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First published online April 22, 2005; 10.1104/pp.104.055483 Plant Physiology 138:142-152 (2005) © 2005 American Society of Plant Biologists Spatial and Temporal Distribution of Polyamine Levels and Polyamine Anabolism in Different Organs/Tissues of the Tobacco Plant. Correlations with Age, Cell Division/Expansion, and Differentiation1Department of Biology, University of Crete, 71409 Heraklion, Greece
Polyamine (PA) titers and biosynthesis follow a basipetal decrease along the tobacco (Nicotiana tabacum) plant axis, and they also correlate negatively with cell size. On the contrary, the titers of arginine (Arg), ornithine (Orn), and arginase activity increase with age. The free (soluble)/total-PA ratios gradually increase basipetally, but the soluble conjugated decrease, with spermidine (Spd) mainly to determine these changes. The shoot apical meristems are the main site of Spd and spermine biosynthesis, and the hypogeous tissues synthesize mostly putrescine (Put). High and low Spd syntheses are correlated with cell division and expansion, respectively. Put biosynthetic pathways are differently regulated in hyper- and hypogeous tobacco tissues: Only Arg decarboxylase is responsible for Put synthesis in old hypergeous vascular tissues, whereas, in hypogeous tissues, arginase-catalyzed Orn produces Put via Orn decarboxylase. Furthermore, Orn decarboxylase expression coincides with early cell divisions in marginal sectors of the lamina, and Spd synthase strongly correlates with later cell divisions in the vascular regions. This detailed spatial and temporal profile of the free, soluble-conjugated, and insoluble-conjugated fractions of Put, Spd, and spermine in nearly all tobacco plant organs and the profile of enzymes of PA biosynthesis at the transcript, protein, and specific activity levels, along with the endogenous concentrations of the precursor amino acids Arg and Orn, offer new insight for further understanding the physiological role(s) of PAs. The results are discussed in the light of age dependence, cell division/expansion, differentiation, phytohormone gradients, senescence, and sink-source relationships.
Polyamines (PAs) are small polycations that represent important intrinsic developmental signals, linked to important developmental phenomena, including cell growth and division (Chattopadhyay et al., 2002  ; Theiss et al., 2002), morphogenesis (Paschalidis et al., 2001; Fos et al., 2003), stabilization of nucleic acids and membranes (Thomas and Thomas, 2001), protein synthesis and chromatin function (Mattheus, 1993), and biotic and abiotic stresses (Kurepa et al., 1998; Perez-Amador et al., 2002; Navakoudis et al., 2003; for review, see Bouchereau et al., 1999). Eukaryotic cells synthesize putrescine (Put) directly from Orn through the activity of Orn decarboxylase (ODC). Plants and some bacteria also synthesize Put indirectly from Arg, by the activity of Arg decarboxylase (ADC). An aminopropyl group derived from decarboxylated S-adenosylmethionine (dSAM) is transferred to Put by spermidine synthase (SPDS) to form spermidine (Spd), and another aminopropyl group is added to Spd by spermine synthase (SPMS) to form spermine (Spm). The dSAM is formed by the activity of S-adenosyl-L-Met decarboxylase (SAMDC; for review, see Wallace et al., 2003).
The positive charge of PAs enables them to interact electrostatically with polyanionic macromolecules within the cell. Spd and Spm can bridge the major and minor grooves of DNA, acting as a clamp holding together either two different molecules or two distant parts of the same molecule (Mattheus, 1993
The complexity in control of cell division, expansion, growth, and senescence in the tobacco (Nicotiana tabacum) plant is well documented (Chen et al., 2001
PA Levels Follow a Basipetal Decrease along Tobacco Plant Axis An inverse correlation is generally observed between leaf ontogenic stage and the titers of all fractions of total PA (Put + Spd + Spm; Fig. 1) independently of growth conditions (data not shown). PA concentrations are extremely high in sink leaves of less than 10 mg in weight and decrease drastically when cell division ceases and cell elongation is the sole process of leaf growth. The trend is same regardless the mode of expression of PA levels (nmol g–1 fresh weight [FW] or nmol mg–1 protein; data not shown). However, when PA levels are expressed per milligram of protein, differences were lower. This could be explained by the fact that the size of the vacuole and water content increase with cell age. The sums of S, SH, and PH fractions of total PA from the apical meristem to the 10th leaf from apex were 3,180 ± 142 and 684 ± 46 nmol g–1 FW, respectively (4.6-fold decrease), or 157 ± 12 and 49 ± 4 nmol mg–1 protein, respectively (3.2-fold decrease). The sum of S, SH, and PH fractions of total PA in leaves (lamina and petiole) also decreases from 2,577 ± 136 in the youngest (circle area of the first leaf) to 124 ± 3 nmol g–1 FW in the oldest (circle area of the 25th) leaf (Fig. 1). The sums of total Put (S + SH + PH), total Spd, and total Spm are high in sink tissues and gradually decrease 34-fold, 22-fold, and 7-fold, respectively, from the first developing (first) to the senescing (25th) leaf (Fig. 2). In petioles and internodes, total-Put, total-Spd, and total-Spm titers remain relatively constant compared to the respective values in leaf margins and centers from the first to the fifth developmental stage (Fig. 2), suggesting that the young vascular tissues mediate basipetal transport of PAs. Furthermore, the internodes contain significantly higher levels of total Put, total Spd, and total Spm than the acropetal and basipetal parts of the petioles (Fig. 2), indicating that PAs are transported and accumulate in shoots. Endogenous PAs were also analyzed using in vitro-grown plants, and the results reinforce the correlation reported herein between PA titers and developmental stages (data not shown).
The Free/Total-PA Ratios Increase Basipetally from Apex to Base of the Plant, whereas the Conjugated Decrease SH-Spd is the most abundant form in the hypergeous (aerial) plant organs (data not shown). The ratios of S-PAs/total PAs (white parts of circles/total circles, Fig. 1) increase 2.4-fold basipetally from shoot apex to the base of the plant, whereas the ratios of SH-PAs/total PAs decrease 1.5-fold (Fig. 1). The ratio S-Spd/total S-PAs increases 1.7-fold with increasing age in hypergeous tissues, whereas the ratios S-Put/total S-PAs and S-Spm/total S-PAs decrease 1.9- and 2.6-fold, respectively (Table I). Furthermore, the ratio S-Spd/total Spd also increases 4-fold, whereas S-Put/total Put increases only 2-fold and S-Spm/total Spm decreases 4-fold. Thus, taking into account also the respective conjugated ratios, it can be concluded that the basipetal increase in the S-PAs/total-PAs ratio is presumably due to decreased conjugation of Put and particularly Spd.
Total-PA levels in primary and secondary root tissues do not differ significantly (Fig. 1), although total Spd is greater and total Put is lower in the secondary roots (Fig. 2), whereas all S fractions and SH-Spd are higher in the secondary roots (data not shown).
Cell expansion in tobacco leaves increases, whereas nuclear DNA synthesis (K. Paschalidis and K. Roubelakis-Angelakis, unpublished data) and cell division (Chen et al., 2001
The adc, samdc, and spds Transcripts and ADC Protein Do Not Coincide with Activity Levels in the Youngest Leaves
The transcripts of odc are consistent with the activity levels in the first leaf; they are high in leaf margin and decrease basipetally to the petiole (Fig. 3, C and D). However, the transcripts of adc, samdc, and spds and the protein level of ADC do not coincide with specific activities, being lower in leaf margin and gradually increasing in leaf center and petiolar parts (Fig. 3, C–E, and data not shown). The basipetally increasing gradients of transcripts continue also to the internode; the spds transcript in the first internode is almost 10-fold higher than in the marginal region of the first leaf (Fig. 3D). The opposite trend of adc transcript on one hand and ADC protein and activity levels on the other could suggest that regulation of ADC in tobacco plants is at the posttranslational level, as has been the case with ADC in oat (Avena sativa; Malmberg and Cellino, 1994
In expanding leaves, the activities of ADC and ODC (Fig. 3A) follow the basipetally decreasing gradient, from leaf margin to petiole, which is also found at the first developmental stage. On the contrary, in leaves of the fifth and 10th developmental stages, SPDS- and SPMS-specific activities increase from leaf margins to petioles following a reverse, to the first developmental stage, gradient (Fig. 3A). Both enzyme and transcript levels of SPDS are almost 2-fold higher in petioles than in leaves at the 10th developmental stage (Fig. 3, A, C, and D). In mammals (Marton and Pegg, 1995
As ODC activity has been correlated with cell divisions in many mammalian and higher plant systems (Marton and Pegg, 1995
The specific activities of all biosynthetic enzymes in the soluble fraction generally decrease with age in tobacco, with the exception of SPMS (Fig. 3, A and B), and are in agreement with the endogenous PA levels. ODC soluble-specific activity is significantly higher in the hypogeous than in the hypergeous organs, including shoot apical meristems, which also exhibit high ODC activity (Fig. 3, A and B). SAMDC activity is also high in roots, whereas ADC is lower in roots than in old and fully developed tissues (Fig. 3, A and B). ADC and ODC activities in the particulate fractions of aerial tobacco tissues (leaves, petioles, and internodes) follow the same trend as the soluble ones (Table II). ODC particulate activity is approximately half of the soluble one, while in young roots it is extremely low as compared to the activity in the soluble fraction (Table II; Fig. 3), which is almost 30-fold higher than the respective ADC (Fig. 3, A and B). In contrary, higher ADC than ODC specific activities are present in the old hypergeous organs. Root apical tissues contain higher soluble but lower particulate ADC and ODC activities than the older root tissues. Furthermore, SAMDC, SPDS, and SPMS activities are absent from the particulate fractions.
Arginase Activities and Arg and Orn Levels Increase with Age; Put Synthesis in Hypogeous Tissues Is Due to Successive Actions of Arginase and ODC In tobacco plant, arginase-specific activities, as well as Arg and Orn levels, increase with increasing organ age (Fig. 4). Differences are more pronounced in internodes, where arginase, Arg, and Orn increase from the youngest to the oldest one, 3.9-, 1.3-, and 1.8-fold, respectively. The above three parameters generally increase from marginal leaf regions to petioles and internodes. The newly formed roots exhibit higher arginase and Orn but lower Arg levels than the primary roots (Fig. 4B). Furthermore, Put biosynthesis in roots (Fig. 2) uses Orn as a substrate and is due to successive actions of arginase (Fig. 4B) and ODC (Fig. 3B).
It is evident that all the studied organs of the tobacco plant are capable of synthesizing PAs (Fig. 5). The highest relative Put biosynthetic capacity is localized in the hypogeous apical meristems followed by the shoot apex and young developing leaves. The highest relative biosynthetic capacity of the higher PAs (Spd and Spm) is localized in the shoot apex and in the youngest aerial tissues, but roots also synthesize Spd and Spm at high rates (Fig. 5). These trends apply to plants of different ages and are independent of photoperiod (data not shown). Spd and Spm synthesis is more uniformly distributed (with lower temporal differences) than Put synthesis throughout tobacco development. Lower but still significant rates of PA biosynthesis are exhibited in expanding leaves and in old vascular tissues.
Tobacco has been used as a model system to study the various events related to primary assimilation or remobilization of carbon and nitrogen during transition of sink to source leaves and senescence (Oparka et al., 1999 ; Masclaux et al., 2000). In addition, PAs are proposed to participate in the plasticity of carbon and nitrogen metabolism in higher plants (Bouchereau et al., 1999). Turano and Kramer (1993) determined the interaction of carbon and nitrogen availability on PA accumulation in soybean (Glycine max) by using a detached leaf system. Aziz (2003) suggests that Spd could regulate fruitlet abscission in grapevine by modulating the levels of sugars and amino acids in inflorescences, whereas, in tobacco, PAs and aromatic amines could be a limiting factor in sexual development (Martin-Tanguy, 1997) or play a role in apical dominance (Geuns et al., 2001). There is a lack of information on the relationship between the PA biosynthetic pathways and the metabolism of carbon and nitrogen or partitioning between source and sink organs in the vegetative plant. In tobacco, PA distribution follows a reduction from sink to source leaves, and the strongest reduction coincides with senescence. In this context, PAs could act either as carbon/nitrogen reserves or as signal molecule(s) regulating senescence and probably sink-source relationships. Also, PAs are present in thylakoid membranes, PSII, and the light-harvesting complex (Kotzabasis et al., 1993b; Sfichi et al., 2004), suggesting a functional role in the photosynthetic activity during senescence or in the regulation of photoassimilate synthesis and/or partitioning between organs. PAs also inhibit ethylene biosynthesis and other degrative processes associated with senescence, including chlorophyll loss, increase in membrane leakage, and rise in RNAse and protease activities (Pandey et al., 2000). PA involvement in delay of senescence was confirmed by elegant transgenic approaches (Mehta et al., 2002; Tucker and Seymour, 2002). However, the underlying molecular basis for this PA implication is still not understood. Other phytohormones may also affect PA metabolism. A transient increase in ADC and ODC activities as well as in the amount of free PAs and a decrease of conjugated PAs during early parthenocarpic fruit development induced by auxins and gibberellins (Alabadí et al., 1996) has been observed; expression of ODC and SPDS genes is also up-regulated (Alabadí and Carbonell, 1998, 1999). Fos et al. (2003) have suggested that parthenocarpy induced by the recessive mutation pat-2 in tomato (Lycopersicon esculentum) activates PA biosynthesis through the ODC pathway leading to elevated free-Spm content in unpollinated ovaries, probably as a result of a higher concentration of active GAs in pat-2 ovaries. Furthermore, jasmonate-induced up-regulation of PA biosynthetic genes (Biondi et al., 2001; Goossens et al., 2003) is differently modified by the presence/absence of exogenously supplied auxins and cytokinins (Biondi et al., 2003).
Although all organs of a tobacco plant are competent to synthesize PAs (Fig. 5), the shoot apical tissues exhibit the highest Spd and Spm synthesis and high Put synthesis. Shoot apical meristems and actively dividing young tissues contain also the highest contents of PAs, which decrease drastically following the completion of their active development and probably the sink-source transition (Fig. 1). Spd titers are generally higher throughout development and Spm exhibits low variation (Fig. 2), suggesting a tighter regulation of cellular Spm metabolism, compared to Put or Spd, in agreement with results from camellia (Camellia sinensis) and grapevine cells (Pedroso et al., 1997
At relatively low level of auxin, it is suggested that the auxin-binding protein 1 acts to mediate cell expansion, whereas high auxin levels stimulate cell division via an unidentified receptor (Chen et al., 2001
Our understanding of the specific PA pathways is still not precise enough to allow a safe correlation of total biosynthetic and catabolic fluxes in all pathways simultaneously. To our knowledge, this is the first exhaustive report on detailed temporal and spatial distribution of the PA anabolic (this work) and catabolic (K. Paschalidis and K. Roubelakis-Angelakis, unpublished data) pathways in plants. The tobacco secondary roots contain significantly higher free Put than other plant tissues (Fig. 6), except the shoot apical tissues, which contain similarly high free Put (data not shown). The root tissues use mainly as the substrate Orn and the ODC pathway for Put production (Fig. 6C). Therefore, the amount of Orn in the tissues could become limiting. The addition of either Orn or Arg to the medium results in increased accumulation of Put in poplar (Populus spp.) cells (Bhatnagar et al., 2001
A constitutive expression of the ODC enzyme in tobacco root cells could lead to a depletion of Orn pools. Since Orn is also the precursor of Arg, its depletion could reduce the availability of Arg and in turn limit the amount of Put synthesized via ADC (Fig. 6). Both low levels of Arg and ADC observed in newly formed roots, as compared to the primary root, together with high Orn, ODC, and arginase levels (Figs. 3 and 4) could support these hypotheses. Thus, most of the Orn in these cells is directly produced from Arg by arginase as part of the urea cycle and not from Glu. This production, however, is insufficient to saturate the available ODC enzyme in these cells. Although little is known about homeostatic regulation of Orn pools in plants (Bhatnagar et al., 2001 ), a homeostatic mechanism must exist to increase Orn production concomitant with its increased use.
The presence of alternative pathways for biosynthesis of PAs in plants (Bouchereau et al., 1999 In conclusion, our data give a clearer picture of the homeostatic characteristics of PA biosynthesis and conjugation in tobacco plant organs during development. Biosynthetic capacity and PA pool size decrease basipetally in the aerial parts during growth and senescence, potentially implicating the sink-source transition. The ratios of soluble and conjugated PAs also change with development (Fig. 1). As the size of the total-PA pool and the relative ratio of SH-PAs/total PAs decrease, the relative ratio of S-PAs/total PAs increases, suggesting that, in addition to biosynthesis, conjugation exerts a significant role in the homeostatic mechanism of cellular PAs. The pool size of PAs in the hypogeous organs is comparative to their size in the mature and old hypergeous tissues (Fig. 1), even though the biosynthetic capacity of roots is severalfold higher (Fig. 5), suggesting different turnover rates between the organs. Moreover, the contribution of the ODC and ADC pathways to Put biosynthesis and the genes of Spd and Spm synthesis are developmentally regulated and exhibit different subcellular localization. Arginase-derived Orn is the substrate for Put production in roots via ODC, whereas Put in old hypergeous tissues is synthesized via the ADC pathway. Furthermore, ODC and SPDS depend on the spatial and temporal status of the plant for a role in the progression of cell division/expansion, whereas a high Spd synthesis response leading to cell division and a low response leading to cell expansion are proposed.
Plant Material Explants were removed from glasshouse-grown (25°C ± 2°C, 16/8 h photoperiod, and total energy of 55 µE m–2 s–1) 6- to 8-week-old tobacco (Nicotiana tabacum L. cv Xanthi) plants and numbered from shoot apex. They were divided in tip and leaf margin, leaf center, acropetal or apical petiole, and basipetal or basal petiole. Furthermore, shoot apex, primary or main root, and secondary or newly formed root explants were also removed.
Free, soluble-conjugated, and insoluble-conjugated PAs were determined according to Kotzabasis et al. (1993a)
Arg and Orn were colorimetrically determined according to Ceriotti and Spandrio (1957)
Total proteins were extracted according to Papadakis et al. (2001)
An isotopic method (Primikirios and Roubelakis-Angelakis, 1999
Arginase activity was colorimetrically measured as the rate of Orn formation, as described by Roubelakis and Kliewer (1978) SPDS and SPMS were assayed by measuring the formation of Spd and Spm, respectively. The assay mixture for SPDS contained 3 mM Put, 0.2 mM dSAM, 100 mM Tris-HCI buffer, pH 9.0, and the enzyme in a total volume of 200 µL. The reaction was performed at 37°C for 1 h and stopped by the addition of 200 µL of 65 mM borate-KOH buffer, pH 10.5, followed with 1 mL of 2 N NaOH and 10 µL of benzoylchloride. The products were separated with an HP 1100 HPLC system. SPMS was assayed similarly to SPDS by replacing Put with Spd in the assay mixture.
Protein samples were electrophoretically resolved, transferred to nitrocellulose membranes, and incubated with an antiserum raised in rabbit against the hybrid protein MBP-ADC (Primikirios and Roubelakis-Angelakis, 2001
A 1,047-bp tobacco adc cDNA clone obtained from suspension cell cultures (K. Primikirios and K. Roubelakis-Angelakis, unpublished data) was used as adc probe. Two degenerate oligonucleotide primers corresponding to two conserved regions in the aligned sequences of oat (Avena sativa; Bell and Malmberg, 1990
Statistical analysis of data concerning endogenous PAs, activities, Arg and Orn levels, and the ratio mRNA/rRNAs was performed with one-way ANOVA to reveal any statistically significant differences (P < 0.01 or P < 0.05) that occurred. Data were logarithmically transformed to homogenize variances when necessary (Bartlett's test). If F values were significant, pairwise comparisons of means were made by Tukey's multiple comparisons test (P < 0.01). Data that contained two groups were compared using t tests. All experiments were carried out three times with similar results and included a minimum of three replicates per sample. Upon request, all novel materials described in this publication will be made available in a timely manner for noncommercial research purposes.
We thank Prof. J. Carbonnel (Universitad Politécnica de Valencia, Spain) for the odc DNA, Dr. A. Michael (Institute of Food Research, Norwich, UK) for the samdc PCR fragment, and Dr. T. Hashimoto (Nara Institute of Science and Technology, Japan) for the spds reverse transcription-PCR fragment. We are grateful to Prof. A. Shirahata and Dr. K. Samejima (University Yyo-Sai, Japan) for the generous gift of dSAM, to Dr. A. Papadakis (University of Crete, Greece) for helpful discussions, and to Dr. I. Corraliza (Department of Biochemistry and Molecular Biology, Cáceres, Spain) for comments on arginase inhibition. Received October 21, 2004; returned for revision December 31, 2004; accepted January 23, 2005.
1 This work was supported by the Interreg II and Pythagoras (Code No. 1945) programs.  Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.055483. * Corresponding author; e-mail poproube{at}biology.uoc.gr; fax 30–2810–394459 or 30–2810–394408.
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ABSTRACT
RESULTS
); P, acropetal petiole (
); I, internode (dotted line); R, primary root; r, secondary root. A, Specific activities in L, P, and I at different developmental stages. B, Specific activities of ADC, ODC, and SAMDC (nmol CO2 h–1 mg–1 protein), SPDS (nmol Spd h–1 mg–1 protein), and SPMS (nmol Spm h–1 mg–1 protein) in shoot apex, primary, and secondary roots. C, RNA gel-blot analysis of the expression of adc, odc, samdc, and spds genes. D, Quantification of mRNA levels (mRNAs/actin mRNAs). E, Western-blot analysis of ADC protein. Numbering started from the apical organ being designated as first. Error bars represent ±SE.
; Orn, 
. B, Arginase activities (µmol Orn h–1 mg–1 protein) and endogenous levels of Arg and Orn (nmol mg–1 protein) in shoot apex, primary root, and secondary root. Error bars represent ±SE.
-difluoromethylornithine does not abolish C. reinhardtii cell divisions but causes an early accumulation of dividing cells (Theiss et al., 2002
-hydroxy-L-Arg (a competitive inhibitor of arginase; Iniesta et al., 2001