JXB Advance Access originally published online on March 23, 2006
Journal of Experimental Botany 2006 57(6):1413-1421; doi:10.1093/jxb/erj121
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RESEARCH PAPER |
Diurnal changes in polyamine content, arginine and ornithine decarboxylase, and diamine oxidase in tobacco leaves
ková1
1Institute of Experimental Botany, Academy of Sciences of Czech Republic, Rozvojová 135, 165 02 Prague 6, Czech Republic
2Department of Plant Physiology, Faculty of Sciences, Charles University, Vini
ná 5, 128 44 Prague 2, Czech Republic
3Department of Biochemistry, Faculty of Sciences, Charles University, Hlavova 2030/8, 128 44 Prague 2, Czech Republic
* To whom correspondence should be addressed. E-mail: cvikrova{at}ueb.cas.cz
Received 29 August 2005; Accepted 13 January 2006
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Abstract |
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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 [EC] and EC 4.1.1.17 [EC] ) and diamine oxidase (EC 1.4.3.6 [EC] ) 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.
Key words: Arginine decarboxylase, diamine oxidase, ornithine decarboxylase, polyamines, tobacco
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Polyamines (PAs) are ubiquitous organic polycations that are involved in the regulation of different cellular processes in animals and plants. The biological activity of PAs is attributed to the cationic nature of these molecules, which enables their interactions with anionic biomolecules (e.g. DNA, RNA, proteins, and phospholipids). In plant cells, PAs are usually present in amounts varying from micromolar to more than millimolar, i.e. levels significantly higher than those of plant hormones. Nevertheless, PAs affect many processes regulated by cytokinins and auxins and, in co-operation with these plant hormones, modulate various morphogenic processes (Altamura et al., 1993
). In plants, PAs participate in the control of such important processes as cell division and growth (Theiss et al., 2002) morphogenesis and differentiation (Paschalidis et al., 2001), programmed cell death (Serafini-Fracassini et al., 2002; Pignatti et al., 2004), stabilization of nucleic acids and membranes (Thomas and Thomas, 2001), and stress responses (Bouchereau et al., 1999).
The polyamine content in cells can be regulated by both metabolic and transport processes. In higher plants PAs are biosynthesized by two main pathways. The diamine putrescine (Put) may be formed directly from ornithine by ornithine decarboxylase (ODC; EC 4.1.1.17
[EC]
) or indirectly from arginine by arginine decarboxylase (ADC; EC 4.1.1.19
[EC]
). S-adenosylmethionine decarboxylase (SAMDC; EC 4.1.1.50
[EC]
) is essential for spermidine (Spd) and spermine (Spm) biosynthesis. Spd is formed from Put as well as Spm from Spd by successive addition of aminopropyl groups derived from decarboxylated S-adenosylmethionine generated by the activity of SAMDC (Wallace et al., 2003). A clear relationship between the diamine cadaverine (Cad) and other PAs has not yet been found (Bagni and Tassoni, 2001). The intracellular concentrations of free PAs may be regulated by the rate of biosynthesis, which is controlled at the transcriptional, post-transcriptional, translational, and post-translational levels (Kumar et al., 1997). Apart from biosynthesis, PA levels may be modulated by conjugation either with small molecules, especially hydroxycinnamic acids (soluble conjugated PAs; Martin-Tanguy, 1985; Bagni and Tassoni, 2001), or with high molecular-mass substances like hemicelluloses, lignin, and, to a lesser extent, proteins as well (insoluble conjugated PAs; Creus et al., 1991). Cytoplasmic levels of PAs are also affected via storage in the vacuoles, mitochondria, and chloroplasts and by extracellular transport (Flores, 1991; Beranger-Novat et al., 1997; Bagni and Tassoni, 2001). The mechanisms which regulate polyamine transport in eukaryotes remain largely unknown. Only translocation of free polyamines has been reported (Antognoni et al., 1998). Another important process of PA level regulation is degradation through oxidative deamination. Two catabolic enzymes were found in plants. Diamine oxidase (DAO; EC 1.4.3.6
[EC]
) which primarily catalyses the conversion of Put to ammonia, H2O2 and pyrroline, and polyamine oxidase (PAO; EC 1.5.3.) with high affinity towards Spd and Spm.
Data on the relationship between the physiological effects of PAs and light are not very extensive. Alterations in the contents and forms of PAs during photoperiodic flower induction were observed in soybean (Caffaro and Vicente, 1994). Photosynthetically active radiation caused Put and Spd accumulation in soybean (Kramer et al., 1992), which is consistent with the hypothesis that phytochrome can regulate the activity of ADC in chloroplasts (Besford et al., 1993). Photo-induction of ADC was also described in leaves of Pharbitis nil (Yoshida and Hirasawa, 1998). Transcription of the SAMDC gene was shown to be under circadian control (Yoshida et al., 1999). Changes in PA content, ADC and ODC activities during light/dark phases in maize calluses did not only respond to light, but they were also found to be regulated by a daily rhythm (Bernet et al., 1999). Photoperiodic control of PA accumulation was observed in petal explants of Araujia sericifera (Moysset et al., 2002).
The purpose of the current work was to identify the daily variation of PA levels in tobacco plants in order to find the optimal time for sample collection. These data will be used in further studies of the role of PAs in abiotic stress response and PA cross-talk with cytokinins using transgenic tobacco plants over-expressing cytokinin metabolic enzymes under different (constitutive as well as inducible) promoters. Moreover, determination of diurnal variation of the levels of individual PAs might contribute to the understanding of mechanisms involved in the regulation of physiologically active PAs.
Data presented here show detailed changes in the contents of free Put, Spd, Spm, and Cad, their soluble and insoluble conjugates in tobacco leaves grown under a long day. The focus was on the evaluation of the effect of light on ADC and ODC activities, as well as on the elucidation of the potential correlation of ADC and/or ODC activities and Put and, subsequently, Spd and Spm accumulation. The role of PA conjugation and oxidative deamination in PA homeostasis during the 24 h cycle was followed, too.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Plant material
Tobacco (Nicotiana tabacum L. cv. Wisconsin 38) plants were grown in soil in a growth chamber for 6 weeks at a 16/8 h photoperiod (130 µmol m–2 s–1) with a day/night temperature of 25/23 °C and relative humidity c. 80%. Mixed samples of leaves (3rd–5th of 7, i.e. with the exception of the youngest and the oldest ones) from three plants were pooled and collected every h during a 24 h period. The sampling during the dark period was realized in dim light and did not exceed 1 min. The samples were immediately frozen in liquid nitrogen.
Polyamine analysis
The leaves were ground in liquid nitrogen and extracted overnight at 4 °C with 5% (v/v) perchloric acid (PCA) (100 mg fresh weight tissue cm–3 5% PCA). 1,7-Diaminoheptane was added as an internal standard. The extracts were centrifuged at 21 000 g for 15 min, and then PCA-soluble free PAs were analysed in one-half of the supernatant. The remaining supernatant and pellet were acid hydrolysed in 6 M HCl for 18 h at 110 °C to obtain PCA-soluble and PCA-insoluble conjugates of PAs as described by Slocum et al. (1989). Standards (Sigma-Aldrich, St Louis, MO, USA), PCA-soluble free PAs, and acid hydrolysed PA conjugates were benzoylated. HPLC analysis of benzoyl-amines was performed on a Beckman-Video Liquid Chromatograph equipped with a UV detector (detection at 254 nm) and C18 Spherisorb 5 ODS2 column (particle size 5 µm, column length 250x4.6 mm) according to the method of Slocum et al. (1989).
Ornithine decarboxylase and arginine decarboxylase assays
Ornithine decarboxylase (ODC; EC 4.1.1.17
[EC]
) and arginine decarboxylase (ADC; EC 4.1.1.19
[EC]
) were determined by a radiochemical method described by Tassoni et al. (2000). Samples were extracted in 3 vols of ice-cold 0.1 M TRIS–HCl buffer, pH 8.5, containing 2 mM ß-mercaptoethanol, 1 mM EDTA and 0.1 mM pyridoxal phosphate, and centrifuged at 20 000 g for 30 min at 4 °C. Aliquots (0.1 cm3) of both supernatant (soluble fraction) and resuspended pellet (particulate fraction) were used to determine ODC and ADC activity. Enzyme activity assays were performed by measuring the 14CO2 evolution from 7.4 kBq L-[1-14C]ornithine (1.92 GBq mmol–1, Amersham Pharmacia Biotech UK) or 7.4 kBq L-[U-14C]arginine (11.5 GBq mmol–1 Amersham Pharmacia Biotech UK), for ODC and ADC, respectively, in the presence of 2 mM unlabelled substrate during a 1.5 h incubation at 37 °C. CO2 was trapped in hyamine hydroxide and the radioactivity was counted by a liquid scintillation analyser (Tri-Carb 2900TR; Packard).
Diamine oxidase assay
Diamine oxidase (DAO, EC 1.4.3.6
[EC]
) activity was assayed by a spectrophotometric method based on detection of the aldehyde with cis-1,4-diamino-2-butene as the substrate (Pe et al., 1991). Samples were homogenized in 0.1 M TRIS–HCl buffer, pH 8.5, containing 2 mM mercaptoethanol and 1 mM EDTA, and centrifuged at 20 000 g for 15 min at 4 °C. The reaction mixture contained 0.1 M TRIS–HCl buffer, pH 8.5, catalase (25 µg) and 0.01 M cis-1,4-diamino-2-butene. The reaction was started by the addition of 0.2 cm3 supernatant, incubated for 1 h at 37 °C and stopped by adding 1 cm3 of Ehrlich's reagent. The reaction mixture was incubated at 50 °C for 5 min, and then chilled in an ice bath before reading the absorbance of produced pyrrol at 563 nm. Enzymatic activity is expressed in pkat mg–1 protein.
Protein content was measured according to Bradford's method using bovine serum albumin as a standard (Bradford, 1976).
Statistical analyses
Means ±SE of two independent experiments with two replicates are shown in the figures. Statistical tests were analysed using the Student's t distribution criteria.
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Results |
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Polyamine contents
Free and bound PAs (PCA-soluble and PCA-insoluble conjugates) were analysed in tobacco leaves each h during a 24 h light/dark cycle. All four PAs (Put, Spd, Spm, and Cad) were present in both free and conjugated forms. Although the content of PAs is usually expressed on a fresh weight basis, it was decided to express the PA contents in nmol g–1 protein as well (Fig. 1) because the focus of this work was on the evaluation of the potential correlation between biosynthetic enzyme activities and the contents of PAs. Differences between the graphs are related to changes in protein in the course of the light/dark cycle, which were higher during the light, especially in the first half of the light period. As a consequence, the peaks of PAs per fresh weight at the end of the light and at the beginning of the dark phase (Fig. 1A) became more pronounced when expressed per protein (Fig. 1B).
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The ratio among PA forms changed during the 24 h cycle (Fig. 2). The most abundant PA in free and PCA-soluble form was Put and the least abundant was Cad. However, Cad prevailed in PCA-insoluble conjugates, followed by Put, Spm, and Spd (Fig. 3). The endogenous level of free Put showed a transient increase at the second and the third hours of the light period (P
0.05 compared with the value at the end of the dark period) and exhibited a maximum after 9 h of light (P 0.005 compared with the value at 7 h). The largest peak occurred at the light/dark transition (P 0.005 compared with the value at 11 h; Fig. 2A). The contents of PCA-soluble Put conjugates showed a first peak 1 h after the middle of the light period (P 0.005 compared to the value at 7 h), and further transient peaks were detected 2 h before the light was switched off and at the beginning of the dark period (statistical significance of both peaks P 0.005 compared with the value at 13 h). A slight rise was observed at the end of the dark phase (Fig. 2A). The peaks in the free Spd content were less marked than those of Put (statistical significance of the increase at 11 h of light was P 0.05 compared with the value at 7 h, which was similar to the peaks at the light/dark transition compared with 13 h). PCA-soluble conjugates of Spd showed a similar trend to those of Put (Fig. 2B). In the peaks, the contents of soluble conjugates of Put and Spd were significantly higher than those of their free forms (PCA-soluble forms represented about 75% of the total content at the midday maximum). The contents of free Spm and Cad and of their soluble conjugates did not change significantly during the diurnal cycle, except that of Spm conjugates, which increased 5 h before the end of the light (P 0.05 compared with the value at 10 h of light) and at the beginning of the dark period (P 0.005 compared to the value at 10 h of light; Fig. 2C, D).
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The time-course of the levels of PCA-insoluble conjugates showed different trends from those observed in free and soluble forms (Fig. 3). Three dominant peaks of insoluble Cad conjugates were detected in tobacco leaves grown under the 16/8 h photoperiod. After the decline at the dark/light transition the first maximum occurred at the beginning (at the second and third hours) of the light period (P
0.005 compared with the value at 1 h of light), the second one 4 h after midday (P 0.005 compared with the value at 10 h of light), and the third one at the beginning of the dark period (2 h after the light was switched off, P 0.005 compared with the value at the light/dark transition). Insoluble conjugates of Put, after the transient decline at the dark/light transition, reached their high value in the middle of the light period (P 0.005 compared with the value at 4 h of light), and then decreased. Their second peak coincided with that of Cad (2 h after the light was switched off, P 0.005 compared with the value at 14 h of light). Relatively high level of insoluble conjugates of Cad and Put were observed during the dark period. However, the amounts of insoluble Put conjugates were much lower than those of Cad. The content of insoluble Spd conjugates was the highest at the first third of the light period. The contents of Spm conjugates followed a trend similar to Cad but the peaks were much less pronounced.
ADC and ODC activities
Biosynthetic enzyme activities were measured both in the soluble and pellet fractions. No increase in ADC activity was observed in light, by contrast, the enzyme activity in the first hour of light decreased. At midday, ADC activity started increasing, reaching a high 1 h later (P 0.005 compared with the value at 7 h of light). After a decline, a second main peak of activity occurred at the light/dark transition (P 0.005 compared with the value at 12 h of light). The enzyme activity in the pellet fraction represented about 45% and 48% of the entire biosynthetic activity in the first and second enzyme peak, respectively (Fig. 4). Light dramatically stimulated the activity of the second PA biosynthetic enzyme, ODC (Fig. 5). The sharp increase at 1 h of light (P 0.005 compared with the value at the dark/light transition) was followed by a transient decrease and then further peaks were observed prior to and after the middle of the light period (statistical significance of both peaks P 0.005 compared with the value at 3 h) and at the beginning (P 0.005 compared with the value at the light/dark transition) and the end of the dark period (P 0.05 compared with the value at 4 h of dark). The ODC enzyme activity was present predominantly in the pellet fraction and was rather low in the soluble fraction except for a peak 1 h after light induction (P 0.005 compared with the value at the dark/light transition), at which time it represented about 35% of total ODC activity. From the third hour of light until the end of the dark period the soluble ODC activity was very low again.
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DAO activity
DAO activity was induced by light in a similar way as the ODC activity. A sharp peak of activity was observed 1 h after the start of light (P
0.005 compared with the value at the dark/light transition), after which the activity declined. The main DAO maximum was found 2 h after midday (P 0.05 compared with the value at 6 h of light) and then the enzyme activity steadily declined. The peak of activity at the light/dark transition (P 0.05 compared with the value at 14 h of light) coincided with the increase in the activities of both biosynthetic enzymes (Fig. 6).
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Discussion |
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Although the number of studies on PAs has been growing constantly, it is still unclear whether ODC or ADC plays a decisive role in Put biosynthesis in plants. The existence of two alternative routes for Put synthesis could be explained by the necessity of specific regulation of different processes affected by Put during growth and development (Koetje et al., 1993
). It has been suggested that the pathway via ADC is generally linked to stress responses and morphogenetic processes, whereas ODC is involved in the regulation of cell division in actively growing plant cell tissue (Koetje et al., 1993; Galston et al., 1997). However, it seems that species and/or tissue specificity should be taken into consideration in plants. In apple, the primary biosynthetic pathway for Put is biosynthesis through ADC (Hao et al., 2005), while in the green alga Chlamydomonas reinhardtii the activity of ODC exceeded that of ADC by orders of magnitude, being highly induced by light (Voigt et al., 2000). On the contrary, in leaves of Pharbitis nil a very rapid photoresponse of ADC with the major activity peak 1 h after illumination was observed, while the activity of ODC did not respond to illumination (Yoshida and Hirasawa, 1998). In tobacco plants, ADC seemed to be responsible for Put synthesis in old hypergenous vascular tissues, whereas ODC expression coincided with early cell divisions and catalysed Put synthesis in hypogenous tissues (Paschalidis and Roubelkis-Angelakis, 2005). In tobacco leaves a different effect of light was observed on the induction of PA biosynthetic enzymes. Marked photoinduction of ODC activity 1 h after the exposure to light was found. Further peaks of ODC were observed prior to and after the middle of the light period with a slight increase at the beginning and the end of the dark period. By contrast, ADC activity decreased at the first hour of the light period and started increasing at midday with the main peak at the light/dark transition.
There is still a lack of knowledge about the tissue and subcellular localization of the enzymes involved in PA metabolism. In animal tissues, despite extensive research, the exact intracellular localization of ODC remains to be clarified. It varies from an exclusively cytoplasmic to both a cytoplasmic and a nuclear localization (Schipper et al., 2004). The assays performed on leaves of Arabidopsis thaliana showed that high particulate ODC activity was associated with the nuclei and chloroplast-enriched fractions (Tassoni et al., 2003). In photosynthetic tissues of tobacco plants ADC was found mainly in chloroplasts, mitochondria, and cytoplasm (Slocum, 1991), whereas in non-photosynthetic tissues of tobacco, the particulate ADC activity appeared to be located mainly in nuclei (Bortolotti et al., 2004). The different compartmentation of biosynthetic enzymes may be related to distinct functions in different cell types (Bortolotti et al., 2004).
The results presented here show that, in tobacco leaves, the total ADC activity was equally divided between the soluble and pellet fractions while the ODC activity was mainly localized in the pellet fraction and only very little activity was present in the cytosolic fraction (there was one peak of activity after light induction in which 35% of the total ODC activity was found).
A significant peak of free and soluble conjugated PA forms observed in tobacco leaves 1–2 h after the middle of the light period coincided with the high activity of ODC pellet fraction. It is well documented that in many plant systems dividing tissues have high PA levels and activities of their biosynthetic enzymes (Pfosser et al., 1990; Bezold et al., 2003). In vitro experiments have shown that free PAs affect many functions of nucleic acids, including transcription and translation (for a review see Kumar et al., 1997). The observed midday peak in the contents of free Put and Spd in tobacco leaves may be related to high physiological activity of the leaf tissue at this time period. This hypothesis might be supported by the above-mentioned fact that ODC expression coincided with early cell divisions observed in hypogenous tobacco tissues (Paschalidis and Roubelkis-Angelakis, 2005). Furthermore, this peak coincided with the main peak of cytokinins and with the maximum of indole-3-acetic acid (IAA) in tobacco leaves cultivated under identical light/dark conditions (Nováková et al., 2005). Since cytokinins, auxin, and PAs are necessary for the progression through the cell cycle, this result leads to the assumption that cell division occurs at this period.
At midday, growth in ADC activity was observed and the main peak of the activity was found at the light/dark transition. This distinct peak of enzymatic activity was accompanied by the accumulation of free PAs and PCA-soluble conjugates. The PA maximum occurring at the light/dark transition coincided with the increase in abscisic acid content determined in tobacco leaves cultivated under the identical light/dark conditions (Nováková et al., 2005). As ABA is a key component of plant defence, the coincidence of its maximum and ADC peak is in a good accordance with the presumption of a potential link of ADC to stress response.
The observed midday and dark phase peaks in Put and Spd contents are in agreement with the accumulation pattern of PAs in maize calli during the light/dark phases (Bernet et al., 1999). Put was the most abundant PA in maize callus, as in tobacco leaves, reaching peaks at 8 h and 12 h in the light and then in the middle of the dark period.
Large intracellular accumulation of free PAs would have harmful effects on the maintenance of cellular pH, ion homeostasis, and on a number of physiological functions where PAs are implied. The endogenous free PA levels could be regulated, apart from the control of PA biosynthesis, also by conjugation with hydroxycinnamic acids in some plant species (Martin-Tanguy, 1985; Creus et al., 1991; Bagni and Tassoni, 2001; Biondi et al., 2001). The detected high levels of PCA-soluble conjugates of Put and Spd (about 75% of the entire Put and Spd pool in the peak 1–2 h after the midday) seem to be the common phenomena in Nicotiana and have been reported by several authors (Martin-Tanguy, 1985; Pfosser et al., 1990; Gemperlová et al., 2005). It has been shown in alfalfa cell suspension cultures and in oak embryogenic cultures that the rate of PA conjugation with hydroxycinnamic acids influenced the endogenous free PA level and, indirectly, cell division (Cvikrová et al., 1999, 2003). The biological function of conjugated PAs remains unclear. It has been speculated that enzymatic hydrolysis of reversible conjugates could supply the cells with an additional amine reserve (Bonneau et al., 1994).
The origin and function of bound PAs are still an open issue. Some reports demonstrated high levels of Put and Spd binding to proteins in actively dividing young tissues. The incorporation of Put, Spd, and Spm into thylakoid and stromal proteins was stimulated by light (Aribaud et al., 1995). Spm was the most efficiently conjugated PA (Della Mea et al., 2004). In tobacco leaves grown under a 16/8 h photoperiod the most abundant PA in PCA-insoluble conjugates was Cad, followed by Put, Spm, and Spd. Cad in animal tissues does not normally participate in any particular metabolic process, but it is completely excreted from cells into the culture medium (Hawel et al., 1994; Hawel and Byus, 2002). Synthesized Cad is, in tobacco leaves, most probably immediately incorporated into the PA-insoluble fraction, as the levels of both free and soluble conjugated forms are very low during the light/dark cycle. In this experiment, the peaks of insoluble Spm conjugates coincided with the peaks of Cad. It may be hypothesized that Cad participates in the protection of chloroplasts from photo damage or stress.
Significant light-affected conjugation of PAs to endogenous proteins was observed in isolated chloroplasts from Helianthus tuberosus (Dondini et al., 2003). It should be noted that light quality is also important for PA metabolism. Lettuce explants cultured under blue light contained a higher proportion of PCA-insoluble conjugated PAs compared with explants grown under white or red light, which contained more of free PA and/or soluble conjugates (Hunter and Burritt, 2005).
The level of free and PCA-soluble PAs seems to be during the light/dark cycle regulated by the activities of biosynthetic enzymes (in tobacco especially ODC) and the catabolic one, DAO, activity of which was found to be substrate inducible (Srivastava et al., 1977). The sharp increase in DAO activity in tobacco leaves 1 h after the exposure to light closely followed or was even concomitant with the light-induced increase in the activity of ODC. This may be the reason why only a slight increase in free Put content was observed after the photoinduction of ODC activity at the beginning of the light phase. The role of both PA conjugation with hydroxycinnamic acids and oxidation in maintaining free PA levels was documented with leaf explants of Chrysanthemum after inhibition of DAO activity. High levels of PAs, especially soluble PA conjugates accumulated in these explants (Aribaud et al., 1994).
Timing of the main peak of DAO activity shortly (1 h) after the middle of the light phase is in agreement with the enzyme maximum observed in isolated etioplasts from Hordeum vulgare after light exposure for 9 h (Andreadakis and Kotzabasis, 1996).
The DAO was found to be localized in cell walls in pea and maize tissues (Slocum and Furey, 1991) and imunohistochemical analyses revealed that DAO expression occurs in cells destined to undergo lignification in tobacco tissues (Paschalidis and Roubelakis-Angelakis, 2005). However, the comparable soluble and cell debris-associated DAO activity was measured in tobacco thin layers (Biondi et al., 2001) and the decline in Put content in the early S-phase of the cell cycle in Helianthus tuberosus was probably due to the high DAO activity occurring in the early S-phase (Serafini-Fracassini, 1991). From the published literature it is clear that the catabolic degradation of Put by DAO is not only a means to regulate the cellular content of this diamine (Kumar et al., 1997; Bagni and Tassoni, 2001). The reaction product of DAO, pyrroline, can be further catabolized to 
-aminobutyric acid (GABA) and subsequently trans-aminated and finally incorporated into the Krebs cycle (Tiburcio et al., 1997) and/or GABA has been shown to play a key role in signal transduction pathways during stress response of many plants (Shelp et al., 1999). The other reaction product of DAO, hydrogen peroxide, might trigger the hypersensitive response, which is thought to be a form of programmed cell death (Walters, 2003) or might be utilized in lignification both during the normal growth and stress response (Angelini et al., 1993).
The results presented here have shown that ADC and ODC activities, as well as putrescine oxidizing activity, correlated well with the alterations in PA content during the day/night period. The data indicate that the parameters studied here were not only light-affected but also appear to be regulated by an internal rhythm.
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Acknowledgements |
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We are grateful to Dr I Machá
ková and Professor M Mok for critical reading of the manuscript. We thank N Hata
ová for skilful technical assistance. This work was supported by grants from the Grant Agency of the Czech Republic No. 206/03/0369. |
Footnotes |
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Abbreviations: ADC, arginine decarboxylase; Cad, cadaverine; DAO, diamine oxidase; EDTA, ethylenediaminetetraacetic acid; GABA,
-aminobutyric acid; ODC, ornithine decarboxylase; PA, polyamine; PAL, phenylalanine ammonia-lyase; PCA, perchloric acid; Put, putrescine; SAMDC, S-adenosylmethionine decarboxylase; Spd, spermidine; Spm, spermine. |
References |
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