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Activity and expression of diamine oxidase in lentil seedling under different growth conditions
Plant Science, 79 (1991) 51-55 Elsevier Scientific Publishers Ireland Ltd.
51
Activity and expression of diamine oxidase in lentil seedlings under different growth conditions Mauro
M a c c a r r o n e a, A n t o n e l l o R o s s i b, L u c i a n a A v i g l i a n o a a n d A l e s s a n d r o F i n a z z i A g r o 'b
"Department o/" Biomedical Bh~technology. University o[' L'Aquiht and hDepartment o/' Exl~erimental Medicine. Tot Vergala, University o/ Rome (Ira/y) (Received March 8th, 1991: revision received June 18th. 1991: accepted July 15th, 1991)
The activity and expression of diamine oxidase during the germination of lentil seeds and in the course of anoxic and thermal stress have been studied. Diamine oxidase activity, as well as the seedlings growth rate, was found to be markedly higher in dark-grown lentil seedlings than in the light-grown ones. The same was true for the respective protein and mRNA amounts. The specific activity of diamine oxidase was decreased by anoxic stress and not affected by thermal stress. The possible physiological meaning of these findings is discussed.
Key words." Lens culinaris: germination: polyamines: plant stress.
Introduction
Diamine oxidases (DAOs; diamine O, oxidoreductase [deaminating], EC 1.4.3.6) are coppercontaining enzymes [1] widespread among eukaryotes [2]. They oxidize only primary amines, producing the corresponding aldehyde, NH 3 and H202. Besides copper, DAOs contain a quinoid organic cofactor [3], which is responsible for their characteristic pink colour. Lentil seedlings DAO is an homodimer, the two subunits having a molecular weight of 80-90 kDa [4] each containing a Cu(ll) atom and an organic cofactor. Plant DAOs have been found mainly in seedlings [5] and though they have been purified and
Correspondence to." Alessandro Finazzi Agro', Dipartimento di Medicina Sperimentale, Universita di Roma, 'Tor Vergata', via O. Raimondo, 00173 Roma, Italia. Abbreviations." BSA, bovine serum albumin: DAO, diamine oxidase: EDTA, ethylenediaminetetraacetic acid: PBS, phosphate-buffered saline: Put, putrescine: SDS, sodium dodecyl sulfate.
characterized from different sources [4,6-8], their physiological role is still poorly understood. The specificity of these enzymes for diamines suggests that they may be involved in regulating their concentration. In view of the role of polyamines as growth regulators or key-elements in various types of stress, a DAO involvement in these processes may be envisaged. Thus it appears that a better understanding of its physiological role would be of great biological and technological relevance. In this paper we report on the effect of light and stress factors, namely anoxia and low temperature, on DAO activity and content and on DAO mRNA level in lentil seedlings. Materials and Methods
Chemicals Chemicals were of the purest analytical grade. [3~-32P] ATP was purchased from Amersham, U.K. Plant material Lentil (Lens culinaris) seeds purchased at a local
0168-9452/91/$03.50 © 1991 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
52 market were swollen overnight at room temperature in tap water. This stage was taken as day 0 of germination: seeds were then grown at 25°C in a greenhouse, either in the dark or under a 16-h photoperiod, and were watered daily with tap water. For each experimental point 75 seedlings were homogenized at 20 000 x g in 0.1 M NaP O 4 (pH 7.0), with a Turrax tip homogenizer. Alter centrifugation at 10 000 x g for 15 rain, the supernatant was recovered and centrifuged again at 12 000 x g for 5 min. Finally the last supernatant was dialyzed overnight, at 4°C, against the homogenization buffer and was kept frozen at -70°C prior to use. Protein assay Protein concentration was determined according to Bradford [9]. Enzyme activity was measured polarographically, at 25°C, on samples containing 1 ml of 0.1 M Na-PO4 buffer (pH 7.2) and 1 mM putrescine as substrate. Measurements were performed by means of an Hansatech CBI (U.K.) oxygen monitor. Anoxia Lentil seedlings were grown in the dark for 5 days, then were kept for 24 h under anaerobic conditions, following the procedure described by Reggiani et al. [10]. Thermal stress Five-day-old, dark-grown seedlings of L. eulinaris were exposed for 24 h to low temperature (4°C) in the dark, in a refrigerator. Determination o[ DA 0 content The amount of diamine oxidase present in the seedlings homogenate was estimated by ELISA, essentially performed according to Abdillahi and Poolman [11]. Antibodies against lentil diamine oxidase, elicited in rabbits and purified following the procedure described elsewhere [12], were used to detect D A O in the samples and were recognized by using a goat anti-rabbit-alkaline-phosphatase conjugate (GAR-AP, BIO RAD), diluted 1:3000 with PBS containing BSA (1%) and Tween 20 (0.01%). p-Nitrophenylphosphate (0.5 mg/ml diethanolamine buffer) was used as substrate of the alkaline phosphatase and the absorbance at
405 nm of the yellow product of the reaction was used to express the amount of D A O detected in the samples. A calibration curve of the E L | S A test was made using different amounts of lentil DAO, purified according to Floris et al. [4]: BSA (1 ~,g/well) was used as a control of specificity. RNA extraction and Northern blot analysis Total R N A was isolated from L. culinaris seedlings following the procedure of Logemann et al. [131. RNA was electrophoresed on a 1% agarose gel containing 6.6% formaldehyde and then transferred to a Hybond-N + membrane (Amersham). The probe in the Northern blot analysis was a 32p-labelled 17 nucleotide-long oligodeoxynucleotide, the sequence of which was derived from a tryptic peptide of lentil DAO. Hybridizations were performed at 50°C in the presence of 0.9 M NaCI, 5 mM EDTA, 50 mM NaPO4 buffer (pH 7.0), 0.3% SDS (w/v) and 100 #g/ml sonicated calf thymus denatured DNA. The filters were then washed four times with 0.9 M NaC1 plus 50 mM Na-PO4 buffer (pH 7.0) and 0.3% SDS (w/v) at the same temperature. Results
Diamine oxidase during growth of" h'ntil seedlings Figure 1 shows the changes in D A O activity during lentil seedlings growth under a 16-h photoperiod (Fig. 1A) or in the dark (Fig. 1B). Plantlets were growing faster in the dark and the enzyme activity was up to 8 times higher in the dark-grown seedlings than in the light-grown ones. Moreover, independently of the light conditions, DAO activity reached a maximum alter 6 days and subsequently sharply declined in parallel with the slower growth rate of the plants (Fig. 1). A phytochrome involvement in the regulation of D A O activity was investigated by irradiating darkgrown seedlings with pulses of red and/or far red light according to Morohashi [14] or Angelini et al. [15]. D A O activity did not show any reduction. Similarly the exposure to blue light did not affect the activity (data not shown). The content of D A O in the different samples
53 600
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Fig. 3. Northern blot analysis of RNA extracted from lentil seedlings. Forty-five #g of total RNA extracted from lightgrown (A) or dark-grown (B) seedlings were hybridized with a 17-mer oligonucleotide as described in Materials and Methods. The arrows mark the positions of ribosomal RNA.
-8 200 6 100
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. . . .
i
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-
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i
. . . .
i
10
. . . .
2
15
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Growth period (days)
Fig. I. Changes in diamine oxidase activity during lentil seedlings growth, under 16-h photoperiod (A) or in the dark (B). Seedlings were harvested at the indicated periods, weighed and homogenized as described under Materials and Methods. O, DAO specific activity; O, fresh weight. In both panels each point represents the mean of three different determinations (S.D. < 4"/,,).
_c
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the e x p o s u r e to a t e m p e r a t u r e (4°C) lower t h a n the o n e at which g r o w t h n o r m a l l y o c c u r r e d (25°C) did n o t affect the e n z y m e activity (Fig. 4C). T h e E L I S A assay s h o w e d n o c h a n g e s in D A O c o n t e n t o f a n o x i a or t h e r m a l l y stressed p l a n t s in c o m p a r i s o n to the c o n t r o l s (Figs. 4 A a n d 4C).
o
< 40
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~ID
was d e t e r m i n e d by E L I S A . It was f o u n d that the increase in activity o f the seedlings g r o w n in the d a r k with respect to those g r o w n u n d e r i l l u m i n a tion was paralleled b y a n i n c r e a s e in the e n z y m e c o n t e n t (Fig. 2). N o r t h e r n blot a n a l y s i s (Fig. 3) s h o w e d that also the D A O m R N A a m o u n t was greater in d a r k - g r o w n plantelets. Anoxie and thermal stress W h e n lentil seedlings were e x p o s e d to a n a e r o b i c stress for 24 h, a decrease o f D A O activity to 30% o f the c o n t r o l value was o b s e r v e d (Fig. 4A), while
100
o
B
20
01. 0
.
0
.
.
.
.
.
5
.
.
.
.
.
.
10
1
Growth period (days)
Fig. 2. Diamine oxidase content in lentil seedlings grown in the light (O) or in the dark (e) for the indicated periods. The amount of DAO present was measured by ELISA. The results are expressed as % of the maximal value and are the mean of three different experiments (S.D. < 3%).
Discussion D A O activity in d a r k - g r o w n lentil seedlings is m u c h h i g h e r t h a n in the l i g h t - g r o w n o n e s (Fig. 1) [5,16]. T h e s a m e is true for the D A O c o n t e n t (Fig. 2) a n d the D A O m R N A level (Fig, 3). These d a t a
54
100" >,,
i " 80-
,<0060a a P 0
A
B SAMPLES
C
Fig. 4. Diamine oxidase activity and content in lentil seedlings under different treatments (details are described in Materials and Methods). The amount of D A O present was measured by ELISA. Both the specific activity (black bars) and the protein content (patched bars) were reported as % of the maximal values. The results are the mean of three different experiments (S.D. < 3%). (A) Seedlings growing in the dark and further 24 h of anoxic treatment; (B) seedlings growing in the dark; (C) seedlings growing in the dark and further 24 h of thermal stress (4°C). All samples refer to 5-day-old seedlings.
indicate that the higher enzyme activity in the dark conditions is regulated at the DAO gene(s) level and is achieved by increasing the concentration of the respective mRNA(s). A possible phytochrome down-regulation was investigated by delivering to dark-grown seedlings pulses of red and/or far-red light [17]. DAO activity, expressed on a protein basis, remained unchanged, at variance with the report of Angelini et al. [15]. The same was found when the samples were exposed to blue light (data not shown). In any case, lentil seedlings are much faster growing in the dark (Fig. 1) and the increase of DAO activity may be correlated to the growth, because the cell proliferation would require a faster metabolism of polyamines [18,19]. Alternatively the increase of DAO activity in etiolated seedlings may be related to processes occurring during stem elongation such as lignification and wall stiffening [20]. In any case phytochrome is not directly involved in the control of DAO activity. Besides the possible role of DAO in plant growth, the involvement of this enzyme in the response to stress factors was investigated. Recent-
ly it has been observed that the level of putrescine, the purported natural substrate of diamine oxidase, increases in rice shoots subjected to anoxic conditions, partly due to an increase of its biosynthesis. However a slower degradation could also be involved. Indeed our experiments with lentil seedlings show a substantial decrease of DAO activity during anoxia (Fig. 4A). Since this enzyme is rather abundant in the coleoptile and because of its very high affinity for 02 ( K m for 02 < 6 IxM) [21], it could be involved in regulating putrescine concentration during occasional periods of low oxygen concentration. Oxygen did not play any role on the expression of the DAO gene(s), because DAO mRNA steady state level (data not shown) and the amount of enzyme (Fig. 4A) were the same in anaerobic and aerobic samples. Therefore anoxia should cause the indirect inhibition of preexisting enzyme itself. As far as the thermal stress is concerned, Fig. 4C shows that lentil seedling diamine oxidase was not involved in the plant response to low temperature exposure. In [3act, both the activity and the amount of enzyme were the same as the controls. Nevertheless, an increase of putrescine levels during chilling injury of fruits from different plants has been reported [22] and it has been proposed that putrescine accumulation can mediate the stress-induced injury [23]. If this hypothesis were of general validity, our data indicate that putrescine accumulation during chilling stress is not due to a reduction of the DAO activity. Acknowledgement
This work has been supported in part by Italian CNR Special Project 'Chimica Fine' Contract 8900804.72. References 1
2
3
B.G. Malmstrom, L.E. Andreasson and B. Reinhammar, in: P.D. Boyer (Ed.), The Enzymes, Vol. 12B, Academic Press, New York, 1975, pp. 507-579. B. Mondovi and A. Finazzi Agro, in: F. Bossa, E. Chiancone, A. Finazzi Agro and R. Strom (Eds.t, Structure and Function Relationships in Biochemical Systems, Plenum Press, New York, 1982, pp. 141-149. S.M. Janes, D. Mu, D. Wemmer, A.J. Smith, S. Kaur, D.
55
4
5 6
7
8
9
10
I1
12
13
Maltby, A.L. Burlingame and J.P. Klinman, A new redox cofactor in eukaryotic enzymes: 6-hydroxydopa at the active site of bovine serum amine oxidase. Science, 248 (1990) 981-987. G. Floris, A. Giartosio and A. Rinaldi, Diamine oxidase from lens esculenta seedlings: purification and properties. Phytochemistry, 22 (1983) 1871 - 1874. T.A. Smith, The di- and poly-amine oxidases of higher plants. Biochem. Soc. Trans., 13 (1985) 319-322. H. Yanagisawa, E. Hirasawa and Y. Suzuki, Purification and properties of diamine oxidase from pea epicotyls. Phytochemistry, 20 ( 1981 ) 2105-2108. H. Matsuda and Y. Suzuki, Purification and properties of the diamine oxidase from Vicia .[aha leaves. Plant Cell Physiol., 22 (1981) 737-741. A. Rinaldi, G. Floris and A. Finazzi Agro, Purification and properties of diamine oxidase from euphorbia latex. Eur. J. Biochem., 127 (1982) 417-422. M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72 (1976) 248-254. R. Reggiani, A. Hoch-Koeppler and A. Bertani, Polyamines in rice seedlings under oxygen-deficit stress. Plant Physiol., 91 (1989) 1197-1201. H. Abdillahi and J.T. Poolman, Whole-cell ELISA for typing Neisseria meningitidis with monoclonal antibodies. FEMS Microbiol. Lett., 48 (1987) 367-371. G. D'Andrea, A. Oratore, L. Avigliano, A. Rossi and A. Finazzi Agro, Characterization and cross-reactivity of antibodies against ascorbate oxidase from green zucchini marrows (Cucurhita pepo me~hdlosa). Plant Sci., 56 (1988) 107-112. J. Logemann, J. Schell and L. Willmitzer, Improved method for the isolation of RNA from plant tissues. Anal. Biochem., 163 (1987) 16-20.
14
15
16
17
18 19
20
21
22
23
Y. Morohashi, Control of mitochondrial activities by phytochrome in cotyledons of dark-grown cucumber seedlings. Plant Cell Physiol., 30 (1989) 81 I-815. R. Angelini, R. Federico and A. Mancinelli, Phytochrome-mediated control of diaminc oxidase level in the epicotyl of etiolated lentil (Lens culinaris Medicus) seedlings. Plant Physiol., 88 (1988) 1207-1209. R. Federico and R. Angelini, Distribution of polyamines and their related catabolic enzyme in etiolated and lightgrown leguminosae seedlings. Planta, 173 (1988) 317-321. J.K. Okamuro and R.B. Goldberg, in: A. Marcus (Ed.), The Biochemistry of Plants, Vol. 15, Academic Press, New York, 1989, pp. 1-82. F. Buffoni, Histaminase and related amine oxidases. Pharmacol. Rev., 18 (1966) 1163-1199. A. Raina and J. Janne, Physiology of the natural polyamines putrescine, spermidine and spermine. Med. Biol., 53 (1975) 121-147. R. Angelini, F. Manes and R. Federico, Spatial and functional correlation between diamine-oxidase and peroxidase activities and their dependence upon de-etiolation and wounding in chick-pea stems. Planta, 182 (1990) 89-96. A. Bellelli, M. Brunori, A. Finazzi Agro, G. Floris, A. Giartosio and A. Rinaldi, Transient kinetics of coppercontaining lentil (Lens culinaris) seedling amine oxidase. Biochem. J., 232 (1985) 923-926. R.E. McDonald and M.M. Kushad, Accumulation of putrescine during chilling injury of fruits. Plant Physiol., 82 (1986) 324-326. R.D. Slocum, R. Kaur-Sawhney and A.W. Galston, The physiology and biochemistry of polyamines in plants. Arch. Biochem. Biophys., 235 (1984) 283-303.
51
Activity and expression of diamine oxidase in lentil seedlings under different growth conditions Mauro
M a c c a r r o n e a, A n t o n e l l o R o s s i b, L u c i a n a A v i g l i a n o a a n d A l e s s a n d r o F i n a z z i A g r o 'b
"Department o/" Biomedical Bh~technology. University o[' L'Aquiht and hDepartment o/' Exl~erimental Medicine. Tot Vergala, University o/ Rome (Ira/y) (Received March 8th, 1991: revision received June 18th. 1991: accepted July 15th, 1991)
The activity and expression of diamine oxidase during the germination of lentil seeds and in the course of anoxic and thermal stress have been studied. Diamine oxidase activity, as well as the seedlings growth rate, was found to be markedly higher in dark-grown lentil seedlings than in the light-grown ones. The same was true for the respective protein and mRNA amounts. The specific activity of diamine oxidase was decreased by anoxic stress and not affected by thermal stress. The possible physiological meaning of these findings is discussed.
Key words." Lens culinaris: germination: polyamines: plant stress.
Introduction
Diamine oxidases (DAOs; diamine O, oxidoreductase [deaminating], EC 1.4.3.6) are coppercontaining enzymes [1] widespread among eukaryotes [2]. They oxidize only primary amines, producing the corresponding aldehyde, NH 3 and H202. Besides copper, DAOs contain a quinoid organic cofactor [3], which is responsible for their characteristic pink colour. Lentil seedlings DAO is an homodimer, the two subunits having a molecular weight of 80-90 kDa [4] each containing a Cu(ll) atom and an organic cofactor. Plant DAOs have been found mainly in seedlings [5] and though they have been purified and
Correspondence to." Alessandro Finazzi Agro', Dipartimento di Medicina Sperimentale, Universita di Roma, 'Tor Vergata', via O. Raimondo, 00173 Roma, Italia. Abbreviations." BSA, bovine serum albumin: DAO, diamine oxidase: EDTA, ethylenediaminetetraacetic acid: PBS, phosphate-buffered saline: Put, putrescine: SDS, sodium dodecyl sulfate.
characterized from different sources [4,6-8], their physiological role is still poorly understood. The specificity of these enzymes for diamines suggests that they may be involved in regulating their concentration. In view of the role of polyamines as growth regulators or key-elements in various types of stress, a DAO involvement in these processes may be envisaged. Thus it appears that a better understanding of its physiological role would be of great biological and technological relevance. In this paper we report on the effect of light and stress factors, namely anoxia and low temperature, on DAO activity and content and on DAO mRNA level in lentil seedlings. Materials and Methods
Chemicals Chemicals were of the purest analytical grade. [3~-32P] ATP was purchased from Amersham, U.K. Plant material Lentil (Lens culinaris) seeds purchased at a local
0168-9452/91/$03.50 © 1991 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
52 market were swollen overnight at room temperature in tap water. This stage was taken as day 0 of germination: seeds were then grown at 25°C in a greenhouse, either in the dark or under a 16-h photoperiod, and were watered daily with tap water. For each experimental point 75 seedlings were homogenized at 20 000 x g in 0.1 M NaP O 4 (pH 7.0), with a Turrax tip homogenizer. Alter centrifugation at 10 000 x g for 15 rain, the supernatant was recovered and centrifuged again at 12 000 x g for 5 min. Finally the last supernatant was dialyzed overnight, at 4°C, against the homogenization buffer and was kept frozen at -70°C prior to use. Protein assay Protein concentration was determined according to Bradford [9]. Enzyme activity was measured polarographically, at 25°C, on samples containing 1 ml of 0.1 M Na-PO4 buffer (pH 7.2) and 1 mM putrescine as substrate. Measurements were performed by means of an Hansatech CBI (U.K.) oxygen monitor. Anoxia Lentil seedlings were grown in the dark for 5 days, then were kept for 24 h under anaerobic conditions, following the procedure described by Reggiani et al. [10]. Thermal stress Five-day-old, dark-grown seedlings of L. eulinaris were exposed for 24 h to low temperature (4°C) in the dark, in a refrigerator. Determination o[ DA 0 content The amount of diamine oxidase present in the seedlings homogenate was estimated by ELISA, essentially performed according to Abdillahi and Poolman [11]. Antibodies against lentil diamine oxidase, elicited in rabbits and purified following the procedure described elsewhere [12], were used to detect D A O in the samples and were recognized by using a goat anti-rabbit-alkaline-phosphatase conjugate (GAR-AP, BIO RAD), diluted 1:3000 with PBS containing BSA (1%) and Tween 20 (0.01%). p-Nitrophenylphosphate (0.5 mg/ml diethanolamine buffer) was used as substrate of the alkaline phosphatase and the absorbance at
405 nm of the yellow product of the reaction was used to express the amount of D A O detected in the samples. A calibration curve of the E L | S A test was made using different amounts of lentil DAO, purified according to Floris et al. [4]: BSA (1 ~,g/well) was used as a control of specificity. RNA extraction and Northern blot analysis Total R N A was isolated from L. culinaris seedlings following the procedure of Logemann et al. [131. RNA was electrophoresed on a 1% agarose gel containing 6.6% formaldehyde and then transferred to a Hybond-N + membrane (Amersham). The probe in the Northern blot analysis was a 32p-labelled 17 nucleotide-long oligodeoxynucleotide, the sequence of which was derived from a tryptic peptide of lentil DAO. Hybridizations were performed at 50°C in the presence of 0.9 M NaCI, 5 mM EDTA, 50 mM NaPO4 buffer (pH 7.0), 0.3% SDS (w/v) and 100 #g/ml sonicated calf thymus denatured DNA. The filters were then washed four times with 0.9 M NaC1 plus 50 mM Na-PO4 buffer (pH 7.0) and 0.3% SDS (w/v) at the same temperature. Results
Diamine oxidase during growth of" h'ntil seedlings Figure 1 shows the changes in D A O activity during lentil seedlings growth under a 16-h photoperiod (Fig. 1A) or in the dark (Fig. 1B). Plantlets were growing faster in the dark and the enzyme activity was up to 8 times higher in the dark-grown seedlings than in the light-grown ones. Moreover, independently of the light conditions, DAO activity reached a maximum alter 6 days and subsequently sharply declined in parallel with the slower growth rate of the plants (Fig. 1). A phytochrome involvement in the regulation of D A O activity was investigated by irradiating darkgrown seedlings with pulses of red and/or far red light according to Morohashi [14] or Angelini et al. [15]. D A O activity did not show any reduction. Similarly the exposure to blue light did not affect the activity (data not shown). The content of D A O in the different samples
53 600
16
500"
14 12
400 A
c
3OO
¢D O
200'
E
100 !
¢-
x
u~
0 6OO
B
16
)< ~N
14
5OO
o O
E
-12
400
c
>
o a
A
-10 300
Fig. 3. Northern blot analysis of RNA extracted from lentil seedlings. Forty-five #g of total RNA extracted from lightgrown (A) or dark-grown (B) seedlings were hybridized with a 17-mer oligonucleotide as described in Materials and Methods. The arrows mark the positions of ribosomal RNA.
-8 200 6 100
'4
0
. . . .
i
•
.'-
-
5
i
. . . .
i
10
. . . .
2
15
2
Growth period (days)
Fig. I. Changes in diamine oxidase activity during lentil seedlings growth, under 16-h photoperiod (A) or in the dark (B). Seedlings were harvested at the indicated periods, weighed and homogenized as described under Materials and Methods. O, DAO specific activity; O, fresh weight. In both panels each point represents the mean of three different determinations (S.D. < 4"/,,).
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P
the e x p o s u r e to a t e m p e r a t u r e (4°C) lower t h a n the o n e at which g r o w t h n o r m a l l y o c c u r r e d (25°C) did n o t affect the e n z y m e activity (Fig. 4C). T h e E L I S A assay s h o w e d n o c h a n g e s in D A O c o n t e n t o f a n o x i a or t h e r m a l l y stressed p l a n t s in c o m p a r i s o n to the c o n t r o l s (Figs. 4 A a n d 4C).
o
< 40
E Q
~ID
was d e t e r m i n e d by E L I S A . It was f o u n d that the increase in activity o f the seedlings g r o w n in the d a r k with respect to those g r o w n u n d e r i l l u m i n a tion was paralleled b y a n i n c r e a s e in the e n z y m e c o n t e n t (Fig. 2). N o r t h e r n blot a n a l y s i s (Fig. 3) s h o w e d that also the D A O m R N A a m o u n t was greater in d a r k - g r o w n plantelets. Anoxie and thermal stress W h e n lentil seedlings were e x p o s e d to a n a e r o b i c stress for 24 h, a decrease o f D A O activity to 30% o f the c o n t r o l value was o b s e r v e d (Fig. 4A), while
100
o
B
20
01. 0
.
0
.
.
.
.
.
5
.
.
.
.
.
.
10
1
Growth period (days)
Fig. 2. Diamine oxidase content in lentil seedlings grown in the light (O) or in the dark (e) for the indicated periods. The amount of DAO present was measured by ELISA. The results are expressed as % of the maximal value and are the mean of three different experiments (S.D. < 3%).
Discussion D A O activity in d a r k - g r o w n lentil seedlings is m u c h h i g h e r t h a n in the l i g h t - g r o w n o n e s (Fig. 1) [5,16]. T h e s a m e is true for the D A O c o n t e n t (Fig. 2) a n d the D A O m R N A level (Fig, 3). These d a t a
54
100" >,,
i " 80-
,<0060a a P 0
A
B SAMPLES
C
Fig. 4. Diamine oxidase activity and content in lentil seedlings under different treatments (details are described in Materials and Methods). The amount of D A O present was measured by ELISA. Both the specific activity (black bars) and the protein content (patched bars) were reported as % of the maximal values. The results are the mean of three different experiments (S.D. < 3%). (A) Seedlings growing in the dark and further 24 h of anoxic treatment; (B) seedlings growing in the dark; (C) seedlings growing in the dark and further 24 h of thermal stress (4°C). All samples refer to 5-day-old seedlings.
indicate that the higher enzyme activity in the dark conditions is regulated at the DAO gene(s) level and is achieved by increasing the concentration of the respective mRNA(s). A possible phytochrome down-regulation was investigated by delivering to dark-grown seedlings pulses of red and/or far-red light [17]. DAO activity, expressed on a protein basis, remained unchanged, at variance with the report of Angelini et al. [15]. The same was found when the samples were exposed to blue light (data not shown). In any case, lentil seedlings are much faster growing in the dark (Fig. 1) and the increase of DAO activity may be correlated to the growth, because the cell proliferation would require a faster metabolism of polyamines [18,19]. Alternatively the increase of DAO activity in etiolated seedlings may be related to processes occurring during stem elongation such as lignification and wall stiffening [20]. In any case phytochrome is not directly involved in the control of DAO activity. Besides the possible role of DAO in plant growth, the involvement of this enzyme in the response to stress factors was investigated. Recent-
ly it has been observed that the level of putrescine, the purported natural substrate of diamine oxidase, increases in rice shoots subjected to anoxic conditions, partly due to an increase of its biosynthesis. However a slower degradation could also be involved. Indeed our experiments with lentil seedlings show a substantial decrease of DAO activity during anoxia (Fig. 4A). Since this enzyme is rather abundant in the coleoptile and because of its very high affinity for 02 ( K m for 02 < 6 IxM) [21], it could be involved in regulating putrescine concentration during occasional periods of low oxygen concentration. Oxygen did not play any role on the expression of the DAO gene(s), because DAO mRNA steady state level (data not shown) and the amount of enzyme (Fig. 4A) were the same in anaerobic and aerobic samples. Therefore anoxia should cause the indirect inhibition of preexisting enzyme itself. As far as the thermal stress is concerned, Fig. 4C shows that lentil seedling diamine oxidase was not involved in the plant response to low temperature exposure. In [3act, both the activity and the amount of enzyme were the same as the controls. Nevertheless, an increase of putrescine levels during chilling injury of fruits from different plants has been reported [22] and it has been proposed that putrescine accumulation can mediate the stress-induced injury [23]. If this hypothesis were of general validity, our data indicate that putrescine accumulation during chilling stress is not due to a reduction of the DAO activity. Acknowledgement
This work has been supported in part by Italian CNR Special Project 'Chimica Fine' Contract 8900804.72. References 1
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