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.     

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.     

Effects of 2-aminoindan-2-phosphonic acid treatment on the accumulation of salidroside and four phenylethanoid glycosides in suspension cell culture of Cistanche deserticola

2-Aminoindan-2-phosphonic acid (AIP), a specific competitive phenylalanine ammonia lyase (PAL) inhibitor was applied to a suspension cell culture of Cistanche deserticola. The effects of AIP treatment on cell growth, PAL activity, contents and yields of total phenolic compound, salidroside and four phenylethanoid glycosides (PheGs) are investigated. The results demonstrated that, 0.5 and 2.0 μM AIP treatments had similar effects on the measurements investigated in this study. AIP treatment resulted in significant decreases in PAL activity, total phenolic compounds content, and PheGs content. Linear regression analysis showed that PAL activity had a high correlation coefficient with the total phenolic compound content and the four PheGs contents. Total PAL activity-time area under curve (AUC) had a high correlation coefficient with the total phenolic compound yield and the yields of five tested compounds in untreated cell samples. In AIP-treated cells, total PAL activity-time AUC retained a high correlation with the total phenolic compound yield and the yields of three tested compounds, echinacoside, acteoside, and tubuloside A, but not salidroside and cistanoside A. The difference could be caused by the different biosynthetic origins of each of the tested compounds. These results demonstrate the important role of PAL in the biosynthesis of PheGs in the suspension cell culture of C. deserticola.     

Significance of phenols in cadmium and nickel uptake

The effects of 2-aminoindane-2-phosphonic acid (AIP), a potent phenylalanine ammonia-lyase (PAL) inhibitor, on the accumulation of cadmium and nickel in chamomile (Matricaria chamomilla) were examined in this study. In vitro assay of AIP effect showed a 90% reduction in PAL activity. In plants cultured for 7 days in Cd or Ni solutions with AIP, PAL activity was higher in both shoots and roots (in comparison with metals without AIP), and was correlated with changes in free phenylalanine content. Individual amino acids were both positively and negatively affected by AIP, with the accumulation of tyrosine and proline showing increases in some variants. Contents of soluble phenols and flavonoids were not considerably affected, while amounts of coumarin-related compounds, cell wall-bound phenols and phenolic acids were substantially reduced in AIP-treated variants. Lignin accumulation decreased in controls and increased in Cd variants in response to AIP. Shoot Cd content was depleted, but shoot Ni was elevated by AIP. Total root content of Cd and Ni decreased in +AIP variants. AIP also caused more expressive changes in hydrogen peroxide and superoxide content in Cd than in Ni variants. Our results indicate that phenols have important roles in the uptake of Cd and Ni. The present findings are discussed in the context of available data regarding AIP's effect on phenols.     

K-Nutrition, Growth Bud Formation, and Amine and Hydroxycinnamic Acid Amide Contents in Leaf Explants of Nicotiana tabacum Variety Xanthi n.c. Cultivated in Vitro

The effects of K-nutrition on growth (increase of fresh weight), bud formation (time of emergence, number of buds), and amine and hydroxycinnamic acid amide contents in foliar explants of Nicotiana tabacum cv Xanthi n.c. cultivated in vitro were examined. In K-deficient medium and in high K medium growth and bud formation were markedly inhibited. Marked changes of amine content (a diamine, putrescine; a phenolic amine, phenethylamine) were observed after a few days of culture. No apparent relationship was found between these amines and growth or bud differentiation. In contrast, changes in hydroxycinnamic acid levels were shown to correlate well with growth and bud formation. The greatest stimulation of budding and growth was correlated with the greatest accumulation of these amides. The highest contents of hydroxycinnamic acid amides were found during the first 15 days in culture when intensive cell division took place. Then they declined sharply after 26 days in culture as the rate of cell division decreased and differentiation occurred.     

Polyamine Sparing May Be Involved in the Prolongation of Cell Division Due to Inhibition of Phenylpropanoid Synthesis in Cytokinin-Starved Soybean Cells

Effects on growth, mostly of an inhibitory nature, have been attributed to phenolic compounds in vivo and in vitro. This suggests that l-α-aminooxy-β-phenylpropionic acid (l-AOPP), a competitive inhibitor of phenylalanine ammonia-lyase (PAL), the enzyme controlling the first step in phenylpropanoid synthesis, might stimulate growth in soybean suspension cultures (Glycine max, cv. Acme). The promotive effect of l-AOPP, measured as an increase in cell number, was more clearly detected in the growth-limiting condition of cytokinin starvation. At least one more cell division cycle was completed in the presence of l-AOPP before growth by division ceased and growth continued by expansion only. Phenolic acids are known to conjugate with polyamines, modulating the free levels of these plant growth substances. Thus, the effect of l-AOPP on the titers of free and conjugated polyamines (putrescine, spermidine, and spermine) was investigated by high performance liquid chromatography in the course of cytokinin starvation. An increased level of free putrescine was detected in the presence of l-AOPP relative to controls, especially in the initial period before growth became restricted to cell expansion. The decrease in free putrescine associated with the cessation of cell division was temporarily delayed, suggesting that an interaction between phenolic acids and polyamines is involved in the mechanism of growth promotion by l-AOPP. Received July 30, 1996; accepted January 28, 1997     

水杨酸对丹参培养细胞中迷迭香酸生物合成及其相关酶的影响

迷迭香酸(RA)是丹参中一种重要的酚酸类次生代谢物。为探讨水杨酸(SA)诱导子对丹参悬浮培养细胞中RA的生物合成及其相关酶的影响,考察了SA诱导子和酪氨酸氨基转移酶(TAT)的竞争性抑制剂(AOPP)对RA合成积累量、苯丙氨酸解氨酶(PAL)和TAT活性的影响。发现在培养的第6天用浓度为6.25 mg/L的SA处理后,PAL活性在诱导后4 h出现高峰,为对照组水平的124%;RA的积累量在诱导后8 h出现峰值(5.914±0.296)mg/g。用浓度为0.1μmol/L的AOPP处理,6 h后AOPP对TAT活性影响较小(与对照组无显著差异),但明显抑制了PAL活性(为对照组水平的44%),且在PAL活性明显降低的同时RA的积累量显著减少(4.709±0.204)mg/g。进一步用0.1μmol/L AOPP和6.25 mg/L SA共处理,AOPP对PAL的抑制作用可得到一定程度的缓解,且RA的积累量较AOPP单独处理的高。表明SA可以诱导丹参悬浮培养细胞中RA积累量的增加,且在RA合成过程中PAL的限速作用比TAT明显。     

Diurnal changes in polyamine content, arginine and ornithine decarboxylase, and diamine oxidase in tobacco leaves

Changes in the contents of polyamines (PAs) in tobacco leaves (Nicotiana tabacum L. cv. Wisconsin 38) grown under 16 h photoperiod were correlated with arginine and ornithine decarboxylase (EC 4.1.1.19 and EC 4.1.1.17) and diamine oxidase (EC 1.4.3.6) activities. The maximum of free and soluble conjugated forms of PAs occurred 1-2 h after the middle of the light period and was followed by two distinct peaks at the end of the light and at the beginning of the dark phase. Putrescine was the most abundant and cadaverine the least abundant PA in both free and PCA-soluble forms. However, cadaverine was predominant in PCA-insoluble conjugates, followed by putrescine, spermidine, and spermine. Both arginine and ornithine decarboxylases are involved in putrescine biosynthesis in tobacco leaves. Light dramatically stimulated the activity of ornithine decarboxylase, while no photoinduction of arginine decarboxylase activity was observed. Ornithine decarboxylase was found mainly in the particulate fraction. Only one peak, just after light induction, occurred in the cytosolic fraction, with 35% of the total ornithine decarboxylase activity. By contrast, the total arginine decarboxylase activity was equally divided between the soluble and pellet fractions. A sharp increase in diamine oxidase activity occurred 1 h after exposure to light, concomitant with the light-induced increase in ornithine decarboxylase activity. After a decline, diamine oxidase activity increased again, together with the rise in the amount of free Put. The roles of both conjugation of PAs with hydroxycinnamic acids and oxidative degradation of putrescine in maintaining free PA levels during the 24 h light/dark cycle are discussed. The presented results have shown that the parameters studied here followed rhythmical changes and were not only affected by light.     

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.     

Effect of inhibition of phenylpropanoid biosynthesison peroxidase and IAA-oxidase activities and auxin contentin alfalfa suspension cultures

The inhibition of phenylpropanoid biosynthesis by 2-aminoindan-2-phosphonic acid (AIP) in alfalfa suspension cultures (Medicago sativa L.) was associated with a marked decline in the content of cinnamic acid derivatives during the whole subculture interval. The levels of all investigated forms of phenolic acids in AIP-treated cells were lower than those of control with the exception of free phenolic acids on days 1 and 2. The dynamics of free IAA content in AIP-treated cells was similar to that of the control but its peak was significantly lower. The auxin content in AIP-treated cells on days 9 and 11 represented only 35 and 40 % of control values, respectively. AIP-treatment stimulated soluble peroxidase (EC 1.11.1.7) activity in the first 2 d after cell inoculation. Time-course of soluble IAA-oxidase activities was similar to that of soluble peroxidases (with the exception of the drop in peroxidase activity on day 3) but beginning day 6, the enzyme activities in AIP-treated cells were significantly lower until the end of the subculture interval.     

Restoration of secondary metabolism in birch seedlings relieved from PAL-inhibitor

Phenylalanine ammonia lyase (PAL) plays a key role in phenylpropanoid metabolism, catalyzing the deamination of phenylalanine (Phe) to form trans-cinnamic acid. Inhibitors of PAL have been used to study the physiological role of the different compounds derived from trans-cinnamic acid, and to test theories about a trade-off between growth and defence in plants. In a previous study with birch (Betula pubescens Ehrh.) seedlings, the PAL inhibitor 2-aminoindane-2-phosphonic acid monohydrate (AIP) caused an accumulation of Phe and a strong decrease in the quantity of simple phenolics, soluble condensed tannins and growth, whereas flavonol glycosides were generally not affected. The present study demonstrates restoration of secondary metabolism in the previously AIP treated birch seedlings. Our results indicate that Phe accumulated during PAL inhibition could be partly used to increase the content of the phenolic acids, flavan-3-ols and to some extent the soluble condensed tannins. Seedling growth also increased when the supply of PAL inhibitor ceased. We thereby show that the inhibition of PAL by AIP in vivo is reversible, at least for moderate AIP concentrations and the rate of restoration is dependent on the inhibitor concentration.     

Protective roles of exogenous polyamines on chromosomal aberrations in <Emphasis Type="Italic">Hordeum vulgare</Emphasis> exposed to salinity

The effects of exogenous polyamines (PAs): spermine (Spm), spermidine (Spd), cadaverine (Cad) and putrescine (Put) on mitotic activity and chromosomal aberrations in root meristem cells of Hordeum vulgare L. (barley) seeds exposed to salinity were analyzed. The PAs significantly inhibited cell division in distilled water. Furthermore, most of these PAs (except for Spd) caused a significant increase in the frequency of chromosomal aberrations as compared to control group. Seeds treated with Put caused the highest percentage of mitotic abnormalities in total. The negative effect of salinity on mitotic index and the frequency of chromosomal aberrations increased with increasing salt concentration. PAs studied could not be successful in ameliorating of the negative effect of salinity on mitotic activity. Particularly, exposure to Cad and 0.40 M NaCl caused a complete block of cell division in total. However, most of the PA studied showed a perfectly performance in alleviating the detrimental effects of increasing salinity on chromosomal aberrations.     

Phenolics during early development of <Emphasis Type="Italic">Betula pubescens</Emphasis> seedlings: inhibition of phenylalanine ammonia lyase

We studied phenolic metabolism and plant growth in birch seedlings at the beginning of their development by inhibiting phenylalanine ammonia lyase (PAL), which is the first committed step in phenylpropanoid metabolism. Betula pubescens (Ehrh.) seeds were germinated in inhibitor-free media and the seedlings were transferred to hydroponic culture at the cotyledon stage. They were 6 days old at the start of the experiment, which lasted for 3 weeks. PAL activity was inhibited by three different concentrations of 2-aminoindane-2-phosphonic acid monohydrate (AIP) in the growing media. At the end of 3 weeks, phenolics in all plant parts (roots, stem, cotyledons, first, second and third true leaves) were determined. AIP inhibited strongly the accumulation of phenolic acids, salidroside, rhododendrins, ellagitannins and their precursors, flavan-3-ols, and soluble condensed tannins. The accumulation of lignin and flavonol glycoside derivatives was moderately inhibited. The accumulation of flavonol glycosides, such as quercetin glycosides and kaempferol glycosides, was not generally inhibited, even in leaves that emerged during the experiment, while the accumulation of insoluble condensed tannins was inhibited only slightly and not in all plant parts. This suggests that flavonol glycosides, which may have a UV-B protective role, and insoluble condensed tannins, which may have structural functions, are prioritized in seedling development. Inhibition of PAL with AIP decreased seedling growth and possible reasons for this are discussed. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Changes in the content of phenolic compounds and IAA-oxidase activity during the growth of tobacco crown gall suspension culture

Phenolic content and IAA-oxidase (IAA-o) activity have been assayed in cells and medium of tobacco crown gall suspension culture in several stages of culture cycle. The highest content of total phenolics in the cells were found prior to cell division and in the middle stage of intensive growth. The beginning of intensive growth is accompanied by temporary reduction in phenolic level in the cells as well as their intensive secretion to the medium. In the second part of the culture cycle, when the phenolic production was weaker, the majority of these compounds were maintained in the cells. The highest activity of IAA-o in the cells was detected in the middle stage of intensive growth, simultaneously with high phenolic content; following it a considerable decrease of IAA-o activity is correlated with maximum of chlorogenic acid (ChA) content (at reduced amount of total phenolics). IAA-o activity increased again at the end of the stage of intensive growth when the level of phenolics was low including ChA. These data suggest that IAA-o in relation to phenolic level determines cell growth in the culture. In the culture medium — fairly distinct negative correlation between IAA-o activity and phenolic content suggests that the latter participates in enzyme activity regulation. During intensive growth IAA-o activity is strongly inhibited. The results prove that phenolic level. IAA-o activity and auxin level are closely correlated and may constitute essential elements of a mechanism of regulation crown gall cell growth in culture.     

Relationship between the enzymatic browning and phenylalanine ammonia-lyase activity of cut lettuce, and the prevention of browning by inhibitors of polyphenol biosynthesis.

Cut lettuce stored at 4 degrees C gradually turned brown on the cut section after several days of storage. Three factors for enzymatic browning, the polyphenol content, polyphenol oxidase activity, and phenylalanine ammonia-lyase (PAL) activity, were examined during the cold storage of cut lettuce. A relationship between the browning and PAL activity was apparent. We tried to prevent this browning by using the two enzyme inhibitors, 2-aminoindane-2-phosphonic acid (AIP), an inhibitor of the phenylpropanoid pathway, and glyphosate, an inhibitor of the shikimate pathway. AIP and glyphosate significantly inhibited the browning of cut lettuce. The polyphenol content and PAL activity were both reduced by the treatment with AIP. These results show that regulating the biosynthesis of polyphenols is essential to prevent the browning of cut lettuce.     

Phenylalanine ammonia-lyase,phenolic acids and ethylene in alfalfa (Medicago sativa L.) cell cultures in relation to their embryogenic ability

Phenylalanine ammonia-lyase (PAL) activity, contents of phenolic acids and ethylene production during the lag-phase, and contents of phenolic acids at the late exponential phase, showed significant differences in embryogenic (EC) and non-embryogenic (NEC) suspension cultures of Medicago sativa L. Maximum PAL activity at 6 h after inoculation was followed by an increase in the level of phenolic acids from 9.6 g g^–1 fresh mass to 21 g g^–1 fresh mass in NEC at 12 h. Thereafter the level of phenolic acids decreased to 5.2 g g^–1 fresh mass at 72 h. The decline was caused predominantly by the decrease of ester-bound cinnamic acid derivatives, the decrease ranging from 83 to 20% of total phenolics. Two maxima of ethylene production were observed in NEC: the first one immediately after inoculation and the second at 6 h, coinciding with the peak of PAL activity. In NEC, most of the phenolic acids occurred in esterified form. Ability to form somatic embryos (EC) was associated with the absence of the second peak of ethylene production as well as of the peak of PAL activity at 6 h. The level of phenolic acids during the lag-phase remained low (7.2 g g^–1 FM) and did not change. The proportion of cinnamic acid derivatives was very low (18% of total phenolics), mostly due to the extremely low level of ferulic acid. In EC, phenolic acids bound to methanol insoluble material formed the major fraction. Loss of embryogenic potential of the embryogenic culture (ECL) was associated with qualitative and quantitative changes in the contents of phenolic acids insignificantly increased PAL activity after inoculation was followed by a moderate increase in the contents of phenolic acids from 9.35 g g^–1 fresh mass to 12.42 g g fresh mass. A high rate of ethylene production was observed only immediately after the transfer of the culture to fresh medium. The loss of embryogenicity correlated also with changes in the relative amounts of the investigated fractions of phenolic acids. A distinct increase in the level of methoxy-substituted phenolic acids is a characteristic feature of the ECL culture.Abbreviations NEC non-embryogenic suspension culture - EC embryogenic suspension culture - ECL embryogenic suspension culture after the loss of embryogenic potential - AA anisic acid - CA cinnamic acid - CaA caffeic acid - pCA p-coumaric acid - FA ferulic acid - pHBA p-hydroxybenzoic acid - SA syringic acid - SaA salicylic acid - SiA sinapic acid - VA vanillic acid - PhA phenolic acids - HPLC High-Performance Liquid Chromatography - GC Gas Chromatography - 2,4-D 2,4-dichlorophenoxyacetic acid - kin kinetin - NAA 1-naphthaleneacetic acid - BL-medium medium of Blaydess - FM fresh mass     

Increases in enzyme levels during the formation of phenolic acids in carrot cell cultures

The levels of glutamic acid dehydrogenase (GDH), phenylalanine ammonia-lyase (PAL), cinnamic acid 4-hydroxylase (CAH) and O-methyltransferase (OMT) were measured during the formation of phenolic acids in carrot cells in suspension culture. Caffeic, ferulic and p-hydroxybenzoic acids were always present as the culture proceded. Total content of these acids increased at the early logarithmic and linear phases. GDH showed high activity at the early logarithmic and stationary phases. PAL activity was much enhanced at the linear and stationary phases. CAH activity was found in actively growing cells, especially at the early and late logarithmic phases OMT behaved similarly to PAL. The increases in GDH and CAH might be responsible for the rapid synthesis of phenolic acid at the early logarithmic phase. The increase in phenolic acid at the linear phase would certainly be due to enhancements of both PAL and OMT. On the other hand, the accumulation of vanillic acid was observed in cells which were transferred and cultured on an agar medium, but not in cells in suspension culture. This accumulation is related to increases in OMT levels and also to changes in the degree of β-oxidation.