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     

Ethylene and polyamine interactions in morphogenesis of <Emphasis Type="Italic">Passiflora cincinnata</Emphasis>: effects of ethylene biosynthesis and action modulators,as well as ethylene scavengers

Ethylene is a plant hormone that is of fundamental importance to in vitro morphogenesis, but in many species, it has not been thoroughly studied. Its relationship with polyamines has been studied mainly because the two classes of hormones share a common biosynthetic precursor, S-adenosylmethionine (SAM). In order to clarify whether competition between polyamines and ethylene influences in vitro morphogenetic responses of Passiflora cincinnata Mast., a climacteric species, different compounds were used that act on ethylene biosynthesis and action, or as ethylene scavengers. Treatment with the ethylene inhibitor, aminoethoxyvinylglycine (AVG) caused a greater regeneration frequency in P. cincinnata, whereas treatment with the ethylene precursor, 1-aminocyclopropane-1-carboxylic-acid (ACC) lessened regeneration frequencies. The data suggested that levels of polyamines and ethylene are not correlated with morphogenic responses in P. cincinnata. It was ascertained that neither the absolute ethylene and polyamine levels, nor competition between the compounds, correlated to the obtained morphogenic responses. However, sensitivity to, and signaling by, ethylene appears to play an important role in differentiation. This study reinforces previous reports regarding the requirement of critical concentrations and temporal regulation of ethylene levels for morphogenic responses. Temporal regulation also appeared to be a key factor in competition between the two biosynthetic pathways, without having any effects on morphogenesis. Further studies investigating the silencing or overexpression of genes related to ethylene perception, under the influence of polyamines in cell differentiation are extremely important for the complete understanding of this process.     

Ethylene and polyamine production patterns during in vitro shoot organogenesis of two passion fruit species as affected by polyamines and their inhibitor

Levels of ethylene and polyamines (PAs) were measured during organogenesis of hypocotyl explants of two species of passion fruit (Passiflora cincinnata Masters and Passiflora edulis Sims f. flavicarpa Degener ‘FB-100’) to better understand the relationships of these regulators and their influence on cell differentiation and morphogenesis. Moreover, histological investigation of shoot ontogenesis was conducted to characterize the different events involved in cell redifferentiation and regulation of PA and ethylene levels. A delay was observed in morphogenic responses of P. edulis f. flavicarpa as compared to P. cincinnata, and these changes coincided with production of elevated levels of polyamine and ethylene levels. During differentiation, cells showed high rates of expansion and elongation, and high ethylene levels were associated with high PA levels, suggesting that the two biosynthesis pathways were highly regulated. Moreover, their interaction might be an important factor for determining cell differentiation. The addition of PAs to the culture medium did not promote organogenesis; however, the incorporation of the PA inhibitor methylglyoxal bisguanylhydrazone in the culture medium reduced shoot bud differentiation, suggesting the need to maintaining a minimum level of PAs for morphogenic events to take place.     

Role of polyamines in adventitious shoot morphogenesis from cotyledons of cucumber <Emphasis Type="Italic">in vitro</Emphasis>

The relationship between polyamines (PAs) metabolism and adventitious shoot morphogenesis from cotyledons of cucumber was investigated in vitro. The endogenous levels of free putrescine (Put) and spermidine (Spd) in the explants decreased sharply, whereas endogenous spermine (Spm) increased during adventitious shoot morphogenesis. The presence of 1–15 mM Put, 1–2 mM Spd, 0.05–1 mM Spm, 5–10 M aminoethoxyvinylglycine (AVG) or 5 M AVG together with 50 M 1-aminocyclopropane-1-carboxylic acid (ACC) in the regeneration medium could promote adventitious shoot formation. Conversely, 1–5 mM D-arginine (D-Arg) or 0.01–0.1 mM methylglyoxal bis-guganylhydrazone (MGBG) inhibited regeneration; and 0.005–0.05 mM ACC displayed little or no evident effects. The explants growing on medium containing 5 M AVG produced higher levels of free Put and Spm, and on medium containing 5 mM Put the explants responded similarly to the AVG-treated explants. However, the exogenous use of 1 mM D-Arg reduced the levels of Put, Spd and Spm, and 0.1 mM MGBG reduced the levels of free Spd and Spm. Moreover, although the explants cultured on medium containing Put and MGBG enhanced ethylene production, AVG and D-Arg inhibited ethylene biosynthesis. This study shows the PAs requirement for the formation of adventitious shoot from cotyledons of cucumber in vitro and the enhanced adventitious shoot morphogenesis may be associated with the elevated level of endogenous free Spm, albeit the promotive effect of PAs on adventitious shoot morphogenesis may not be related to ethylene metabolism.     

Polyamines and the Cell Cycle of Catharanthus roseus Cells in Culture

Investigation was made on the effect of partial depletion of polyamines (PAs), induced by treatment with inhibitors of the biosynthesis of PAs, on the distribution of cells at each phase of the cell cycle in Catharanthus roseus (L.) G. Don. cells in suspension cultures, using flow cytometry. More cells treated with inhibitors of arginine decarboxylase (ADC) and ornithine decarboxylase (ODC) were accumulated in the G₁ phase than those in the control, while the treatment with an inhibitor of spermidine (SPD) synthase showed no effect on the distribution of cells. The endogenous levels of the PAs, putrescine (PUT), SPD, and spermine (SPM), were determined during the cell cycle in synchronous cultures of C. roseus. Two peaks of endogenous level of PAs, in particular, of PUT and SPD, were observed during the cell cycle. Levels of PAs increased markedly prior to synthesis of DNA in the S phase and prior to cytokinesis. Activities of ADC and ODC were also assayed during the cell cycle. Activities of ADC was much higher than that of ODC throughout the cell cycle, but both activities of ODC and ADC changed in concert with changes in levels of PAs. Therefore, it is suggested that these enzymes may regulate PA levels during the cell cycle. These results indicate that inhibitors of PUT biosynthesis caused the suppression of cell proliferation by prevention of the progression of the cell cycle, probably from the G₁ to the S phase, and PUT may play more important roles in the progression of the cell cycle than other PAs.     

Agrobacterium-mediated Transfer of Arginine Decarboxylase and Ornithine Decarboxylase Genes to Datura innoxia Enhances Shoot Regeneration and Hyoscyamine Biosynthesis

The genes (adc and odc) for two enzymes, arginine decarboxylase and ornithine decarboxylase involved in polyamine biosynthesis, were introduced into anther-derived calli of Datura innoxia through Agrobacterium tumefaciens. The transformed calli exhibited increased regeneration frequency as compared to control. Transgenic lines showed higher polyamine levels, mainly in the putrescine titre, and such lines also yielded a high level of the alkaloid, hyoscyamine. The results suggest that polyamines can modulate in vitro morphogenesis and polyamine biosynthetic pathway can be exploited for enhancement of polyamine-derived alkaloids of pharmaceutical importance.     

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.     

Cross-talk of polyamines and nitric oxide in endophytic fungus-induced betulin production in Betula platyphylla plantlets

Key message

This paper showed that NO, PAs, PA-induced NO, and NO-induced PAs mediate fungus-induced betulin accumulation in birch plantlets.

Abstract

The aim of this study was to investigate the relationship between nitric oxide (NO) and polyamines (PAs) and to determine their roles in betulin accumulation induced by the endophytic fungus Phomopsis in Betula platyphylla. Treatment of birch plantlets with the endophytic fungus Phomopsis promoted an NO burst and accumulation of PAs and betulin. Birch plantlets were treated with the NO-specific scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt (cPTIO) and the PA synthesis inhibitor d-arginine (d-arg). cPTIO and d-arg inhibited the fungus-induced NO burst and accumulation of PAs and betulin. The exogenous NO donor sodium nitroprusside promoted PA production and betulin accumulation, whereas an exogenous PA, putrescine, promoted an NO burst and betulin accumulation. In addition, d-arg inhibited NO production and cPTIO decreased PA production during fungus-induced betulin accumulation. Our results indicate that NO, PAs, PA-induced NO, and NO-induced PAs mediate fungus-induced betulin accumulation in birch plantlets.     

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.     

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.     

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.     

Polyamines, polyamine oxidases and nitric oxide in development, abiotic and biotic stresses

Nitric oxide (NO), polyamines (PAs), diamine oxidases (DAO) and polyamine oxidases (PAO) play important roles in wide spectrum of physiological processes such as germination, root development, flowering and senescence and in defence responses against abiotic and biotic stress conditions. This functional overlapping suggests interaction of NO and PA in signalling cascades. Exogenous application of PAs putrescine, spermidine and spermine to Arabidopsis seedlings induced NO production as observed by fluorimetry and fluorescence microscopy using the NO-binding fluorophores DAF-2 and DAR-4M. The observed NO release induced by 1 mM spermine treatment in the Arabidopsis seedlings was very rapid without apparent lag phase. These observations pave a new insight into PA-mediated signalling and NO as a potential mediator of PA actions. When comparing the functions of NO and PA in plant development and abiotic and biotic stresses common to both signalling components it can be speculated that NO may be a link between PA-mediated stress responses filing a gap between many known physiological effects of PAs and amelioration of stresses. NO production indicated by PAs could be mediated either by H₂O₂, one reaction product of oxidation of PAs by DAO and PAO, or by unknown mechanisms involving PAs, DAO and PAO.     

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     

Role of Ethylene in Axillary Shoot Proliferation of Lavandin--Interaction with Benzyladenine and Polyamines

The role of ethylene in in vitro axillary proliferation of lavandin(Lavandula officinalis Chaix Lavandula latifolia Villars)was investigated. Basal ethylene production was modulated bythe addition of exogenous growth regulators (BA and polyamines),ethylene precursor (ACC) and inhibitors (AVG and SA). The resultsindicate that BA action is mediated by ethylene and, furthermore,that ethylene can substitute for BA in shoot proliferation.Among tested polyamines (Put, Spd, Spn) only Put was effectivein stimulating both the differentiation process and relatedethylene production. Moreover, Put appears to have a regulatoryfunction similar to BA. The data obtained in the present workoutline the complexity of axillary budding which is under multihormonalcontrol, ethylene playing an essential role. Key words: Axillary proliferation, benzyladenine, ethylene, lavandin, putrescine     

Promoter strength influences polyamine metabolism and morphogenic capacity in transgenic rice tissues expressing the oat adc cDNA constitutively

We analyzed molecularly and biochemically a series of transgenic rice lines expressing the oat adc (arginine decarboxylase) cDNA under the control of the constitutive maize ubiquitin 1 promoter. We established baseline biochemical parameters to elucidate the role of polyamines (PAs) during morphogenesis. We measured mRNA levels, ADC enzyme activity and cellular PAs in dedifferentiated callus. Polyamine levels were also quantified in two subsequent developmental stages – regenerating tissue and differentiated shoots. We observed significant (P<0.05) differences in the levels of individual PAs at the three developmental stages. The amounts of putrescine (Put) and spermidine (Spd) in dedifferentiated transgenic callus were lower than those in the wild type or in hpt (hygromycin resistant)-controls, whereas the amount of spermine (Spm) was increased up to two-fold. In regenerating tissue, this trend was reversed, with significantly higher levels of Put and Spd (P<0.05), and lower levels of Spm (P<0.05) compared to non-transformed or hpt-control tissues at the same developmental stage. In differentiated shoots, there was a general increase in PA levels, with significant increases in Put, Spd, and Spm (P<0.05); on occasion reaching six times the level observed in wild type and hpt-control tissues. These results contrast those we reported previously using the weaker CaMV 35S promoter driving adc expression. mRNA measurements and ADC enzyme activity were consistently higher (P<0.01) in all tissues expressing pUbiadcs compared to equivalent tissues engineered with 35Sadc. Our findings are consistent with a threshold model which postulates that high adc expression leading to production of Put above a basal level is necessary to generate a big enough metabolic pool to trigger PA flux through the pathway leading to an increase in the concentration of Spd and Spm. This can be best accomplished by a strong constitutive promoter driving adc. We discuss our results in the context of flux through the PA pathway and its impact on morphogenesis.     

Exogenous polyamines influence root morphogenesis and arbuscular mycorrhizal development of <Emphasis Type="Italic">Citrus limonia</Emphasis> seedlings

To investigate the influence of exogenous polyamines (PAs) on the root morphogenesis and arbuscular mycorrhizal development of Citrus limonia Osbeck seedlings, experiments were carried out in pots or specially designed containers. Analysis using WinRHIZO^® sofware indicated that exogenous PAs significantly reduced the average root diameter, which was due to the increased percentage of fine roots and the decreased percentage of coarse roots. Root length and the 1st lateral number were also slight increased. The increase in the mitotic index of root tip cells revealed using hydroponic culture indicated the involvement of the mitosis process in the PA-induced changes in root morphogenesis. These changes finally led to the increases in the relative chlorophyll contents (SPAD) and the biomass of seedlings. Moreover, exogenous PAs affected the mycorrhizal colonization in a concentration-dependent manner, with low concentration (0.2 × 10^?4 M) increasing but high concentration (1.0 × and 5.0 × 10^?4 M) decreasing it. These results suggest that exogenous PAs can affect positively the growth of C. limonia seedlings via the pathway of altered root morphogenesis and mycorrhizal development.     

Influence of putrescine on anthocyanin production in callus cultures of <Emphasis Type="Italic">Daucus carota</Emphasis> mediated through calcium ATPase

The influence of Putrescine (Put) on the growth and elicitation of anthocyanin in callus cultures of Daucus carota var. Nantes scarlet was investigated through the use of α-DL-difluoromethylarginine (DFMA), the polyamine (PA) biosynthetic inhibitor. It was observed that the addition of Put (0.05 mM) resulted in enhancement of growth and anthocyanin content. The anthocyanin content was found to be enhanced by 1.68 fold on the 21^st day as compared to the untreated controls. The PA inhibitor was found to result in lowering of the growth and the anthocyanin accumulation, which could be partially restored by the addition of Put in combination with this inhibitor. The levels of Ca²⁺ ATPase were also found to be elevated in treatment with Put suggesting the involvement of calcium in the elicitation of anthocyanin. The endogenous titres of PAs and the ethylene production under these treatments were also studied. The treatment with DFMA resulted in lower levels of endogenous PAs and higher levels of ethylene. Lowering of ethylene by putrescine treatment shows that PA treatment also inhibited ethylene formation, which would also imply that endogenous ethylene does not influence anthocyanin production in carrot callus cultures.