension cultures was studied in a young cell culture line (H2 line) and in a synchronized cell line (>3 years; H1 line). The effect of two exogenous PAs, putrescine (Put) and spermidine (Spd), and a number of metabolic inhibitors (L-canavanine, DL-α-difluoromethylornithine, methylglyoxal-bis-guanylhydrazone, cyclohexylamine) on the production of Sm during the growth cycle were analyzed. The results suggest that PAs are not directly involved in the Sm synthesis pathway. In our cell culture system, Sm production and PA contents were determined by the age of the suspension culture cells: with increasing age, the suspension culture cells showed a decreasing capacity to reach the stationary phase during prolonged subculture that was associated with a decreased production of Sm, a steady increase in PA content, and a constant Put/Spd ratio. The synchronization of dividing cells from the S. marianum H1 line did not modify this behaviour. In young cell suspensions, maximum Sm production occurred in the stationary phase, concurrent with the cellular PA contents reaching their minimum value. At the start of the stationary phase, the high percentage of cells in the growth phases (G₀/G₁) and a transient increase in the Put/Spd ratio were accompanied by maximum Sm production and a blockade of cell division.     

Stress-dependent accumulation of spermidine and spermine in the halophyte Mesembryanthemum crystallinum under salinity conditions

We studied the effects of chloride salinity (300 and 500 mM NaCl) on the content of free polyamines (PAs) from putrescine (Put) family in Mesembryanthemum crystallinum L. leaves and roots. The contents of Put and spermidine (Spd) in leaves increased temporarily, achieving the highest values on the third day of salinity treatment; thereafter (by days 7–14), they dropped sharply. The content of spermine (Spm) increased gradually, and its high level was maintained until the end of experiment. The dynamics of Spm accumulation in leaves under salinity conditions resembled that of phosphoenolpyruvate carboxylase (PEPC), a key enzyme of the water-saving CAM pathway of photosynthesis. This indicates indirectly the involvement of Spm in the common ice plant adaptation to salinity. A decrease in the molar ratios of Spd to Spm in the leaves under salinity conditions could point to the acceleration of Spm biosynthesis (accumulation) during plant adaptation, whereas the levels of Spm precursors, Put and Spd, did not increase. This phenomenon could be explained by an accelerated conversion of Spd into Spm, an active liberation of free Spm from its conjugates, or changes in the rates of studied PA biosynthesis and degradation under salinity. At the same time, the intracellular concentration of ethylene rose under these conditions. It was supposed and then demonstrated, that the pathway of ethylene biosynthesis and that of the synthesis of Put family PAs compete under severe salinity conditions. This competition might be based on the disturbances in sulfur metabolism and a decrease in the methionine content, an immediate precursor of S-adenosyl-L-methionine.     

Polyamine and nitric oxide levels relate with morphogenetic evolution in somatic embryogenesis of <Emphasis Type="Italic">Ocotea catharinensis</Emphasis>

In this study we examined the effect of polyamines (PAs) putrescine (Put), spermidine (Spd) and spermine (Spm) on growth, morphology evolution, endogenous PAs levels and nitric oxide (NO) release in Ocotea catharinensis somatic embryo cultures. We observed that Spd and Spm reduced culture growth, permitted embryo morphogenetic evolution from the earliest to last embryo development stages, increased endogenous PAs levels, and induced NO release in O. catharinensis somatic embryos. On the other hand, Put had little effect on these parameters. Spd and Spm could successfully be used to promote somatic embryo maturation in O. catharinensis. The results suggest that Spd and Spm have an important role during the growth, development and morphogenetic evolution of somatic embryos, through alterations in the endogenous nitric oxide and PAs metabolism in this species.     

Polyamines promote root elongation and growth by increasing root cell division in regenerated Virginia pine (Pinus virginiana Mill.) plantlets

Polyamines have been demonstrated to play an important role in adventitious root formation and development in plants. Here, we present a detailed analysis of influence of exogenously added polyamines on adventitious root development and its relationship to cold tolerance in Virginia pine (Pinus virginia Mill.). Our results demonstrated that polyamines putrescine (Put), spermidine (Spd), and spermine (Spm) at 0.001 mM improve rooting frequency and promote root elongation. Put, Spd, and Spm at 0.01–1 mM decrease rooting frequency and reduce root elongation root elongation. Measurements of diamine oxidase (DAO, EC 1.4.3.6) and polyamine oxidase (PAO, EC 1.4.3.4) activities showed that higher DAO and PAO enzyme activities were obtained when high concentrations of polyamines were applied and when plantlets were treated for 5–7 week at 4°C and 16°C. Survival rate of plantlets increased with the treatment of polyamines at low temperature. Polyamines increased mitotic index of cells in root tips of regenerated plantlet cultured on medium containing 0.001 μM Put, Spd, or Spm, but did not increase mitotic index in tissues of needle tips of the same plantlets. These results demonstrated that polyamines promote root elongation and growth by increasing root cell division in regenerated Virginia pine plantlets.     

Abscisic acid increases the content of free polyamines and delays mitotic activity induced by spermine in isolated embryonic axes of chick-pea seeds

The effects of abscisic acid (ABA) on the free polyamine: spermine (Spm), spermidine (Spd), putrescine (Put) and cadaverine (Cad) content, mitotic index (MI) and DNA synthesis have been studied in embryonic axes isolated from chick-pea ( Cicer ariennum L.) seeds. Spermine and spermidine decreased in controls during the first 24 h of germination, the former by 70% and the latter by 20%. This decrease was slowed down by ABA (5 μ M or 25 μ M ) in response to its concentration; at 24 h the values for Spm and Spd were 3-fold and 1.5-fold those of the controls, respectively. Exogenous ABA induced the synthesis of Put from 0 to 24 h, by which time the content of this polyamine had doubled. The controls at 24 h registered the same Put content as those with 25 μ M ABA, suggesting that ABA had ceased to induce Put synthesis. The pattern for cadaverine ABA-induced synthesis was similar to that of Put. From 18 to 24 h, however, the level of Cad in the controls was 3 times higher than in the treatments. The mitotic index in meristem cells reached a maximum at 30–36 h, when cell elongation stopped in the sub-apical region. This maximum was lowered by exogenous ABA in response to the concentration and occurred 12 h earlier in the presence of Spm (0.1–1.0 m M ), which induced high MI levels during the period studied. With 10 m M Spm, two complete waves of mitosis were observed, but when Spm and ABA were added together, the tetramine had no observable effect. Spermine stimulated ³H-thymidine uptake as well as DNA synthesis, with a maximum at 12 h, but did not counteract the inhibitory effect of ABA. Our results suggest that ABA causes changes in mitotic activity that Spm cannot overcome.     

Effect of NaCl and Polyamines on Plasma Membrane Lipids of Wheat Roots

Caryopses of a salt sensitive wheat cultivar (Triticum aestivum L. cv. Giza 163) were presoaked in 2.5 mM putrescine (Put), 5 mM spermidine (Spd) or 2.5 mM spermine (Spm) for 24 h and then subjected to 150 mM NaCl added to the growth medium for 15 d. Effects of NaCl and polyamines (PAs) on plasma membrane (PM) lipids, phospholipids, fatty acids, and free sterols were determined. NaCl treatment caused a decrease in total phospholipids, increase in saturated fatty acids and altered distribution of sterols and phospholipids. NaCl also induced increase in sterol/phospholipid ratio. PAs treatments (particularly Put and Spd) counterbalanced the NaCl deleterious effects on PM lipids.     

Long-sized oligogalacturonides inhibit,whereas spermidine enhances,xylogenesis in tobacco leaf explants

Abstract

Long-sized oligogalacturonides (OGs) are plant cell wall fragments involved in defence responses and developmental processes. A hormone/OG interaction in the control of different organogenic processes is known. However, hormones also modulate polyamine (PA) effects on organogenesis. Furthermore, OGs are known to affect mitotic activity leading to specific morphogenic events, and PAs are known to affect mitotic activity leading to xylogenesis. Thus, it may be reasonable to assume that OGs and PAs affect mitotic activity in the same cell types, and in the same hormone-induced morphogenic processes, e.g., xylogenesis. To gain further insight into this aspect, the effects of OGs, and of putrescine (Put) and spermidine (Spd), on auxin (indoleacetic acid, IAA) plus benzyladenine (BA)-induced morphogenesis in tobacco leaf explants were investigated histologically. The effect of PA biosynthetic inhibitors in the culture medium was also monitored, as well as the combined application of the inhibitor with the corresponding PA. Results show that vascular mitoses consistently occurred in the control (IAA+BA-treated) explants, leading exclusively to xylogenic nodule formation. The application of OGs resulted in an inhibition of vascular mitoses, and into a strong reduction of vascular nodule formation. By contrast, Spd enhanced both vascular mitoses and nodule formation, and Put was less effective than Spd on both events. Taken together, the results reveal a new biological activity of OGs and Spd in morphogenesis, obtained under the same hormonal conditions, and in the same tissue (i.e., the vascular parenchyma), namely the inhibition of xylogenesis by OGs, and its promotion by Spd. The fact that the effects of Spd and OGs on this morphogenic event may involve a different relationship with auxin is discussed.     

Effect of polyamines on in vitro somatic embryogenesis in <Emphasis Type="Italic">Momordica</Emphasis><Emphasis Type="Italic">charantia</Emphasis> L.

The effects of exogenous polyamines (PAs) on enhancement of somatic embryogenic calli was investigated in Momordica charantia L. in vitro. Induction of somatic embryogenesis (SE) in leaf explants of M. charantia after 21 days of culture in Murashige and Skoog (MS) medium was determined using scanning electron microscopy. During induction of SE there were high titers of Putrescine (Put) as compared to Spermidine (Spd) and Spermine (Spm), a prerequisite for cell division. Addition of PAs to the embryogenic media resulted in an increase in fresh weights and number of somatic embryos of 21-day old embryogenic calli. Put at a concentration of 1 mM showed maximum increase in fresh weights of embryogenic calli (5 fold) and number of somatic embryos produced per 0.2 g of callus (2.5 fold). Moreover addition of PAs to the embryogenic media resulted in lowering of endogenous free PA level of 21-day old embryogenic calli. Thus, when the media was supplemented with exogenous PAs a positive correlation was found to exist between Somatic Embryogenesis enhancement and decrease in endogenous free PA levels.     

离体黄瓜子叶节花芽分化与内源激素及多胺的关系

用高效液相色谱法(HPLC)测定了黄瓜子叶节花芽分化期(0-6天)内源激素及多胺的变化。结果显示,子叶培养0-2天生长素(IAA)、赤霉素(GA_3)、玉米素(ZT)、脱落酸(ABA)等4种内源激素均明显下降,4-5天略有上升,表明0-2天IAA、GA_3和ABA的剧降有利于花原基形成,3-5天较高的ZT含量有利于花器官原基的形成。除腐胺(Put)外,精胺(Spm)、亚精胺(Spd)、尸胺(Cad)在0-1天均下降,1-4天上升,4-5天再下降,Put在0-1天急剧上升,而后持续下降,表明高水平的内源多胺总量和Put可能有利于花原基分化,2天后Spm含量上升有利于花器官原基分化,而Cad含量变化可能是区别花芽和营养芽分化的特征之一。     

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.     

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.     

Exogenous Putrescine Reduces Sodium and Chloride Accumulation in NaCl-Treated Calli of the Salt-Sensitive Rice Cultivar I Kong Pao

In order to gain information on the putative involvement of polyamines (PAs) in the response of rice cells to salinity, mature embryo-derived calli issued from the salt-sensitive cultivar I Kong Pao were exposed for 3 months to the simultaneous presence of NaCl (0, 150 and 300 mM) and exogenous polyamines (putrescine (Put): 1 and 10 mM; spermidine (Spd): 1 and 10 mM; spermine (Spm): 1 mM). Callus growth, endogenous PAs, Na⁺, K⁺ and Cl⁻ concentrations were quantified and analysed in relation to cell viability based on 2,3,5-triphenytetrazolium chloride (TTC) reduction. All exogenous PAs were efficiently absorbed from the external medium. Exogenous Put 1 mM clearly stimulated growth of salt-stressed calli in relation to a decrease in both Na⁺ and Cl⁻ accumulation. In contrast, Spd 10 mM and Spm 1 mM exacerbated the deleterious impact of NaCl on callus growth and induced a decrease in K⁺ concentration. While Put helped in the maintenance of cell viability, Spd 10 mM and Spm 1 mM decreased cell viability, mainly in relation to an inhibition of the alternative respiratory pathway. It is proposed that Put may assume positive functions in salt stress resistance in rice.