Putrescine Influences Growth and Production of Coumarins in Hairy Root Cultures of Witloof Chicory (Cichorium intybus L. cv. Lucknow Local)

The effect of putrescine (Put) on the growth and production of two coumarins, esculin and esculetin, in hairy roots of chicory (Cichorium intybus L. cv. Lucknow local) was examined. To study the role of Put on growth and production of coumarins, polyamine inhibitors, namely α-dl-difluromethylornithine and α-dl-difluromethylarginine were used at 1 mM concentration. Put treatment at 1.5 mM produced a 1.9-fold increase in the growth of hairy roots, as well as the production of esculin and esculetin. The treatments with polyamine (PA) inhibitors resulted in much lower growth and production of coumarins compared with both 1.5-mM Put treatment and the control. Both free and conjugated PAs were studied over the whole culture period, and conjugates of all three PAs, namely Put, spermidine, and spermine, were higher than free PAs throughout the culture period. The treatments with PA inhibitors showed lower levels of endogenous PAs compared with Put-treated samples. The treatment with 1.5 mM Put showed maximum accumulation of endogenous conjugated Put (2,098 ± 157 nmoles gm⁻¹ fresh weight). The production of esculin and esculetin was strictly correlated with growth in all treatments. Put at 1.5 mM resulted in greater length of primary root (18.3 ± 1.4 cm) as compared with the control (11 ± 0.9 cm) and larger numbers of secondary and tertiary roots. Received July 14, 1999; accepted October 5, 1999     

Influence of Polyamines on Growth of Hairy Root Cultures of Witloof Chicory (Cichorium intybus L. cv. Lucknow Local) and Formation of Coumarins

The effect of polyamines (putrescine, spermidine, and spermine) was examined for growth and production of two coumarins, esculetin and esculin, in the hairy roots of chicory (Cichorium intybus L. cv. Lucknow local). Of the polyamines administered, 1.5 mm putrescine alone resulted in a 2.3-fold higher increase in the growth of hairy roots as well as in the production of esculetin and esculin, which was 3.37 times more than that of the control on day 21. The endogenous level of conjugated putrescine was more than fivefold that of free putrescine levels in untreated samples. The production of esculetin and esculin in hairy root cultures strictly correlated with growth in all of the treatments. Putrescine at 1.5 mm resulted in a greater length of primary root (18.29 ± 1.37 cm) compared with the control (10.96 ± 0.82 cm) and more secondary and tertiary roots. This study also provides insight into the morphogenetic changes that occur in roots in response to the external supply of polyamines. Received July 20, 1998; accepted January 19, 1999     

Putrescine and Silver Nitrate Influences Shoot Multiplication, In Vitro Flowering and Endogenous Titers of Polyamines in Cichorium intybus L. cv. Lucknow Local

Abstract The influence of putrescine (Put) and AgNO₃ on shoot multiplication, in vitro flowering and endogenous titers of polyamines in Cichorium intybus L. cv. Lucknow local was investigated. Exogenous administration of Put at a concentration of 40 mM resulted in maximum tissue response in terms of shoot numbers (34.6 ± 2.61) and shoot lengths (7.6 ± 0.57 cm) on MS media supplemented with 2-iP (2.0 mg L⁻¹) and GA₃ (0.5 mg L⁻¹) as observed on the 35^th day. Exogenous application of 40 μM AgNO₃ resulted in maximum shoot number (36.8 ± 2.63) and shoot lengths (7.9 ± 0.76 cm) on day 35 on the same media. Endogenous titers of conjugated spermidine decreased sharply from day 7–21, whereas endogenous conjugated spermine levels peaked on day 28 (1265 ± 94.9 nmoles g⁻¹ FW), after treatment with 40 mM Put. Whereas, AgNO₃ (40 μM) fed samples resulted in higher titers of endogenous conjugated spermine (1405 ± 105.6 nmoles g⁻¹ FW, 3.62 fold over control) on day 14. All other treatments showed decreasing endogenous levels during the whole culture period. Both Put (40 mM) and AgNO₃ (40 μM) resulted in floral initiation and floral development on day 28 and 14 (3.76 ± 0.16, 4.2 ± 0.21 flowers per shoot apices), respectively. To investigate the role of Put (40 mM) and AgNO₃ (40 μM) on morphogenetic response and endogenous conjugated polyamine titers in shoots of chicory, polyamine inhibitors (DFMA and DFMO) were used. The morphogenetic response and the endogenous conjugated pool of polyamines were diminished in DFMA and DFMO treatments, but could be restored by addition of Put (40 mM) and AgNO₃ (40 μM). Under exogenous Put feeding, ethylene production was reduced in shoot cultures of chicory. This study shows for the first time the influence of polyamines on multiple shoot initiation from axillary buds of C. intybus L. cv. Lucknow local and also indicates the promotive effect of Put and AgNO₃ on autoregulation of polyamine biosynthesis, thereby regulating in vitro flowering, the endogenous pool of polyamines and shoot multiplication. Received 12 November 1999; accepted 11 February 2000     

Oxidative stress and fluctuations of free and conjugated polyamines in the halophyte Mesembryanthemum crystallinum L. under NaCl salinity

The accumulation of conjugated and free polyamines in plants is very important for their protection against oxidative stress induced by abiotic factors. In the present study, the species halophytic plant Mesembryanthemum crystallinum L. was used as a model system in which the process of Crassulacean Acid Metabolism induction is linked with oxidative stress, especially under salinity conditions. A comparative analysis of the content of free polyamines, perchloric (PCA)-soluble and PCA-insoluble conjugated polyamines in mature leaves and roots was carried out with plants exposed to salinity. It was found that adult leaves and roots under normal conditions or salinity (400 mM NaCl) contained all types of free polyamines (putrescine, spermidine, spermine, and cadaverine). In leaves only PCA-insoluble conjugates were found, which showed a tendency to grow with increased duration of salt action (1.5–48 h). In contrast to leaves, in roots all forms of polyamine conjugates (PCA-soluble and -insoluble) were detected. However, the formation of all conjugates, especially PCA-soluble forms in roots, was sharply inhibited by salt shock (400 mM NaCl, 1.5 h) or exogenous cadaverine (1 mM) treatment. PCA-soluble conjugates of cadaverine in roots were found only when the treatment was carried out in combination with aminoguanidine (1 mM), as a result of diamine oxidase inhibition and consequently a decreasing of H₂O₂ production in plant cells. The activation of diamine oxidase and guaiacol peroxidase by NaCl or exogenous cadaverine was observed in leaves and roots. Thus, the activation of oxidative degradation of polyamines combined with H₂O₂–peroxidase reaction in cells are involved in the regulation of free and conjugated polyamines titers under salinity.     

Oxidative stress and fluctuations of free and conjugated polyamines in the halophyte Mesembryanthemum crystallinum L. under NaCl salinity

The accumulation of conjugated and free polyamines in plants is very important for their protection against oxidative stress induced by abiotic factors. In the present study, the species halophytic plant Mesembryanthemum crystallinum L. was used as a model system in which the process of Crassulacean Acid Metabolism induction is linked with oxidative stress, especially under salinity conditions. A comparative analysis of the content of free polyamines, perchloric (PCA)-soluble and PCA-insoluble conjugated polyamines in mature leaves and roots was carried out with plants exposed to salinity. It was found that adult leaves and roots under normal conditions or salinity (400 mM NaCl) contained all types of free polyamines (putrescine, spermidine, spermine, and cadaverine). In leaves only PCA-insoluble conjugates were found, which showed a tendency to grow with increased duration of salt action (1.5–48 h). In contrast to leaves, in roots all forms of polyamine conjugates (PCA-soluble and -insoluble) were detected. However, the formation of all conjugates, especially PCA-soluble forms in roots, was sharply inhibited by salt shock (400 mM NaCl, 1.5 h) or exogenous cadaverine (1 mM) treatment. PCA-soluble conjugates of cadaverine in roots were found only when the treatment was carried out in combination with aminoguanidine (1 mM), as a result of diamine oxidase inhibition and consequently a decreasing of H₂O₂ production in plant cells. The activation of diamine oxidase and guaiacol peroxidase by NaCl or exogenous cadaverine was observed in leaves and roots. Thus, the activation of oxidative degradation of polyamines combined with H₂O₂–peroxidase reaction in cells are involved in the regulation of free and conjugated polyamines titers under salinity.     

Influence of putrescine, silver nitrate and polyamine inhibitors on the morphogenetic response in untransformed and transformed tissues of Cichorium intybus and their regenerants

The addition of 40 mM putrescine (Put) to Murashige and Skoog's (MS) medium resulted in increased shoot multiplication and shoot growth in untransformed plants relative to transformed plants of Cichorium intybus L. Put at a concentration of 40 mM also resulted in flowering in both systems on the 28th day, with elevated titers of endogenous conjugated Put and spermine (Spm) in both untransformed and transformed plants. The addition of 40 µM AgNO₃ to untransformed axillary buds of C. intybus L. cultured on MS media resulted in increased shoot multiplication (36.9DŽ.63 shoots per culture) and increased shoot growth (7.82ǂ.76 cm) as compared to transformed ones (11.6ǂ.89 shoots per culture; 3.20ǂ.24 cm). Moreover, cultures treated with 40 µM AgNO₃ showed in vitro flowering on the 28th day in both systems, with the endogenous levels of conjugated spermine being higher in untransformed plants than in transformed ones. The morphogenetic response and the endogenous conjugated pool of polyamines were lower following !-DL-difluromethylarginine and !-DL-difluromethylornithine treatments; the addition of put (40 mM) and AgNO₃ (40 µM) restored these to normal levels. Under exogenous put feeding, ethylene production was lower in both the untransformed and transformed cultures. We believe that an interplay between polyamine and ethylene biosynthesis is involved in regulating the morphogenetic response in both transformed and untransformed shoots of C. intybus. The response to AgNO₃ and Put treatment was not altered by the transformation process.     

Levels of Polyamines and Kinetic Characterization of Their Uptake in the Soybean Pathogen Phytophthora sojae

Polyamines are ubiquitous biologically active aliphatic cations that are at least transiently available in the soil from decaying organic matter. Our objectives in this study were to characterize polyamine uptake kinetics in Phytophthora sojae zoospores and to quantify endogenous polyamines in hyphae, zoospores, and soybean roots. Zoospores contained 10 times more free putrescine than spermidine, while hyphae contained only 4 times as much free putrescine as spermidine. Zoospores contained no conjugated putrescine, but conjugated spermidine was present. Hyphae contained both conjugated putrescine and spermidine at levels comparable to the hyphal free putrescine and spermidine levels. In soybean roots, cadaverine was the most abundant polyamine, but only putrescine efflux was detected. The selective efflux of putrescine suggests that the regulation of polyamine availability is part of the overall plant strategy to influence microbial growth in the rhizosphere. In zoospores, uptake experiments with [1,4-¹⁴C]putrescine and [1,4-¹⁴C]spermidine confirmed the existence of high-affinity polyamine transport for both polyamines. Putrescine uptake was reduced by high levels of exogenous spermidine, but spermidine uptake was not reduced by exogenous putrescine. These observations suggest that P. sojae zoospores express at least two high-affinity polyamine transporters, one that is spermidine specific and a second that is putrescine specific or putrescine preferential. Disruption of polyamine uptake or metabolism has major effects on a wide range of cellular activities in other organisms and has been proposed as a potential control strategy for Phytophthora. Inhibition of polyamine uptake may be a means of reducing the fitness of the zoospore along with subsequent developmental stages that precede infection.     

Synergistic effect of auxins and polyamines in hairy roots of <Emphasis Type="Italic">Cichorium intybus</Emphasis> L. during growth,coumarin production and morphogenesis

Hairy roots of Cichorium intybus obtained by infecting with different Agrobacterium rhizogenes strains (LMG-150 and A20/83) were studied for total endogenous indole-3-acetic acid (IAA) levels and indole-3-acetic acid oxidase (IAAO) activity. The roots initiated by LMG- 150 showed higher endogenous IAA levels as well as IAAO activity as compared to the roots from A20/83. Coumarin production in roots obtained by both of these strains strictly correlated with growth, with higher content in the roots obtained by LMG- 150. Moreover roots from LMG-150 showed increased growth index, length of primary roots and number of secondary and tertiary roots. The roots derived from LMG-150 were studied for total endogenous IAA and IAAO activity under the exogenous administration of polyamines and fungal elicitors. The treatment with putrescine (Put) at 1.5 mM level showed maximum endogenous IAA levels and IAAO activity as compared to the control and other polyamine administration, it also supported faster growth in terms of biomass accumulation, and total coumarin production. Of the various treatments, mycelial extract (ME) and culture media filtrate (CMF) of Pythium aphanidermatum and Phytopthora parasitica var. nicotiana, the treatment with 1 % CMF of P. parasitica var. nicotiana, resulted in maximum IAA levels and IAAO activity, which was supported by maximum biomass, coumarin production as compared to the control and other elicitor treatments. Two different regenerants of chicory obtained through A. rhizogenes LMG-150 designated as T-I and T-II, were studied for total endogenous IAA levels and IAAO activity. T-II showed higher titers of IAA with higher activity of IAAO as compared to T-I. Endogenous titer of IAA and IAAO activity was found to be maximum in transformed roots as compared to T-I, T-II, normal roots and normal plants. Our work showed a variation in endogenous auxin levels in these transformed plants. There exists a synergistic effect of endogenous IAA titers and polyamines in regulating root morphogenesis. Fungal elicitors influenced growth and coumarin production and an elicitor preparation of 1 % CMF of P. parasitica var. nicotiana gave spontaneous regeneration of shoots. The implications of these results are discussed.     

Influence of polyamines on growth and formation of secondary metabolites in hairy root cultures of Beta vulgaris and Tagetes patula

Growth of hairy roots of Beta vulgaris, which produces betalaines, and of Tagetes patula, which produces thiophenes, was studied under the influence of externally treated polyamines. Of the three polyamines, viz. putrescine, spermidine and spermine, administered singly at 1.5 mM concentration, putrescine and spermidine at 0.75 mM concentration influenced increase in the accumulation of biomass of B. vulgaris and T. patula hairy roots by 1.42 and 1.30 fold over the control. Whereas, the treatment of spermine (1.5 mM) alone resulted in decrease in the biomass in both the systems. Combined administration of putrescine (0.75 mM) and spermidine (0.75 mM) enhanced growth in both B. vulgaris and T. patula than that observed in individual treatments. Polyamines administered alone or in combination did alter production of betalaine and thiophene content. Dose response experiments showed that, when putrescine and spermidine was administered at 0.75 mM concentration, it resulted in maximum biomass and production of beta-laine and thiophene in B. vulgaris and T. patula respectively as compared to the control and the media treated with double and triple strength of nitrates and in combination with putrescine and spermidine at equimolar concentration. In B. vulgaris and T. patula hairy root cultures, endogenous spermine titers were maximum in putrescine and spermidine 0.75 mM each treated, cultures, which was 1.63 and 2.0 fold higher than in control on 28^th and 35^th days respectively.     

Levels of polyamines and kinetic characterization of their uptake in the soybean pathogen Phytophthora sojae

Polyamines are ubiquitous biologically active aliphatic cations that are at least transiently available in the soil from decaying organic matter. Our objectives in this study were to characterize polyamine uptake kinetics in Phytophthora sojae zoospores and to quantify endogenous polyamines in hyphae, zoospores, and soybean roots. Zoospores contained 10 times more free putrescine than spermidine, while hyphae contained only 4 times as much free putrescine as spermidine. Zoospores contained no conjugated putrescine, but conjugated spermidine was present. Hyphae contained both conjugated putrescine and spermidine at levels comparable to the hyphal free putrescine and spermidine levels. In soybean roots, cadaverine was the most abundant polyamine, but only putrescine efflux was detected. The selective efflux of putrescine suggests that the regulation of polyamine availability is part of the overall plant strategy to influence microbial growth in the rhizosphere. In zoospores, uptake experiments with [1,4-(14)C]putrescine and [1,4-(14)C]spermidine confirmed the existence of high-affinity polyamine transport for both polyamines. Putrescine uptake was reduced by high levels of exogenous spermidine, but spermidine uptake was not reduced by exogenous putrescine. These observations suggest that P. sojae zoospores express at least two high-affinity polyamine transporters, one that is spermidine specific and a second that is putrescine specific or putrescine preferential. Disruption of polyamine uptake or metabolism has major effects on a wide range of cellular activities in other organisms and has been proposed as a potential control strategy for Phytophthora. Inhibition of polyamine uptake may be a means of reducing the fitness of the zoospore along with subsequent developmental stages that precede infection.     

Role of polyamines in the cytokinin-dependent physiological processes. I. Effect of benzyladenine on polyamine levels during chloroplast differentiation in the tissue culture of Dianthus caryophyllus

Tissue culture of Dianthus caryophyllus L. (cv. William Sim.) obligatory requiring N⁶-benzyladenine for greening provides a good system to study the interactions between cytokinins and polyamines. Polyamines were analyzed as dansyl derivatives which are separated by thin layer chromatography and detected by fluorescence spectrophotometry. Green callus growing on benzyladenine — containing medium showed decrease in the contents of free, conjugated and bound putrescine and spermidine in comparison to chlorophyll-less callus (control callus) growing on cytokinin-free medium. The level of spermine free, conjugated and bound forms increased about 6 %, 77 % and 28 % respectively in tissue culture growing in the presence of cytokinin. Spermidine was dominant polyamine bound to chromatin isolated from control callus. Chromatin isolated from green callus was characterized by a lower level of each polyamine in comparison to chlorophyll-less callus. Polyamines were found in plastid membrane fraction isolated from chlorophyll-less and green callus. A significant increase the levels of polyamines (putrescine, spermidine and spermine) bound to plastid membranes in green callus (+ benzyladenine) in comparison to chlorophyll-less callus (− benzyladenine) was observed. Additionaly, methylglyoxal-bis(guanylhydrazone) an inhibitor of S-adenosylmethionine decarboxylase depressed the greening process. Our results suggest that cytokinin-induced chloroplast differentiation in carnation tissue culture may be partly mediated through the polyamines bound to thylakoid membranes. A possible role of polyamines during cytokinin-induced formation of photosynthetic apparatus is discussed.     

Growth kinetics, polyamine pattern and biosynthesis in hairy root lines of Nicotiana tabacum

The possibility of a relation between the expression of root inducing (Ri) T-DNA genes of Agrobacterium rhizogenes and changes in polyamine metabolism has been explored in fast-growing tobacco hairy roots. Transformed root cultures have been established on hormone-fee medium; they came from transgenic plants of Nicotiana tabacum L. cv. Xanthi with different altered phenotypes, designated transformed (T) and supertransformed (T'). T and especially T' roots developed more rapidly both by elongation and lateral branching, and showed a higher growth rate than the untransformed control. After 3 weeks in culture, normal roots showed a very reduced meristematic zone, and flow cytometric analysis indicated that 2C nuclei were predominant in the apical parts in contrast to T and T' roots, in which endopolyploidisation also appeared. Putrescine, spermidine and traces of spermine were present in all the samples, both in free and in conjugated forms. Putrescine was the major polyamine detected in controls and in transformed roots. At the time of excision, the polyamine levels were similar in normal, T and T' roots. Significant differences were found during the progression of growth, particularly in the TCA-insoluble fraction in which polyamines varied differently according to the type of roots, increasing considerably in T roots on day 8, then decreasing. The lower polyamine contents found in growing transformed roots were concomitant to low arginine (EC 4.1.1.19) and ornithine (EC 4.1.1.17) decarboxylase activities. It is suggested that polyamine levels and related enzyme activities are linked to growth kinetics rather than being a consequence of foreign gene expression.     

Proline controls the level of polyamines in common sage plants under normal conditions and at UV-B irradiation

Common sage (Salvia officinalis L.) plants grown in water culture to the stage of 4–5 true leaves were treated for 12, 24, 36, or 48 h with proline added to nutrient medium to a final concentration of 5 mM, or irradiated with UV-B light (12.3 kJ/m² for 10 min), or subjected to combined action of these factors. In these plants, activity of proline dehydrogenase (PDH), the content of proline, and the contents of free and conjugated polyamines were determined in the leaves and roots. It was shown that, in control plants, the content of endogenous proline was close to zero. In the presence of proline in medium, its total content in the roots was 9 μmol/g fr wt in 12 h of exposure, whereas in the leaves the content of proline increased only in 24 h and achieved only 1 μmol/g fr wt. The content of free putrescine increased in the leaves and especially in the roots after 10-min irradiation with UV-B light. The biosynthesis of putrescine was induced in the presence of proline in medium and was observed earlier than after UV-B irradiation. UV-B irradiation affected not only the synthesis of putrescine but also that of spermidine and spermine; it also induced accumulation of their soluble conjugates. Exogenous proline enhanced putrescine synthesis but inhibited the formation of polyamine soluble conjugates. At combined treatment of the two factors, the content of free putrescine in the leaves displayed a tendency to the rise and in the roots, to the decrease. At the same time, the content of polyamine free conjugates increased in both leaves and roots. All these facts could be considered as an indirect indication of relationship between proline and polyamine biosyntheses. We can also state that an artificially created high proline concentration in common sage tissues characterized of its low constitutive level resulted in disturbances in the homeostasis of low-molecular cell metabolites and induced a requirement in its restoration by diverse ways. This agrees with activation of PDH, a key enzyme of proline degradation. Induction of polyamine biosynthesis and changes in the content of their soluble conjugates might be one of the ways for such restoration. Under stress conditions, the high proline concentration is not toxic for plants because polyamines and proline are the components of the plant defense system, thus weakening damaging effects of abiotic stressors.     

In vivo nuclear-magnetic-resonance analysis of polyamine and alkaloid metabolism in transformed root cultures of Datura stramonium L.: evidence for the involvement of putrescine in phytohormone-induced de-differentiation

The utilisation and accumulation of ¹⁵N-labeled metabolites by a ¹⁵N-labeled transformed root culture of Daturastramonium L. was investigated by in vivo ¹⁵N-nuclear-magnetic-resonance (NMR) spectroscopy. After resuspension in spent growth medium, the pools of [¹⁵N]glutamate and [¹⁵N]glutamine were rapidly depleted and there was an increase in the ¹⁵N-NMR signals from conjugated putrescines and hyoscyamine. The signal from the conjugated putrescines passed through a maximum 2 d after the roots were resuspended, and it was concluded that putrescine could be stored as putrescine conjugates prior to its utilisation in other pathways. The transient accumulation of ¹⁵N-label in the hydroxy-cinnamoylputrescines was reduced when the de-differentiation of the root cultures into a suspension culture was initiated by exposure to a medium containing α-napthaleneacetic acid and kinetin. This led to the hypothesis that phytohormone-induced de-differentiation of the root cultures required the presence of free polyamines, and this was tested using two potent inhibitors of putrescine biosynthesis, dl-α-difluoromethylarginine and dl-α-difluoromethylornithine. In-vivo ¹⁵N-NMR spectra of roots grown in ¹⁵N-enriched medium supplemented with these inhibitors showed that the ¹⁵N-labelling of the conjugated polyamines and hyoscyamine was markedly reduced. dl-α-difluoromethylarginine also prevented the phytohormone induced de-differentiation of the root cultures, and this effect could be reversed by the supply of exogenous putrescine. Thus the supply of putrescine appears to play a crucial role in mediating the phytohormone induced de-differentiation of the root culture. Received: 13 September 1997 / Accepted: 12 November 1997     

Changes in endogenous polyamine levels are associated with differentiation in Dictyostelium discoideum.

This is the first report correlating levels of polyamines and its fractions with differentiation in Dictyostelium discoideum. Temporal changes in endogenous levels of free, conjugated and bound putrescine, spermidine and spermine were analysed at critical stages of morphogenesis in this organism. No spermine was found at any given stage and putrescine was the most abundant polyamine. There was a sharp increase in the levels of both free (and total) and conjugated forms of putrescine and spermidine at the slug stage as compared to the growth phase. The levels of putrescine and spermidine were found to be higher in isolated prespore cells as compared to the prestalk cells. Remarkably, the levels of polyamine decreased at the early culminant stage. Data suggest that a moderate level of polyamines is needed for growth but it is important to have high levels of polyamines at the time of differentiation.     

NaCl处理下大麦根系质膜微囊结合多胺与Na+/H+逆向运输的关系

NaCl10 0mmol/L处理结合外施Spd和Put以及多胺代谢抑制剂邻二氮杂菲和MGBG ,以改变大麦根系质膜结合多胺种类和数量 ,研究了大麦根系质膜上两种形态多胺与质子泵和Na /H 逆向运输活性的关系。结果发现 ,NaCl处理后大麦根系质膜微囊上存在Na /H 逆向运输活性。质膜H ATPase活性与膜上非共价键结合多胺数量间呈显著正相关 ,其中 ,Spd对H ATPase的激活程度大于Put。膜蛋白上共价键结合多胺数量与Na /H 逆向运输活性间呈极显著正相关关系 ,说明大麦根系质膜Na /H 逆向运输的盐诱导似乎与Na /H 逆向运输蛋白的从头合成有关。此外 ,质膜Na /H 逆向运输活性仅与膜蛋白上共价键结合多胺数量有关 ,而与多胺种类关系不大。     

Properties of putrescine uptake by PotFGHI and PuuP and their physiological significance in Escherichia coli

Properties of putrescine uptake by PotFGHI and PuuP and their physiological significance were studied using a polyamine biosynthesis and uptake deficient Escherichia coli KK3131 transformed with pACYC184 containing potFGHI or puuP. Putrescine uptake activity of E. coli KK3131 transformed with pACYC184-PotFGHI was higher than that of E. coli 3131 transformed with pACYC-PuuP when cells were cultured in the absence of putrescine. Putrescine uptake by PotFGHI was both ATP and membrane potential dependent, while that by PuuP was membrane potential dependent. Feedback inhibition by polyamines occurred at the PotFGHI uptake system but not at the PuuP uptake system. Expression of PuuP was reduced in the presence of PuuR, a negative regulator for PuuP, and expression of PuuR was positively regulated by glucose, which reduces the level of cAMP. The complex of cAMP and CRP (cAMP receptor protein) inhibited the expression of PuuR in the absence of glucose. Thus, the growth rate of E. coli KK3131 in the presence of both 0.4 % (22.2 mM) glucose and 10 mM putrescine was in the order of cells transformed with pACYC-PotFGHI > pACYC-PuuP > pACYC-PuuP + PuuR, which was parallel with the polyamine content in cells. The results indicate that PotFGHI is necessary for rapid cell growth in the presence of glucose as an energy source. When glucose in medium was depleted, however, PuuP was absolutely necessary for cell growth in the presence of putrescine, because accumulation of putrescine to a high level by PuuP was necessary for utilization of putrescine as an energy source.     

Fluctuation in free and conjugated polyamines in Scots pine seedlings after changes in temperature and daylength

Free, soluble and insoluble conjugated polyamines from the needles, roots and stem of five month old Scots pine (Pinus sylvestris L.) seedlings inoculated with Suillus variegatus (Fr.) O. Kuntze and seedlings without inoculation were analysed during decrease in daylength and temperature. Temporary changes in free, soluble and insoluble conjugated polyamine pools caused by a decrease in daylength or temperature were observed. Inoculation of pine seedlings affected significantly the polyamine levels of five month old pine seedlings. The roots of inoculated seedlings contained significantly higher levels of free and soluble conjugated purtrescine and free, soluble conjugated and insoluble conjugated spermidine than the roots of noninoculated seedlings. The needles of inoculated seedlings contained significatly higher concentrations of free putrescine and soluble conjugated spermidine but lower amount of free spermine than the needles of noninoculated seedlings. The stems of inoculated seedlings contained higher concentrations of free putrescine but lower amounts of insoluble conjugated spermine. Changes in polyamine levels in noninoculated seedlings were observed after shortening of the daylength, whereas in inoculated ones changes were induced mainly by the decrease in temperature. The possible role of polyamines in the initial stage of cold hardening process is discussed.     

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.     

Polyamines in grapevine microcuttings cultivated in vitro. Effects of amines and inhibitors of polyamine biosynthesis on polyamine levels and microcutting growth and development

The main free amines identified during growth and development of grapevine microcuttings of rootstock 41 B, (Vitis vinifera cv. Chasselas × Vitis berlandieri) cultivated in vitro were agmatine, putrescine, spermidine, spermine, diaminopropane and tyramine (an aromatic amine). Amine composition differed according to tissue, with diaminopropane the major polyamine in the apical parts, internodes and leaves. Putrescine predominated in the roots. There was also a decreasing general polyamine and specific tyramine gradient along the stem from the top to the bottom. Conjugated amines were only found in roots. The application of exogenous amines (agmatine, putrescine, spermidine, tyramine) stimulated development and growth of microcuttings, suggesting that the endogenous concentrations of these amines can be growth limiting. Diaminopropane (the product of oxidation of spermidine or spermine by polyamine oxydases) strongly inhibited microcutting growth and development. -DL-difluoromethylarginine (DFMA), a specific and irreversible inhibitor of the putrescine-synthesizing enzyme, arginine decarboxylase (ADC), led to inhibition of microcutting development. Application of agmatine or putrescine to the inhibited system resulted in a reversal of inhibition indicating that polyamines are involved in regulating the growth and development of grapevine microcuttings. -DL-difluoromethylornithine (DFMO), a specific and irreversible inhibitor of putrescine biosynthesis from ornithine decarboxylase (ODC), had no effect on microcutting development and growth. We propose that ADC regulates putrescine biosynthesis during microcutting development.