Journal of Experimental Botany, Vol. 52, No. 355, pp. 231-242,
February 2001
© 2001 Oxford University Press
Original Papers |
Methyl jasmonate upregulates biosynthetic gene expression, oxidation and conjugation of polyamines, and inhibits shoot formation in tobacco thin layers
1 Dipartimento di Biologia, Università di Bologna, Via Irnerio 42, 40126 Bologna, Italy
2 Dipartimento di Biologia Vegetale, Università di Roma ‘La Sapienza’, Rome, Italy
Received 10 April 2000; Accepted 13 September 2000
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Abstract |
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The effect of methyl jasmonate (MJ) on de novo shoot formation and polyamine metabolism was investigated in thin layer explants of tobacco (Nicotiana tabacum L. cv. Samsun). A relatively low concentration of MJ (0.1 µM) enhanced explant fresh weight, but had no effect on the final number of shoots per explant while higher concentrations (1 and 10 µM) significantly inhibited organogenesis. The histological study revealed that, with increasing concentrations of MJ, the formation of meristemoids and shoot domes declined and the incidence of cell hypertrophy increased. In explants cultured with 0.1, 1 or 10 µM MJ, the endogenous levels of free putrescine, spermidine and spermine generally declined compared with controls, after 7 and 15 d. Perchloric acid-soluble conjugated polyamines accumulated dramatically during culture, but much more so in the presence of MJ than in controls. Acid-insoluble conjugated spermidine alone increased in response to the elicitor. Activities of the putrescine biosynthetic enzymes arginine decarboxylase (ADC, EC 4.1.1.19) and ornithine decarboxylase (ODC, EC 4.1.1.17) in the soluble fraction of MJ-treated explants displayed up to 3-fold increases relative to control explants. However, the most relevant increases in these enzyme activities occurred in the particulate fraction. The activity of S-adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.21), an enzyme involved in spermidine and spermine biosynthesis, was also stimulated by exposure to MJ. Northern analyses revealed MJ-induced, generally dose-dependent, increases in the mRNA levels of all three enzymes. Diamine oxidase (DAO, EC 1.4.3.6) activity was stimulated by MJ mainly in the cell wall fraction. The upregulation of polyamine metabolism is discussed in relation to the morphogenic behaviour of MJ-treated explants.
Key words: Nicotiana tabacum, thin layers, shoot formation, methyl jasmonate, polyamine metabolism.
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Introduction |
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Jasmonic acid (JA), a derivative of linolenic acid synthesized via the octadecanoid pathway and its methyl ester (MJ) are ubiquitous cyclopentanone compounds which are claimed to represent a new class of plant growth regulators (Creelman and Mullet, 1997
). Like other phytohormones, they exert numerous biological effects ranging from promotion of leaf senescence and abscission, tendril coiling, and stomata closure to inhibiton of root growth and germination of non-dormant seeds (Creelman and Mullet, 1997). Jasmonates seem to be involved in specific morphogenic events, such as tuberization and bulb formation (Koda, 1997), by regulating cell division (Ueda and Kato, 1982) and expansion (Takahashi et al., 1994). As far as in vitro-cultured tissues are concerned, effects of JA and/or MJ on cytokinin-induced soybean callus growth (Ueda and Kato, 1982) and on growth and development of isolated tomato roots (Tung et al., 1996) have been hitherto reported.
Jasmonates, however, seem to play a dual role in plant development and defense (Creelman and Mullet, 1997). Indeed, a large body of evidence has accumulated indicating that, by altering the expression of a number of genes, they may be implicated in the signal transduction cascade that mediates a vast array of wounding and elicitor-induced responses (Blechert et al., 1995). In fact, jasmonate-induced proteins include proteinase inhibitors, antifungal proteins such as thionin, and hydroxyproline- and proline-rich cell wall proteins (Creelman and Mullet, 1997, and references therein). Another line of evidence for the role of jasmonates in disease resistance comes from their stimulatory effect on secondary metabolite production (Gundlach et al., 1992).
It has recently been shown (Lee et al., 1997; Mader, 1999; Biondi et al., 2000) that treatment with jasmonates can increase the accumulation of another class of secondary metabolites, namely polyamines (putrescine, spermidine and spermine) covalently bound to hydroxycinnamic acids, i.e. hydroxycinnamoyl amides (HCAs). HCAs are particularly abundant in Solanaceae where they constitute most of the acid-soluble conjugated polyamine pool; in tobacco they accumulate dramatically before and during flowering and upon infection by pathogens (Flores and Martin-Tanguy, 1991). Tissues having no or very scarce amounts of HCAs, rapidly accumulate them during in vitro culture in response to exogenous hormones (Torrigiani et al., 1987; Burtin et al., 1989; Scoccianti et al., 2000). In their free form, putrescine, spermidine and spermine are positively implicated in many aspects of plant growth and differentiation (Cohen, 1998), whereas a role for HCAs in regulating growth is doubtful (Wyss-Benz et al., 1990).
In some cases (Lee et al., 1996; Imanishi et al., 1998; Mader, 1999; Biondi et al., 2000) altered levels of free polyamines, and changes in the activities and/or transcript levels of the main biosynthetic enzymes, arginine decarboxylase (ADC, EC 4.1.1.19), ornithine decarboxylase (ODC, EC 4.1.1.17) and S-adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.21), have been reported to occur in response to treatment with jasmonates. Nevertheless, a complete picture of the effects of jasmonates on polyamine metabolism is still lacking, and data are as yet partly contradictory. In particular, effects on diamine oxidase (DAO, EC 1.4.3.6) activity have not been investigated.
Tobacco thin layers represent a useful in vitro model system for the study of organogenesis in so far as the response is efficient and versatile (Tran Thanh Van et al., 1974). The action of exogenous polyamines, and the pattern of endogenous free and conjugated polyamine content and metabolism have been extensively investigated in these explants in relation to all the organogenic programmes, i.e. shoot formation, flower formation and rhizogenesis (Torrigiani et al., 1987; Tiburcio et al., 1988; Altamura et al., 1991, 1995). The aim of the present study was to analyse the effect of exogenously supplied MJ on organogenesis and on the polyamine metabolic status, including expression of biosynthetic genes, in the course of shoot formation in tobacco thin layers.
Present results indicate that ADC, ODC and SAMDC activities and gene expression, as well as putrescine oxidising activity, were all strongly up-regulated by MJ. This led to a conspicuous accumulation of mainly soluble conjugated polyamines. In parallel, the elicitor promoted cell hypertrophy and inhibited de novo shoot formation in a dose-dependent manner.
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Materials and methods |
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Plant material and tissue culture
Thin layer explants were excised from plants of Nicotiana tabacum L. cv. Samsun grown under controlled light and temperature regimes with a 12 h photoperiod, a light intensity of 1.87 W m-2, and a day/night temperature of 25/19 °C. Medium composition and explant culture conditions were as described previously (Biondi et al., 1998
). Methyl jasmonate (Boehringer Ingelheim Bioproducts, Heidelberg, Germany) was dissolved in 99% ethanol. The stock solution (1 mM) in ethanol was diluted with distilled water up to 100 times the final concentration. The 100-fold concentrated stock solution was added to autoclaved media (1 ml in 100 ml medium) after filter-sterilization to give final concentrations of 0.001, 0.1, 1, and 10 µM. Ethanol controls were made by supplementing the medium with an amount of ethanol equivalent to that present in 10 µM MJ treatments. The time-course of morphogenesis was evaluated macroscopically up to the end of culture. At each sampling time (5, 7 and 15 d), approximately 150 explants were examined for each treatment. At the end of culture, the number of shoots was counted under a dissecting microscope. Differences between means were compared using Student's t-test, and differences between the percentages using the 
2 test.
Histological analysis
For each concentration of MJ (0, 0.1, 1, and 10 µM), five randomly chosen explants were fixed in FAA (70% ethanol-glacial acetic acid-formalin, 18 : 1 : 1, v/v) on day 12, dehydrated, embedded in paraffin (melting point 52–54 °C, Merck) and sectioned at 8 µm thickness with a Top-Super S-150 microtome (Pabisch, Milano, Italy). Sections were stained using a Top-Stainer LX-100 (Pabisch) with eosin and Carazzi's haemalum, as previously described (Altamura et al., 1995, and references therein).
TLC analysis of polyamines
Free polyamines were separated by thin layer chromatography after extraction of 200–400 mg explants in 5 vols of 4% (v/v) perchloric acid (PCA), centrifugation for 15 min at 18 000 g and dansylation of 0.1 ml aliquots of the supernatant. Precoated plates of Silicagel 60 (Merck) were used and run with ethylacetate : cyclohexane (2 : 3, v/v) as the eluent (Torrigiani et al., 1987). Spots were visualized under UV light and those corresponding to putrescine, spermidine and spermine were identified by comparison with dansylated standards. They were scraped, eluted with anhydrous acetone and their relative fluorescence measured using a spectrofluorometer (Jasco FP 770, Tokyo, Japan). Polyamines released from PCA-soluble and -insoluble conjugates were recovered after hydrolysis (with 6 N HCl at 110 °C for 18 h) of aliquots of the supernatant and resuspended pellet (washed twice with the initial volume of PCA), respectively, and quantified following the protocol described above. Experiments were repeated at least once (3–4 replicates each). Differences between mean values for controls and MJ-treated samples were analysed statistically using Student's t-test.
Enzyme assays
For ADC and ODC activity assays, explants were homogenized on ice with 5 vols of 0.1 M TRIS-HCl buffer, pH 8.5, containing 50 µM pyridoxal 5-phosphate (PLP), and centrifuged at 18 000 g for 30 min at 4 °C. Aliquots (0.3 ml) of the supernatant and of the resuspended pellet were used for enzyme assays. These were performed essentially as described earlier by measuring the 14CO2 evolution from 7.4 kBq L-[1-14C]ornithine (2.11 TBq mol-1, Amersham Pharmacia Biotech Italia, Milano, Italy) or DL-[U-14C]arginine (11 TBq mol-1, Amersham Pharmacia Biotech Italia) for ODC and ADC, respectively (Scaramagli et al., 1995).
To determine SAMDC activity, explants were homogenized on ice with 5 vols of 0.1 M TRIS-HCl buffer, pH 7.6, containing 50 µM EDTA and 25 µM PLP. The homogenate was centrifuged at 18 000 g for 30 min at 4 °C; 0.2 ml aliquots of the supernatant were incubated with 3.7 kBq S-adenosyl-L-[carboxyl-14C]methionine (2.07 TBq mol-1, Amersham Pharmacia Biotech Italia) and the rate of 14CO2 evolution from the labelled substrate was measured as described elsewhere (Scaramagli et al., 1999b).
DAO activity was assayed essentially as described previously (Torrigiani et al., 1989) except that it was measured separately in different fractions of the crude extract prepared according to two different protocols. In both cases, explants were ground on ice with a mortar and pestle in 3 vols of 100 mM potassium phosphate buffer, pH 8, containing 2 mM dithiothreitol. In the first procedure (Method 1), the homogenate was filtered through four layers of cheesecloth and activity was assayed separately in the cell debris remaining on the cheesecloth, and in the filtrate. The second procedure (Method 2) was performed essentially as described previously (Laurenzi et al., 1999). The homogenates were centrifuged at 12 000 g for 20 min at 4 °C. Pellets were resuspended in the initial volume of extraction buffer supplemented with 0.01% Triton X-100 and filtered through nylon filters (pore size 70 µm). After repeating this step three times, the washed pellets were resuspended in 5 ml buffer g-1 FW. The supernatant was re-centrifuged at 100 000 g for 60 min at 4 °C. DAO activity was determined in the 12 000 g and 100 000 g supernatants, and in the washed pellets to determine wall-bound activity. Activity in the different fractions was assayed by a radiometric method based on the production of 
1-[14C]pyrroline from [1,4–14C]putrescine (4.03 TBq mol-1, Amersham Pharmacia Biotech Italia) during a 30 min incubation at 37 °C (Scaramagli et al., 1999b). The [14C]pyrroline was immediately extracted in 1 ml toluene and 0.5 ml aliquots were placed in scintillation cocktail (Ultima Gold, Beckman Analytical, Milano, Italy) and counted in a Beckman LS 6500 scintillation counter. Experiments were repeated at least once (3–4 replicates each). Controls and MJ-treated samples were compared using Student's t-test to check for significant differences in activity levels.
Protein content was measured according to Bradford, using bovine serum albumin as standard (Bradford, 1976).
RNA extraction and Northern blot
Total RNA was extracted from c. 200–300 mg fresh weight explants using an RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. RNA (15 µg per track) was size-fractionated on a 1.2% agarose formaldehyde gel and transferred in 10xSSC (20xSSC: 0.3 M sodium citrate, 3.0 M NaCl, pH 7.0) onto nylon membranes (Hybond-N, Amersham, Milano, Italy) overnight according to standard methods (Sambrook et al., 1989). RNA was cross-linked to the membrane by exposure to UV at 312 nm (Vilber Lourmat, Marné La Vallée, France) for 4 min.
The tobacco probes (kindly supplied by Anthony J Michael, Institute of Food Research, Norwich, UK) were obtained as described previously (Michael et al., 1996). RNA blots were pre-hybridized at 42 °C for 2 h and hybridized at 42 °C for 18–20 h (Scaramagli et al., 1999b) with [32P]dCTP-labelled PCR fragments (random priming with a Rediprime DNA Labelling Kit, Amersham Pharmacia Biotech Italia) of SAMDC, ADC and ODC amplified by using specific oligonucleotide primers homologous to the 5' and 3' ends of the ORFs of the respective genes. The following primers were used to obtain c. 1.10 kb, 2.17 kb and 1.30 kb PCR products of SAMDC, ADC and ODC, respectively: 5'-CTAATGGATTCGGCCTTGCCTGTC-3' (sense) and 5'-CACAGCCCTCAAGACACTACTCC-3' (antisense) for SAMDC; 5'-ATGCCGGCCTTAGGTTGTTGTGTAG-3' (sense) and 5'-ACAACTTCAAGCGGTGCAATAGGACCA-3' (antisense) for ADC; 5'-GGATGGCCGGCCAGACAGTCATCG-3' (sense) and 5'-TAGAGGTGGTTCATCAGCTTGG-3' (antisense) for ODC.
Following hybridization, membranes were washed as described previously (Scaramagli et al., 1999b) and then exposed to X-ray film at -80 °C for 24 h (ADC, SAMDC) or 7 d (ODC) with intensifying screen (DuPont, Wilmington, DE, USA). Equal loading of total RNA on gels was verified by ethidium bromide staining. Band densities were quantified in each sample using the image analysis Phoretix programme (Phoretix International Ltd, Newcastle upon Tyne, UK) and data are shown as relative intensities, normalised to the loading controls.
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Results |
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Growth and morphogenesis
In a preliminary dose–response experiment, thin layer explants were cultured for 15 d on media containing either ethanol alone or 0.001, 0.1 or 10 µM MJ. At the end of culture, results showed that, compared with controls grown in the absence of either ethanol or MJ, the lowest concentration tested (as well as ethanol alone) had no effect either on explant fresh weight or on the number of shoots per explant. The highest concentration significantly (P<0.01) inhibited the latter (6.3±0.7 versus 15.0±0.7 in controls), while 0.1 µM enhanced explant fresh weight (0.57±0.05 versus 0.38±0.02 g in controls) at a low significance level (P<0.05) and had, apparently, no effect on organogenesis (data not shown). In subsequent experiments, a 10x higher concentration was also found to increase explant fresh weight compared with controls at the same significance level, whereas a 100x higher concentration did not alter final fresh weight (Fig. 1A
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By day 7 in culture (Table 1
), the percentage of explants which exhibited macroscopic clumps of callus was significantly (P<0.01) higher with 0.1 µM MJ, but not with 1 µM; with the highest concentration of MJ, the percentage of ‘swollen’ explants, without macroscopic callus, was significantly (P<0.05) higher than in controls whereas there were significantly (P<0.01) fewer explants with macroscopic callus. By day 15, explants treated with the two lower concentrations of MJ no longer displayed differences compared with controls. By contrast, the highest concentration yielded a higher percentage of explants in the swollen stage or with macroscopic callus, resulting in a significantly (P<0.01) lower percentage (about 50%) of shoot-forming explants (Table 1). The number of shoots per explant at the end of culture (day 15) was also unaffected by 0.1 µM MJ, but was significantly (P<0.01) inhibited by the two higher concentrations (Fig. 1B
). However, due to the increase in explant size, when organogenesis was expressed as number of shoots g-1 explant fresh weight, a significant (P<0.01) reduction was also observed in the presence of the lowest concentration of MJ (Fig. 1B). This reduction was further accentuated by 1 µM MJ which also promoted a fair amount of disorganized growth (Fig. 2A). The sum of callus and shoots formed under the latter treatment resulted in the highest explant fresh weight at the end of culture. The highest concentration of MJ significantly (P<0.01) impaired shoot formation (Table 1; Fig. 1B) without altering explant fresh weight (Fig. 1A). In fact, at the end of culture, the morphology of explants treated with this concentration of MJ was quite different from all other treated or control explants insofar as they formed macroscopic callus and very few shoots (Fig. 2A).
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Histological analysis
At day 12 in culture, numerous meristemoids and shoot domes with leaf primordia protruded from the surface of control explants (Fig. 2B). In these explants, cell hypertrophy rarely occurred, whereas it increased with increasing MJ concentration; in parallel, the presence of both meristemoids and shoot domes decreased. In fact, in the treatment with 0.1 µM MJ, cell proliferation and meristemoid formation was similar to controls. However, hypertrophic cells, belonging to the superficial proliferated areas and separating meristemoids and domes, were more frequent than in controls (data not shown). In the presence of 1 µM MJ, cell hypertrophy was observed in large groups of cells, again belonging only to the most superficial proliferated layers (Fig. 2C). Furthermore, in the regions showing extensive cell hypertrophy, meristemoids and domes were never observed. In the presence of the highest concentration of MJ, explants showed a lower level of cell proliferation than in the other treatments (Fig. 2D, in comparison with Fig. 2B, C), and the cells of the most superficial layer were almost all hypertrophic. The only superficial zones not showing cell hypertrophy were those in which rare meristemoids and domes were observed (Fig. 2D). Furthermore, only in the latter treatment did a high frequency of hypertrophic cells characterize the middle and deep layers of the poorly proliferated explants (Fig. 2E).
Free and conjugated polyamines
As shown in Fig. 3A, free putrescine increased markedly (about 16-fold) during time in culture in control explants. Free spermidine titres also rose sharply between days 1 and 7 and then declined by about 30% on day 15 (Fig. 3B); spermine accumulated slightly on day 15 (Fig. 3C). After 24 h, treatment with MJ had no effect on free polyamine levels, while by day 7 all three amines had declined significantly (P<0.01) compared with untreated explants. The effect was dose-dependent in the case of spermidine, but not putrescine and spermine. A similar significantly inhibitory effect on free putrescine (P<0.01, except with 0.1 µM MJ) and spermine (P<0.05) levels was registered on day 15; however, given the high levels accumulated in control explants, the relative reduction in explants exposed to 10 µM MJ was even more marked: putrescine 69%, spermidine 51%, spermine 68%.
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Soluble conjugated polyamines accumulated in control shoot-forming explants during time in culture, so that by day 15 their levels were well above those of the free fraction (Fig. 4
). In MJ-treated explants, no relevant changes were observed after 24 h in culture (Fig. 4, insets). Instead, the overall decline in free amine levels observed on days 7 and 15 was accompanied by a dramatic increase in the amount of PCA-soluble putrescine (Fig. 4A) and spermidine (Fig. 4B) conjugates (spermine conjugates were not detectable) in a generally dose-dependent manner. The effect was highly significant (P<0.01) with 1 and 10 µM MJ. The largest stimulation (up to 23-fold for putrescine and 20-fold for spermidine) relative to controls was observed on day 7. However, the highest absolute amount was reached on day 15 in explants treated with the highest concentration of MJ, where soluble conjugated putrescine and spermidine concentrations were approximately 10 and 3.5 millimolar, respectively.
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Insoluble conjugated putrescine and spermidine levels were markedly lower than those of the soluble conjugates (Fig. 5A
, B). Insoluble spermine conjugates were not detected. Only the spermidine conjugates were significantly (P<0.05) affected by exposure to 1 and 10 µM (day 7) or 10 µM (day 15) MJ, with a marked (up to 12-fold) and dose-dependent increase on day 7 (Fig. 5B).
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Although putrescine was always the predominant amine in all three fractions, the free and soluble conjugated putrescine-to-spermidine ratio declined compared with controls in explants treated with 1 or 10 µM MJ. In the acid-insoluble fraction, while putrescine levels were 3-fold (day 15) to 10-fold (day 7) higher than spermidine levels in controls, this ratio fell to approximately one in explants treated with 10 µM MJ.
Biosynthetic enzyme activities and gene expression
ADC, ODC and SAMDC activities were determined in 7- and 15-d-old explants because these displayed relevant changes in polyamine content. The putrescine synthesizing enzyme (ADC and ODC) activities were detected in both soluble and particulate fractions. On the contrary, SAMDC is known to be a cytosolic enzyme (Cohen, 1998), consequently its activity was measured only in the supernatant fraction.
An ADC transcript was revealed by Northern analysis in controls and with all the MJ concentrations tested. After 7 d in culture, signal intensity in the presence of the highest concentration of MJ was about double compared with controls (Fig. 6A, B). On day 15, ADC mRNA levels were higher in MJ-treated explants than in controls, and were highest with the 10 µM concentration (Fig. 6D, E). Soluble ADC activity was essentially the same in 7- and 15-d-old explants grown in the absence or in the presence of 0.1 µM MJ (Fig. 6C, F). In 7-d-old explants, both 1 and 10 µM MJ significantly (P<0.01) enhanced this activity (about 2- and 4-fold, respectively) relative to controls (Fig. 6C). Also in 15-d-old explants, the same concentrations of MJ significantly (P<0.01) stimulated (about 4- and 6-fold, respectively) soluble ADC activity (Fig. 6F). ADC activity was not detectable in the pellet of control and 0.1 µM MJ-treated explants. It appeared on day 7 in the presence of the two higher concentrations of MJ, at levels higher than those measured in the supernatant fraction; at the same concentrations, on day 15, this activity, though still present, decreased and was lower than in the soluble fraction (Fig. 6C, F).
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On day 7, a weak and comparable ODC mRNA signal was revealed in controls and with the lowest MJ concentration (Fig. 7A
, B). An even weaker signal was detected on day 15 (Fig. 7D, E). In both cases, however, the signal increased upon exposure to MJ in a dose-dependent manner. A moderate but significant (P<0.05) increase in soluble ODC activity was observed in MJ-treated explants compared with controls after 7 d in culture (maximum increase 3-fold in the presence of 10 µM MJ, Fig. 7C). The strongest stimulation of this activity was observed on day 15 in the presence of 1 µM MJ (Fig. 7F). As with ADC, particulate ODC activity was significantly (P<0.01) enhanced by the two higher concentrations of MJ (Fig. 7C, F). Maximum stimulation (24-fold) occurred in 7-d-old explants grown in the presence of 10 µM MJ.
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The SAMDC transcript also accumulated with increasing MJ concentration in both 7- and 15-d-old explants (Fig. 8A
, B, D, E). Similar to the other two biosynthetic enzymes, SAMDC activity was strongly stimulated especially in response to the two higher concentrations of MJ (Fig. 8C, F). In fact, increases were in the order of 6–7 times compared with controls on both days 7 and 15.
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DAO activity
When DAO activity was measured in the two fractions obtained by using Method 1 (see Materials and methods), a differential recovery of the activity depending on time in culture and treatment was observed (Fig. 9). In control explants, a cell debris-associated DAO activity was detected which was comparable to the extractable activity measured in the filtrate. At 24 h and 7 d MJ had no effect on extractable activity compared with controls, but caused a strong and significant (P<0.01) increase (about 6- and 5-fold with 1 and 10 µM, respectively) in the activity associated with the cell debris (Fig. 9A, B). Both in relative and absolute terms, the highest cell debris-associated activity was observed on day 7 in explants treated with the highest concentration of MJ (Fig. 9B). On the contrary, both extractable and cell debris-associated DAO activities followed an opposite trend in MJ-treated explants compared with controls on day 15 (Fig. 9C).
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In order to gain further insight into the cell debris-bound activity, Method 2 was applied on 7-d-old explants. Results (Fig. 10
) show that at 1 µM concentration MJ significantly enhanced extractable DAO activity, but not at 10 µM. Wall-bound activity was not detectable in control explants. It was induced to very high levels, relative to extractable activity, by 10 µM MJ. Although the lower concentration of MJ enhanced total DAO activity, this treatment resulted in a different recovery of the enzyme activity (equally distributed in the supernatants and cell wall fraction).
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Discussion |
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The present study examines the effects of jasmonates on the morphogenic response in a shoot-forming tissue culture system. Although information on the effects of jasmonates on the growth and development of in vitro-cultured explants is scanty, there is sufficient evidence suggesting that these compounds interact with most of the traditional plant growth regulators. As regards cytokinins, jasmonates were found to nullify the senescence-retarding effect of kinetin in oat leaf segments completely and to be powerful inhibitors of cytokinin-induced soybean callus growth (Ueda et al., 1981
; Ueda and Kato, 1982). The proposition that jasmonates exert anti-cytokinin effects is in accord with present results regarding the inhibitory effect of MJ on shoot formation, insofar as this organogenic process is mainly under cytokinin control (Altamura et al., 1995). Conversely, a high concentration of MJ favoured callus formation, even though extensive proliferative growth was impaired by the occurrence of cell hypertrophy which increased with increasing MJ concentration. Jasmonates are strong promoters of tuberization in potato plants, which is initiated mainly by cell expansion (Takahashi et al., 1994, and references therein). The assumption that jasmonates may favour cell expansion was confirmed by Takahashi and co-workers in potato tuber discs and could account for the induction of hypertrophic cells observed in MJ-treated tobacco thin layers (Takahashi et al., 1994). If indeed MJ exerts an anti-cytokinin-like effect, then the auxin-to-cytokinin balance optimal for shoot formation would be shifted in favour of IAA which induces cell hypertrophy and the formation of callus with hypertrophic cells, as previously described in this system (Altamura et al., 1995).
MJ is also known to promote ethylene production in various plants (Saniewski et al., 1998, and references therein). It has previously been shown that when shoot-forming tobacco thin layers are exposed to an ethylene-releasing compound (2-chloroethylphosphonic acid, CEPA), callus growth is favoured and shoot meristemoid formation inhibited (Biondi et al., 1998). This response is reminiscent of the one induced by 10 µM MJ. In fact, shoot formation is inhibited and callus growth enhanced in both cases, although the type of callus produced is different. The callus induced by CEPA grew mainly through active cell division (Biondi et al., 1998) whereas that induced by 10 µM MJ grew mainly through cell expansion leading to cell hypertrophy.
The present study also describes a time-course analysis of the effects of MJ on polyamine content and metabolism during an in vitro organogenic process. An adequate complement of free amines is necessary to sustain de novo organ formation (Tiburcio et al., 1988; Altamura et al., 1991; Scaramagli et al., 1995). In a previous article, a negative relationship between shoot formation and the over-accumulation of acid-soluble polyamine conjugates induced by a putative inhibitor of SAMDC activity was proposed (Scaramagli et al., 1999a). In fact, present results indicate that organogenesis was severely reduced in the presence of a relatively high concentration of MJ possibly due to the depletion of the free polyamine pool, but more likely to the over-accumulation of HCAs. The depletion of the free pool occurred in spite of an activation of all the biosynthetic enzymes, presumably because free amines were utilized to form conjugates.
Earlier reports point to an involvement of jasmonates in altering cellular titres of free putrescine, spermidine and spermine. In whole rice plants, MJ induced the accumulation of putrescine and spermine while spermidine levels decreased (Lee et al., 1996), whereas in tobacco BY-2 cell cultures, levels of putrescine increased while spermidine and spermine did not change (Imanishi et al., 1998). It may be that differentiated tissues respond differently from actively dividing cells, and that there are complex interactions with other plant growth regulators. In fact, whole plants and cell suspensions, in addition to being very different from each other, are also different from thin layers which consist of differentiated cells induced to actively divide prior to de novo organ formation.
The first paper dealing with jasmonate effects on conjugated forms of polyamines reported a MJ-induced accumulation of coumaroyl conjugates of putrescine in barley leaf segments (Lee et al., 1997). In roots and shoots of micropropagated potato plants treated with JA, Mader found that the amount of free polyamines was reduced by this treatment, whereas acid-soluble polyamine conjugates increased up to 10-fold (Mader, 1999). Similar results with regard to polyamine conjugates, but an opposite trend with regard to the free ones, were obtained by Biondi et al. in cultured roots of Hyoscyamus muticus (Biondi et al., 2000). Consequently, no clear-cut pattern emerges in relation to jasmonate effects on levels of free amines, whereas results regarding acid-soluble conjugates point to a substantial stimulation of their levels.
Jasmonates are known to enhance the amount of phenolic compounds, the conjugation partners for polyamines, by stimulating the phenylpropanoid pathway. Thus, exposure to MJ of cell cultures from various species led to an increase in phenylalanine ammonia lyase (PAL) activity (Gundlach et al., 1992). Conversely, treatment with an inhibitor of PAL activity reduced the JA-induced increase in amine conjugates in potato (Mader, 1999). Since acid-soluble amine conjugates are mainly HCAs (Flores and Martin Tanguy, 1991), their massive accumulation in response to MJ provides additional support to the contention (Gundlach et al., 1992) that elicitation by jasmonates is not specific to any one type of secondary metabolite, but affects a wide range of small molecular weight substances involved in responses to stress and pathogen attack, as indeed are HCAs (Flores and Martin-Tanguy, 1991). It has been suggested that HCAs in elicited tissues may function in detoxifying cells from excess phenolics; they may also act as free radical scavengers (Schraudner et al., 1996). Finally, since biosynthetic activity was also stimulated by MJ, conjugation may help to regulate the size of the free amine pool.
An interesting role for polyamine conjugates may reside in the cell wall. Wall-bound amides of hydroxycinnamic acids (e.g. coumaroyl- and feruloyltyramine) appear to be involved in resistance reactions and developmental processes in Solanaceae (Hahlbrock and Scheel, 1989). In addition, phenylpropanoids also function as complex polymeric constituents of the cell wall (lignin, suberin). Consequently, most of the acid-insoluble polyamine conjugates released after hydrolysis of the PCA-precipitable material are likely associated with the cell wall. It has been reported that hydroxycinnamic acids produced on strong stimulation (by MJ and/or fungal elicitors) of the phenylpropanoid pathway appear to become preferentially esterified to cell wall polymers (Kauss et al., 1992). Not surprisingly, therefore, the amount of at least one of the amines (spermidine) present in this fraction was also enhanced in MJ-treated tobacco thin layers.
Although information regarding the effect of jasmonates on polyamine biosynthesis are still contradictory, there is no doubt that, in the few cases examined, one or more of the enzymes in this pathway are activated by these elicitors. In rice plants ADC and SAMDC activities were enhanced by exposure to MJ (Lee et al., 1996), while that of ODC remained unchanged. On the contrary, Imanishi and co-workers reported rapid induction by MJ of mRNAs encoding for ODC in tobacco cells, while levels of mRNA for ADC and SAMDC were not affected by the elicitor treatment (Imanishi et al., 1998).
In the present study, a conspicuous enhancement by MJ of the activity and gene expression of all the biosynthetic enzymes assayed was observed. ADC and ODC are particularly active in organogenic tissues such as shoot- and flower-forming tobacco thin layers where they are activated within 24 h (Tiburcio et al., 1988; Scaramagli et al., 1995). They are also required during elongation and differentiation processes. Several workers have advanced the hypothesis that ODC is preferentially implicated in cell division while ADC is involved in cell elongation and stress responses (Flores and Martin-Tanguy, 1991; Cohen, 1998). On the other hand, the relative contribution of ADC and ODC to HCA formation is still uncertain (Burtin et al., 1989; Wyss-Benz et al., 1990; Robins et al., 1991). An interesting feature reported here was the marked increase in particulate ADC and ODC activities suggesting that MJ acts on organelle-associated activity. In fact, both enzyme activities have been found in isolated mitochondria and chloroplasts (Torrigiani et al., 1986). ODC was found associated to chromatin in barley nuclei (Panagiotidis et al., 1982), and in situ hybridization studies have shown ADC to be associated with thylakoid membranes (Borrell et al., 1995). Work is in progress to establish whether or not MJ indeed acts preferentially on organelle-associated putrescine biosynthetic activities, and which subcellular compartments are involved.
ADC and ODC are known to be post-translationally regulated (Cohen, 1998). In the present work, although RNA levels and the respective enzyme activities were not altered to the same extent, a generally dose-dependent stimulation in response to increasing concentrations of MJ was observed for both parameters. Thus, the pattern of transcript accumulation followed a similar trend to that of the respective enzyme activity. SAMDC, whose trancript levels accumulated dramatically in response to MJ, is also post-translationally regulated in plant cells (Xiong et al., 1997), although a developmental control has been proposed. It too is required during cell division and elongation (Mad-Arif et al., 1994), but is also stimulated in response to treatments inducing HCA over-accumulation (Scaramagli et al., 1999b).
It is well known that DAO enzymes are, at least in part, compartmented in cell walls. Since peroxidases are responsible for the cross-linking of cell wall components, it has been proposed that the role of DAO in the apoplast is to generate the hydrogen peroxide needed in cell expansion and/or lignification during both normal growth and in response to wounding and pathogens (Angelini et al., 1993). Thus, treatment with MJ, which mimics the latter events and induces cell hypertrophy in tobacco thin layers caused, not unexpectedly, marked changes in wall-bound DAO activity. In fact, jasmonates seem to interfere with the main components of plant cell walls (e.g. extensin, polysaccharides, Miyamoto et al., 1997; Merkouropoulos et al., 1999) and, together with their effects on wall-bound DAO activity, may determine the extent and/or quality of cell wall modifications.
In conclusion, in shoot-forming tobacco thin layers the morphogenic effects of MJ mainly includes the induction of cell hypertrophy and inhibition of shoot formation, thus mimicking some of the effects induced by auxin alone and counteracting those of cytokinin. Parallel to these morphogenic effects, MJ strongly upregulates polyamine biosynthesis, conjugation and oxidation. Thus, ADC, ODC and SAMDC can be included amongst the jasmonate-responsive genes. In fact, stimulation of polyamine biosynthetic enzyme activities with increasing MJ concentrations can be related to most of the events taking place in elicited explants: cell division/expansion, and conjugate accumulation/stress response. Furthermore, the major rise in wall-bound DAO activity also suggests that, in this system, MJ may induce DAO/peroxidase-mediated alterations in the cell wall possibly associated with cell hypertrophy.
Since elicitation can provoke not only quantitative but also qualitative changes in the spectrum of secondary metabolites formed (Sheludko et al., 1999), exogenous jasmonates can be helpful tools for studying what determines the activation of one or another secondary pathway. In addition, free and conjugated polyamines offer a useful approach towards the elucidation of the regulation of the primary/secondary metabolism interface, with putrescine as the focal point from which polyamines, tropane alkaloids and HCAs are derived.
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We are grateful to Dr AJ Michael for kindly supplying the tobacco molecular probes. The authors wish to thank E Zega (Dipartimento di Biologia Vegetale, Università La Sapienza, Rome, Italy) for technical support and G Bugamelli for growing the plants. The work was supported by funds of the University La Sapienza (Rome, Italy), Progetti Ateneo to MMA, of the MURST (ex-60%) to SB and of the University of Bologna for the project ‘Molecular Signals in Differentiation’ to PT.
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3 To whom correspondence should be addressed. Fax: +39 051 242576. E-mail: sbiondi{at}alma.unibo.it
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Altamura MM, Torrigiani P, Capitani F, Scaramagli S, Bagni N.1991. De novo root formation in tobacco thin layers is affected by inhibition of polyamine biosynthesis. Journal of Experimental Botany 42, 1575–1582.
Altamura MM, Capitani F, Falasca G, Gallelli G, Scaramagli S, Bueno M, Torrigiani P, Bagni N.1995. Morphogenesis in cultured thin layers and pith explants of tobacco. I. Effect of putrescine on cell size, xylogenesis and meristemoid organization. Journal of Plant Physiology 147, 101–106.
Angelini R, Bragaloni M, Federico R, Infantino A, Porta-Puglia A.1993. Involvement of polyamines, diamine oxidase and peroxidase in resistance of chick-pea to Ascochyta rabiei. Journal of Plant Physiology 142, 704–709.[Web of Science]
Biondi S, Fornalé S, Oksman-Caldentey K-M, Eeva M, Agostani S, Bagni N.2000. Jasmonates induce over-accumulation of methyl putrescine and conjugated polyamines in Hyoscyamus muticus L. root cultures. Plant Cell Reports 19, 691–697.
Biondi S, Scaramagli S, Capitani F, Marino G, Altamura MM, Torrigiani P.1998. Ethylene involvement in vegetative bud formation in tobacco thin layers. Protoplasma 202, 134–144.
Blechert S, Brodschelm W, Hölder S, Kammerer L, Kutchan TM, Mueller MJ, Xia Z-Q, Zenk MH.1995. The octadecanoid pathway: signal molecules for the regulation of secondary pathways. Proceedings of the National Academy of Sciences, USA 92, 4099–4105.
Borrell A, Culianez-Macià FA, Altabella T, Besford RT, Flores D, Tiburcio AF.1995. Arginine decarboxylase is localized in chloroplasts. Plant Physiology 109, 771–776.[Abstract]
Bradford MM.1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248–254.[Web of Science][Medline]
Burtin D, Martin-Tanguy J, Paynot M, Rossin N.1989. Effects of the suicide inhibitors of arginine and ornithine decarboxylase activities on organogenesis, growth, free polyamine and hydroxycinnamoyl putrescine levels in leaf explants of Nicotiana Xanthi n.c. cultivated in vitro on a medium producing callus formation. Plant Physiology 89, 104–110.
Cohen SS.1998. A guide to the polyamines. New York: Oxford University Press.
Creelman RA, Mullet JE.1997. Biosynthesis and action of jasmonates in plants. Annual Review of Plant Physiology and Plant Molecular Biology 48, 355–381.[Web of Science][Medline]
Flores HE, Martin-Tanguy J.1991. Polyamines and plant secondary metabolites. In: Slocum RD, Flores HE, eds. Biochemistry and physiology of polyamines in plants. Boca Raton, Florida: CRC Press. 57–76.
Gundlach H, Müller MJ, Kutchan TM, Zenk MH.1992. Jasmonic acid is a signal transducer in elicitor-induced plant cell cultures. Proceedings of the National Academy of Sciences, USA 89, 2389–2393.
Hahlbrock K, Scheel D.1989. Physiology and molecular biology of phenylpropanoid metabolism. Annual Review of Plant Physiology and Plant Molecular Biology 40, 347–369.[Web of Science]
Imanishi S, Hashizume K, Nakakita M, Kojima H, Matsubayashi Y, Hashimoto T, Sakagami Y, Yamada Y, Nakamura K.1998. Differential induction by methyl jasmonate of genes encoding ornithine decarboxylase and other enzymes involved in nicotine biosynthesis in tobacco cell cultures. Plant Molecular Biology 38, 1101–1111.[Web of Science][Medline]
Kauss H, Krause K, Jeblick W.1992. Methyl jasmonate conditions parsley suspension cells for increased elicitation of phenylpropanoid defense responses. Biochemical and Biophysical Research Communications 189, 304–308.[Web of Science][Medline]
Koda Y.1997. Possible involvement of jasmonates in various morphogenic events. Physiologia Plantarum 100, 639–646.
Laurenzi M, Rea G, Federico R, Tavladoraki P, Angelini R.1999. De-etiolation causes a phytochrome-mediated increase of polyamine oxidase expression in outer tissues of the maize mesocotyl: a role in the photomodulation of growth and cell wall differentiation. Planta 208, 146–154.[Web of Science]
Lee J, Vogt T, Schmidt J, Parthier B, Löbler M.1997. Methyl jasmonate-induced accumulation of coumaroyl conjugates in barley leaf segments. Phytochemistry 44, 589–592.
Lee T-M, Lur H-S, Lin Y-H, Chu C.1996. Physiological and biochemical changes related to methyl jasmonate-induced chilling tolerance of rice (Oryza sativa L.) seedlings. Plant, Cell and Environment 19, 65–74.
Mad Arif SA, Taylor MA, George LA, Butler AR, Burch LR, Davies HV, Starck MJR, Kumar A.1994. Characterization of the S-adenosylmethionine decarboxylase gene. Plant Molecular Biology 26, 327–338.[Web of Science][Medline]
Mader JC.1999. Effects of jasmonic acid, silver nitrate and L-AOPP on the distribution of free and conjugated polyamines in roots and shoots of S. tuberosum in vitro. Journal of Plant Physiology 154, 79–88.
Merkouropoulos G, Barnett C, Shirsat AH.1999. The Arabidopsis extensin gene is developmentally regulated, is induced by wounding, methyl jasmonate, abscisic and salicylic acid, and codes for a protein with unusual motifs. Planta 208, 212–219.[Web of Science][Medline]
Michael AJ, Furze JM, Rhodes MJC, Burtin D.1996. Molecular cloning and functional identification of a plant ornithine decarboxylase cDNA. Biochemical Journal 314, 241–248.
Miyamoto K, Oka M, Ueda J.1997. Update on the possible mode of action of the jasmonates: focus on the metabolism of cell wall polysaccharides in relation to growth and development. Physiologia Plantarum 100, 631–638.
Panagiotidis CA, Georgatsos JG, Kyriakidis DA.1982. Superinduction of cytosolic and chromatin-bound ornithine decarboxylase activities of germinating barley seeds by actinomycin D. FEBS Letters 146, 193–196.
Robins RJ, Parr AJ, Walton NJ.1991. Studies on the biosynthesis of tropane alkaloids in Datura stramonium L. transformed root cultures. 2. On the relative contributions of L-arginine and L-ornithine to the formation of the tropane ring. Planta 183, 196–201.
Sambrook J, Fritsch EF, Maniatis T.1989. Molecular cloning: a laboratory manual. Cold Spring Harbor: Cold Spring Harbor Laboratory Press.
Saniewski M, Miyamoto K, Ueda J.1998. Gum formation by methyl jasmonate in tulip shoots is stimulated by ethylene. Journal of Plant Growth Regulation 17, 179–183.
Scaramagli S, Bueno M, Torrigiani P, Altamura MM, Capitani F, Bagni N.1995. Morphogenesis in cultured thin layers and pith explants of tobacco. II. Early hormone-modulated polyamine biosynthesis. Journal of Plant Physiology 147, 113–117.
Scaramagli S, Biondi S, Capitani F, Gerola P, Altamura MM, Torrigiani P.1999a. Polyamine conjugate levels and ethylene biosynthesis: inverse relationship with vegetative bud formation in tobacco thin layers. Physiologia Plantarum 105, 367–376.
Scaramagli S, Biondi S, Torrigiani P.1999b. Methylglyoxal (bis-guanylhydrazone) inhibition of organogenesis is not due to S-adenosylmethionine decarboxylase inhibition/polyamine depletion in tobacco thin layers. Physiologia Plantarum 107, 353–360.
Schraudner M, Langebartels C, Sandermann Jr H.1996. Plant defence systems and ozone. Biochemical Society Transactions 24, 456–462.[Web of Science][Medline]
Scoccianti V, Sgarbi E, Fraternale D, Biondi S.2000. Organogenesis from Solanum melongena L. (eggplant) cotyledon explants is associated with hormone-modulated enhancement of polyamine biosynthesis and conjugation. Protoplasma 211, 51–63.
Sheludko Y, Gerasimenko I, Unger M, Kostenyuk I, Stoeckigt J.1999. Induction of alkaloid diversity in hybrid plant cell cultures. Plant Cell Reports 18, 911–918.
Takahashi K, Fujino K, Kikuta Y, Koda Y.1994. Expansion of potato cells in response to jasmonic acid. Plant Science 100, 3–8.
Tiburcio AF, Kaur-Sawhney R, Galston AW.1988. Polyamine biosynthesis during vegetative and floral bud differentiation in thin layer tobacco tissue cultures. Plant and Cell Physiology 29, 1241–1249.
Torrigiani P, Serafini-Fracassini D, Biondi S, Bagni N.1986. Evidence for the subcellular localization of polyamines and their biosynthetic enzymes in plant cells. Journal of Plant Physiology 124, 23–29.[Web of Science]
Torrigiani P, Altamura MM, Pasqua G, Monacelli B, Serafini-Fracassini D, Bagni N.1987. Free and conjugated polyamines during de novo floral and vegetative bud formation in thin cell layers of tobacco. Physiologia Plantarum 70, 453–460.
Torrigiani P, Serafini-Fracassini D, Fara A.1989. Diamine oxidase activity in different physiological stages of Helianthus tuberosus tuber. Plant Physiology 89, 69–73.
Tran Thanh Van M, Dien NT, Chlyah A.1974. Regulation of organogenesis in small explants of superficial tissue of Nicotiana tabacum L. Planta 119, 149–159.[Web of Science]
Tung P, Hooker TS, Tampe PA, Reid DM, Thorpe TA.1996. Jasmonic acid: effects on growth and development of isolated tomato roots cultured in vitro. International Journal of Plant Science 157, 713–721.
Ueda J, Kato J.1982. Inhibition of cytokinin-induced plant growth by jasmonic acid and its methyl ester. Physiologia Plantarum 54, 249–252.
Ueda J, Kato J, Yamane H, Takahashi N.1981. Inhibitory effect of methyl jasmonate and its related compounds on kinetin-induced retardation of oat leaf senescence. Physiologia Plantarum 52, 305–309.
Wyss-Benz M, Streit L, Ebert E.1990. Feruloylputrescine and caffeoylputrescine are not involved in growth and floral bud formation of stem explants from Nicotiana tabacum L. var Xanthi nc. Plant Physiology 92, 924–930.
Xiong H, Stanley BA, Tekwani BL, Pegg AE.1997. Processing mammalian and plant S-adenosylmethionine decarboxylase proenzymes. The Journal of Biological Chemistry 272, 28342–28348.
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