Correlation between Ornithine Decarboxylase and. Putrescine in Tomato Plants lnfected by Citrus Exocortis. Viroid or Treated with Ethephon'. Jose M. Belles ...
Plant Physiol. (1993) 102: 933-937
Correlation between Ornithine Decarboxylase and Putrescine in Tomato Plants lnfected by Citrus Exocortis Viroid or Treated with Ethephon' Jose M. Belles, Miguel A. Pérez-Amador, Juan Carbonell, and Vicente Conejero* Laboratorio de Bioquímica y Biologia Molecular, Departamento de Biotecnologia, Universidad Politécnica de Valencia, 46022 Valencia, Spain (J.M.B., V.C.); and Instituto de Agroquimica y Tecnologia de Alimentos, Consejo Superior de lnvestigaciones Científicas, Jaime Roig 11, 46010 Valencia, Spain (M.A.P.-A., J.C.)
other plants (Smith, 1985; Suzuki et al., 1990). Only recently, Rastogi and Davies (1990, 1991) have suggested that diamine and polyamine oxidizing enzymes are present in tomato pericarp, although their activities were not measured in vitro. To elucidate the link between the enhancement of ethylene and the decrease of putrescine level in pathogen signal transduction (Bellés et al., 1991), we have focused our attention on ADC and ODC activities and the levels of conjugated PAs in tomato plants infected with CEVd or treated with ethephon. We also studied the effect of the inhibition of ethylene action on either ADC or ODC activities and the levels of conjugated PAs after the treatment of plants with ethephon.
We have investigated the arginine decarboxylase (ADC, EC 4.1.1.19) and ornithine decarboxylase (ODC, EC 4.1.1.17) activities and the levels of conjugated polyamines to explain the decrease of free putrescine level caused by citrus exocortis viroid (CEVd) and ethephon treatment in tomato (Lycopersicon esculentum Mill. cv Rutgers) plants (J.M.Bellés, J.Carbonell, V. Conejero [1991] Plant Physiol 9 6 1053-1059). This decrease correlates with a decrease in O D C activity in CEVd-infected or ethephon-treated plants; ADC activity was not altered. CEVd infection had no effect on polyamine conjugates, and ethephon produced a decrease in putrescine conjugates. lnterference with ethylene action by silver ions prevented the decrease in O D C activity and in free and conjugated putrescine. It is suggested that changes in putrescine level after CEVd infedion and ethephon treatment are regulated via O D C activity and that conjugation is not involved.
MATERIALS AND METHODS Plant Material and Treatments
Tomato (Lycopersicon esculentum Mill. cv Rutgers) plants were grown from seeds (kindly supplied by Professor H.L. Sanger, Max Planck Institut fiir Biochemie). Plant cultivation and inoculation methods were similar to those previously described; inoculation of tomato seedlings was camed out by puncturing the stems with a needle dipped in either buffer or the 2 M LiC1-soluble fraction of nucleic acids from CEVdinfected Gynura (Bellés et al., 1991). Ethephon treatment was performed as follows: 35-d-old plants were sprayed until as an ethylene run-off with a solution of ethephon (15 m) generator, containing 0.1% (v/v) Tween 80 as a wetting agent. One millimolar STS was prepared by mixing silver nitrate and sodium thiosulfate solutions at a concentration ratio of 1:4. The concentrations reported are those of the silver component.
The widespread occurrence of putrescine, spermidine, and spermine in higher plants and its implication in growth and development is now well established. A considerablenumber of studies have been published on the association of PAs with a variety of physiological processes in plants (Bagni, 1989; Evans and Malmberg, 1989; Galston, 1989; Smith, 1990; Tiburcio et al., 1990). However, the involvement of PAs in plant-pathogen interaction has been studied in only a few cases (Rajam et al., 1985; Walters and Wylie, 1986; Bakanashvili et al., 1987). In an earlier paper (Bellés et al., 1991), we reported on the levels of free PAs in leaves of tomato (Lycopersicon esculentum) plants infected by CEVd or treated with ethephon (2-chloroethylphosphonicacid). The results strongly suggested that a decrease in putrescine is a signaling step between the increase of ethylene synthesis as a consequence of the pathogenic signal and the response of the plant. Therefore, it was of considerable interest to investigate how the level of putrescine in tomato leaves subjected to different kinds of stresses was regulated. It is thought that in solanaceous tissues synthesis and conjugation rather than catabolism are responsible for the regulation of PA levels. In tomato tissues, the presence of the diamine and polyamine oxidases has not been as well established as it has been in
Analysis of Free PAs and PA Conjugates
Free PAs were extracted from apical leaves (apex + four youngest leaves) and analyzed according to Flores and Galston (1982a) as reported in detail (Bellés et al., 1991). Soluble and insoluble PA conjugates of the same tissues were determined essentially as described by Tiburcio et al. (1985).
' This work was supported by grants PB87-O662 and PB87-0353 from DirecciÓn General de Investigación Científica y Técnica (Spain). * Corresponding author; fax 34-6-3877429.
Abbreviations: ADC, arjjnine decarboxylase; CEVd, citrus exocortis viroid; ODC, omithine decarboxylase; PA(s), polyamine(s);STS,
silver nitrate:sodium thiosulfate. 933
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Bellés et al.
Assays of A D C and O D C Activities
The procedure for enzyme extraction from apical leaves was performed according to Tiburcio et al. (1985) except that extraction buffer contained 20 m 2-mercaptoethanol instead of sodium ascorbate. The activities of ODC and ADC were determined according to Birecka et al. (1985). The enzymic assay for ADC contained 100 pL of crude enzyme, 40 pL of unlabeled Arg to give a final concentration of 7 mM, and 10 pL of 10 pCi/mL ~-[u-'~C]Arg (305 mCi/mmol, Amersham). ODC activity was similarly measured by using ~ - [ l - ' ~ C l O m (55 mCi/mmol, Amersham) as the substrate. The assay consisted of 100 pL of crude enzyme, 40 pL of unlabeled Orn to give a final concentration of 15 m, and 10 p L of 10 pCi/mL DL-[ 1-'4C]Om. The reaction mixtures, which progressed linearly for 90 min, were incubated for 45 min at 35OC in glass vials. Vials were capped with rubber stoppers pierced by a pin bearing an 11-mm-diameter disc of Whatman No. 1 paper impregnated with 20 p L of 4 N KOH. Reactions were stopped by injecting 0.2 mL of cold 10% TCA (v/v) through the rubber. After an additional45-min incubation, the discs were dried at 25OC and immersed in 0.2 mL of water with 2 mL of liquid scintillation cocktail (OptiPhase HiSafe 3, LKBPharmacia). The radioactivity trapped onto the discs was determined by counting in a Wallac 1410 scintillation counter. Enzyme activity is expressed as nmol I4CO2released h-' (mg protein)-'. Enzyme activity was proportional to the crude extract volume. Assays with or without boiled extract did not liberate CO1. Protein concentration in the soluble extracts was determined according to Bradford (1976), using BSA as a standard. The data presented are for single experiments with triplicate samples and are representative of a group of three or more experiments.
Plant Physiol. Vol. 102, 1993
both soluble and insoluble PA conjugates were analyzed. Only soluble PA conjugates were found in a11 the experiments. CEVd infection did not produce any significant change in the content of conjugated putrescine (Table I). Changes obseived (about three times the initial value both in control and infected plants) were associated with tlevelopment. Ethephon treatment produced a marked decrease only in putrescine conjugates, which was evident 24 h after the treatment. The amount of putrescine conjugates 48 h later was less than one-fourth that of the control (Table 11). As in CEVd-infected tomato plants, ethephon treatment did not significantlly affect the leve1 of spennidine conjugates. In control plants, putrescine and spermidine conjugates did not change during the period of the assay (48 h) (Table 11). Spennine conjugates were practically undetectable under the conditions used in the assay. Effect of STS ain Decarboxylase Activities and Putrescine Conjugates in Ethephon-Treated Plants
Figure 1, A and B, show that CEVd infection and ethephon treatment produced a decrease in the ODC activity that Percent a ge I
I
I
25tA
,
I
7
14
O O
Days P e r c e nt a g e
RESULTS A D C and O D C Activities in Healthy and CEVd-lnfected or Ethylene-Treated Plants
The activities of ADC and ODC were detennined in apical leaves of Rutgers tomato plants infected with CEVd or treated with ethephon and the results are shown in Figure 1, A and B, respectively. Figure 1A shows the decrease in ODC activity induced by CEVd infection, which clearly paralleled the decrease in free putrescine content. The decrease was evident at 7 d and more pronounced at 14 d after the appearance of symptoms. In contrast, no decrease in ADC 'activity was found (Fig. 1A). Figure 1B shows the time course of ODC activity along a 48-h period after ethephon treatment. A marked decrease in ODC activity is observed as soon as 12 h after treatment. After 48 h, only 20% of the activity remained. Again, the decrease in ODC activity clearly matched the decrease in putrescine. As in CEVd-infected tomato plants, no change in ADC activity was observed in ethephon-treated plants (Fig. 1B). PA Conjugates in CEVd-lnfected and Ethephon-Treated Plants
To determine whether conjugation contributed to the decrease of free putrescine (Bellk et al., 1991), the levels of
2 5 1
B
- 0
O
12
24
36
48
Hours
of CEVd infection on ADC (O) and ODC (O) activities and free putrescine (A) in Rutgers tomato leaves. ADC and ODC activities and free putrescine were measured in extracts from apical leaves (apex + four youngest leaves) in CEVd-inl'ected plants 7 and 14 d after the appearance of symptoms, which 'corresponded approximately with d 35 and 42 after sowing. Values of free putrescine and ADC and ODC activities at d O were 3.2 -C 0.4 nmol (mg protein)-', 7.8 f 0.3 and 10.0 zt 0.2 nmol COZ h-.' (mg protein)-', respectively. B, Effect of ethephon on ADC (O)and ODC (O)activities and free putrescine (A) in tomato leaves. Five-weekold plants were sprayed with 15 mM ethephon or water with 0.1% Tween 80. ADC: and ODC activities and free putrescine were measured in extracts from apical leaves at the indicated time!; after the treatment. Values of free putrescine and ADC and ODC activities at O h were 4.1 f 0.6 nmol (mg protein)-', 6.5 f 0.3 and 12.7 zt 0.4 nmol COz h-' (mg protein)-', respectively. Figure 1. A, Effect
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Ornithine Decarboxylase in Tornato Leaves
Table 1. Effect of CEVd rnfectron on polyamrne conpgates in tomato leaves
Conjugated polyamines were measured in extracts from apical leaves from plants analogous to those used in the experiments shown in Figure 1 . Values are mean f SE, n = 3. Days after the Appearance of Symptoms
PA Conjugates Putrescine Control
Spermidine
CEVd-infected
Control
CEVd-infected
nmol (mg proteh-’
O
7 14
42.7 f 3.8 40.6 f 3.3 7.5 f 0 . 5 46.7 f 3.1 44.4 f 2.3 8.3 f 0 . 6 126.4 f 6.5 130.5 f 11.9 1 0 . 8 f 1.2
8.3 f 0 . 8 6.6f0.5 7.3 f 0.7
paralleled that of putrescine. To test the hypothesis that inhibition of ethylene action would prevent the expected lowering of the ODC activity in ethephon-treated tomato plants, we treated the plants with silver ion, a potent inhibitor of ethylene action in tomato (Beyer, 1976), at a concentration that did not affect PA synthesis (BellCs et al., 1991). Five hours later, these plants were also treated with ethephon. We carried out this experiment using tomato plants treated with ethephon because of the rapidity of the response: the decrease in free putrescine occurred in a few hours, whereas the decrease took severa1 days in plants infected with CEVd. Table I11 shows that STS applied 5 h before the treatment with ethephon completely prevented the strong decrease of ODC activity in apical leaves, but it did not affect ADC activity. In addition, STS inhibited the lowering of putrescine conjugates caused by ethephon (Table IV). STS alone had no effect on either decarboxylase activity (ADC or ODC) or on the level of putrescine conjugates (Tables 111 and IV). DISCUSSION
The decrease in free putrescine level in tomato leaves after CEVd infection or ethephon treatment (Bellés et al., 1991) could be due mainly to a decrease in the activity of ADC and/or ODC and/or an increase in putrescine conjugates, as well as to an increase of putrescine catabolism. There are different reports on the contribution of ODC and ADC in the control of PA synthesis. In some stages of fruit development in tomato (Cohen et al., 1982), avocado (Kushad et al., 1988),
Table li. Effect of ethephon treatment on polyamine conjugates in tomato leaves
Conjugated polyamines were measured in extracts from apical leaves of 5-week-old plants at the indicated time after the ethephon treatment (15 m d . Values are mean f SE. n = 3. PA Conjugates Time after Treatment
Putrescine Control
Ethephon
35.9 f 1.8 3 7 . 4 f 1.6 35.1 f 2.2
40.3 f 2 . 1 23.7 f 1.2 7.9 f 0 . 3
h
O 24 48
Table 111. Effect of STS on ADC and ODC activities in ethephontreated tomato leaves Five-week-old plants were treated with 1 mM STS 5 h before the treatment with ethephon (15 mM). Measurements were made in apical leaves prior to treatment with ethephon and 48 h later. Values are mean f SE. n = 3. Time after boxylase TreatActivity” ment
ADC
O
ODC
48
ADC
4.7f0.5 6 . 8 f 0.4 5.7f0.5
STS
+
6.4 f 0.7 5 . 7 f 0.5 7.1 f 0 . 8 6.8 f 0 . 6 13.6 f 0.5 13.3 f 0 . 6 12.1 f 0 . 4 11.9 f 0 . 3 6.3 f 0.5 6.0 f 0.6 7.3 0.3 6.1 f 0.4 12.7 f 0 . 4 3.4 f O . 3 1 3 . 0 f 0 . 6 11.4 f 0 . 8
*
and tobacco (Slocum and Galston, 1985), changes in the level of putrescine are correlated with changes in ODC activity. The elevated putrescine levels in Alcobaca (alc) tomato fruits (Rastogi and Davies, 1991) and in oat exposed to an osmotic shock (Flores and Galston, 1982b) have been correlated with an increased activity of ADC. However, in oat seedlings, a strong inhibition of ADC activity had little effect on the content of putrescine (Birecka et al., 1991). ADC and ODC activities have been reported to be affected by ethylene. Apelbaum et al. (1985) showed that ethylene considerably reduced ADC activity in etiolated pea seedlings. Also in pea seedlings, ethephon strongly depressed ADC activity but had reverse effects on ODC activity (Palavan et al., 1984). In Rutgers tomato green fruits, ethylene decreased both ADC and ODC activities (Bakanashvili et al., 1987). More recently, it has been shown that ethylene induced an increase in putrescine in rice, apparently mediated by an enhanced ADC activity (Lee and Chu, 1992). Our results show that ethephon treatment and CEVd infection, which induce an increase in ethylene production and
Table IV. Effect of STS on polyamine conjugates in ethephon-
treated tomato leaves Conjugated polyamines were measured in extracts from apical leaves of 5-week-old plants that had been pretreated with STS (1 mM) and treated with ethephon (15 mM) 5 h later. Measurements were made prior to treatment with ethephon and 48 h later. Values are mean f SE, n = 3. Time after the
Treatment
Treatment
Spermidine
nmol (mg protein)-’
Control Ethephon
Ethephon + STS
STS
48
PA Conjugates Putrescine
h
Ethephon
5 . 0 f 0.3 4.4 f 0.5 6.1 f 0 . 6
Ethephon STS
ODC a nmol of COz h-’ (mg proteinl-’.
Spermidine
nmol (mg protein)-’
Ethephon
h
O
Control
Control
Control Ethephon
Ethephon + STS
STS
50.8 f 2.5 48.0 f 2.0 45.3 f 1.7 52.0 f 3.0 53.7 f 2.2 11.6 f 1.0 50.3 f 2.4 47.9 f 2.9
6.2 f 0.5 5.3 f 0.4 5.7 f 0.4 7.1 f 0.9 7.2 f 0.7 6.4 0.6 6.9 f 0.5 7.0 f 0.6
*
Bellés et al.
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Plant Physiol. Vol. 102, 1993
a decrease in free putrescine level in Rutgers tomato plants
LITERATURE ClTED
(Bellés et al., 1991), produce a reduction in ODC activity, whereas ADC is not affected, suggesting that ODC may play an important role in regulating putrescine biosynthesis in tomato plants subjected to different kinds of stress. Furthermore, our finding that inhibition of ethylene action by STS prevents the decrease of ODC activity supports the view that ethylene mediates the control of the level of ODC (Table 111). Other workers (Negrel et al., 1984) found that ODC dramatically increased in tobacco mosaic virus-infected tobacco plants, but the level of putrescine did not increase concomitantly. PAs can occur as bound forms conjugated to phenol metabolites, which in some plant organs account for the major part of the PA pools (Slocum and Galston, 1985; Tomgiani et al., 1987). The function of these conjugates is currently unknown, but a considerable number of published studies suggest that they can play an important function in many aspects of plant development (Smith et al. 1983; MartinTanguy et al., 1987). The decrease in putrescine conjugates found in this work after ethephon treatment (Table 11) is probably due to a diminished content of putrescine as a substrate for conjugation. The stronger decrease in free putrescine in ethephon-treated plants as compared with that in viroid-infected plants (Bellés et al., 1991) parallels the stronger impairment of ODC activity induced by ethephon. This diminished content of conjugated putrescine indicates that conjugation does not contibute to an explanation of the lowering of the putrescine level. Consistent with this idea is the fact that CEVd infection did not significantly alter the putrescine conjugate levels (Table I). The fact that STS counteracts the effect of ethephon treatment strongly suggests that ethylene, either released from or induced by ethephon, is mediating the decrease in conjugated putrescine. The accumulation of putrescine conjugates in apical leaves as the plant develops (Table I) is in agreement with that found in tobacco (Martin-Tanguy, 1985). We had previously demonstrated (Bellés et al., 1991) that the enhancement of ethylene level and the subsequent lowering of putrescine content are critica1 steps in the transduction of pathogenic and stress signaling. Based on the results obtained by Apelbaum et al. (1985), we suggested that ADC activity could be the enzyme controlling the putrescine level after ethephon treatment. The results presented in this paper indicate that ODC, rather than ADC, is involved in the link between ethylene and putrescine. These results also suggest that putrescine conjugation is not involved as a primary factor in this controlling system.
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ACKNOWLEDCMENTS
The authors wish to thank Dr. R. Serrano for reading and correcting the manuscript and Dr. I. Rodrigo for his assistance with the figure. Received December 30; 1992; accepted March 31, 1993. Copyright Clearance Center: 0032-0889/93/102/0933/05.
Ornithine Decarboxylase in Tomato Leaves
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