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Mitodepressive and clastogenic effects of some aminophosphonates, inhibitors of phenylalanine ammonia-lyase. I. 2-aminoindan-2-phosphonic acid
Plant Physiol. Bioc/zem., 1998, 36 (1 l), 805-808
Mitodepressive and clastogenic effects of some aminophosphonates, inhibitors of phenylalanine ammonia-lyase. I. 2-Aminoindan-2-phosphonic acid Regina Osiecka’*, Krystyna M. Janas ’ Department of Plant Cytology and Cytochemistry, *Department of Plant Growth Regulation, University oflbdi, ul. Banacha 12/16, 90-237t6di, Poland. * Author to whorn correspondence should be addressed (fax +48 42 35 44 23; email [email protected]) (Received June 16, 1998; accepted July 28, 1998)
Abstract - The phosphonic analogue of L-phenylalanine, 2-aminoindan-2-phosphonic acid (AIP) decreased the growth of seedlings and the mitotic activity of root tip cells of bean (Viciafabu L. ssp. minor var. Dino) in a dose-dependent manner. AIP at a concentration of 100 pM induced micronuclei and chromosomal aberrations in Mcia root tip meristems. 0 Elsevier, Paris 2-Aminoindan-2:-phosphonic acid I chromosomal aberrations 1 maleic hydrazide I micronuclei I mitotic index I growth I viciu fuba AIP, 2-aminoindan-2-phosphonic acid / MH, maleic hydrazide / PAL, phenylalanine ammonia-lyase / PheP, I-amino-2phenylethylphosphonic acid / PhPP, 1-amino-3-phenylpropylphosphonic acid
1. INTRODUCTION Phenylalanine ammonia-lyase (PAL, EC 4.3.1 S) is a key enzyme which catalyses the deamination of phenylalanine to truns-cinnamic acid which is further converted to a large range of phenylpropanoid-derived secondary products such as coumarins, lignin, flavonoids etc. Inhibitors of PAL are useful tools in the study of these metabolic pathways as well as in the elucidation of the physiological roles of their products. Aminoalkanephosphonic acids are broadly defined as amino acid1 analogues in which the carboxylic group is replaced by a phosphonic or related group which has the same function (phosphonous, phosphinic, phosphine oxide, etc.). These structural analogues of amino acids usually bind as their carboxylic counterparts to the active sites of enzymes or other cell receptors. As inhibitors of metabolic processes, they are used as anticancer drugs, pesticides, etc. [6]. Most PAL inhibitors are derivatives of phenylalanine. Some of the phosphonic analogues of phenylalanine have been screened as putative inhibitors of PAL in vitro and in vivo, e.g. 2-aminoindan-2-phosphonic Plant Physiol.
Biochem.,
0981-9428/98/l
l/O
Elsevier,
Paris
acid (AIP), 1-amino-2-phenylethylphosphonic acid l-amino-3-phenylpropyl-phosphonic acid WW, (PhPP) [3,5, 171. AIP is a potent PAL inhibitor (Ki value of 0.08 yM, whereas the Km value for phenylalanine is 45 pM for buckwheat PAL). This compound effectively inhibits anthocyanin synthesis in buckwheat seedlings with an I,, of ca. 1.5 @I [17]. AIP blocks lignification [7, 141 and accumulation of phenylalanine-derived phenolic compounds in elicitor-treated cultured tomato cells [2]. However, different seedlings react with different sensitivity when treated with AIP. Usually 20-30 pM of AIP have been used [ 1,7]. Sometimes much higher concentrations of AIP up to 50-100 @I are needed [9-l 1, 14, 151. However, most experiments showed that this compound also decreased the growth of plants [I, 4, 131. Knowledge on the side-effects of enzyme inhibitors is very important if we want to use these compounds in physiological investigations. Because AIP is a PAL inhibitor which is often used in physiological studies, our first experiment was undertaken to examine the influence of this compound on the growth and mitotic activity in Vicia root tip cells and to determine the
806
R. Osiecka, K.M. Janas
potential clastogenic action by means of micronucleus and chromosomal aberration tests. 2. RESULTS AND DISCUSSION AIP decreased root elongation of Kcia jiba seedlings, determined after 4 days of cultivation in darkness, as a function of its concentration. Hypocotyl length increased at lower concentrations of AIP (25 @VI), but at 50 pM, it had no significant effect on the growth of hypocotyls (table Z). An inhibitory effect on hypocotyls was observed when seedlings were cultivated at the highest concentration of AIP. Fresh weight was reduced in both hypocotyls and roots (table Z). In roots of V fuba seedlings growing in the presence of AIP, an increase in dry mass from 70 mg in control roots to 76.5 mg and 74.5 mg in the presence of 25 pM and 50 pM AIP, respectively, was observed. In roots cultivated at the highest concentration of AIP (100 @VI), dry mass decreased. The growth retarding effect was accompanied by a decrease in root tip mitotic activity figure 1). The mitotic indices in meristems were 63 %, 54 % and 33 % of the negative control value for AIP at concentrations of 25, 50 and 100 pM, respectively @gure I). Sato et al. [15] and Nakashima et al. [l I] found that low concentrations of AIP did not inhibit lignification of tracheary elements in isolated Zinnia eleguns L. mesophyll cells. Therefore, they used AIP concentration as high as 100 pM in the culture medium. Hence. we also used this concentration in further experiments. Because maleic hydrazide (MH) decreases growth of roots, as does AIP, we also compared the activity of MH with that of AIP. MH has been used to retard the growth of several plants, e.g. grass, hedges, trees. It is known that MH in plants primarily acts on dividing cells. This compound’s interference with nucleic acid metabolism is the main reason for its inhibitory effects on plant cell division and growth. MH is known as a chromosome-
breaking agent (clastogcn). lirst described 111 195 I ] 161. MH-induced chromosomal aberrations are preferentially localized in heterochromatic segments. AIP at all tested concentrations decreased mitotic activity to a similar degree as MH. At low concentration (25 pM). its action is not as pronounced as that of MH (100 pM) (f&l/~ /). Tub10 II shows that AIP at a concentration of IO0 pM induces a significant increase in the frequency of chromosomal aberrations similar to MH. The frequency of chromosomal aberrations induced by AIP is &fold higher than that of spontaneous damages (negative control). but lower than that observed in the positive control (with MH) (table II). We observed chromosomal breaks (86 %) and NUpd type isochromatid breaks ( 16 %) (data not shown).
AIP induces four times more micronuclei in comparison to the negative control (Sorensen’s buffer) in Viciu root tip meristems (table II). The number of micronuclei induced by AIP is 13.2/l 000 cells. while in meristems treated with MH, the number of micronuclei is 17.4/l 000 cells in comparison to 3/l 000 cells in the negative control (ruble UJ.
Figure 1. Effect of AIP (25 FM, 50 FM and 100 PM) on mitotic ity of Vicia root tip cells. 0, Control;
Table I. The effect of 2-aminoindan-2-phosphonic grown
on Srensen’s
buffer
(control,
AIP (PM)
Length Roots
0
64.7 54.4 50.6 49.7
25 50 100
Plunt
Phvsiol.
Biochem
+ + + *
acid on growth of Vi& fuba seedlings. Growth was measured 0) or in the presence of various concentrations of AIP at 25 “C in darkness.
59.9 64.6 55.9 36.9
f 10.6 f 15.4 + 13.6 z!z 9.1
maleic
Roots I .2 I .o I.1 I .o
A f + +
0.30 0.05 0.06 0.1
Dry Hypocotyls 2.26 f 0.20 1.96 + 0.08 1.77 * 0.15
1.72 _t 0.15
hydrazide
in 4-d old seedlings
Fresh weight k)
of seedlings (mm) Hypocotyls
17.7 12.7 10.9 13.5
MH.
Roots 70.0 + 2.9 16.5 k 3.5 74.5 * 2.1 60.0 t 0.01
activ( 100 yM).
which
mass (mg) Hypocotyls 139+ 19 15222 139k 12 115 A 3.5
were
Clastogenicity of 2-aminoindan-2-phosphonic acid
Table II. Effect of AIP on frequency of chromosomal aberrations per cell and number of micronuclei (per 1 000 cells) in Vicia root tip cells. MH, Maleic hydrazide. Series
Control MH (100 PM) AIP (100 PM)
Number of micronuclei per 1 000 cells 3 17.4 13.2
Frequency of aberrations 0.005 0.045 0.04
These results show that AIP at the tested concentration (100 @vI) has a clastogenic effect manifested in the induction of chromosomal aberrations and micronuclei. It also has a mitodepressive effect, decreasing in a dose-dependent manner the mitotic activity of Vicia root tip cells. This may be the reason for the inhibition of the growth of Kcia seedlings. We suppose that the mitodepressive and clastogenic effects were not due to inh:ibition of PAL in vivo because another, but weak, inhibitor of PAL - D,L-PhPP (Ki value 3 @I for potato tuber PAL) - also exerts an effect similar to that of AIP, and a stronger inhibitor - D,L-PheP (Ki value 1.6 l&I for potato tubers) - did not affect micronuclei (in preparation). The way aminophosphonates act at the cellular level is still poorly understood. No data are available in the literature on the mutagenic activity of AIP or other aminophosphonates. p-Fluorophenylalanine, a known inhibitor of PAL, at concentrations of 25, 100 and 400 l&I, reduces the mitotic index, depending on the dose used, and inhibits the initial growth of cultured shoot tips of Vitis Zabruscana. Abnormal mitoses, including C-mitosis, lagging chromosomes, multipolar mitosis and micronuclei, have also been observed [ 121. Our results suggest that AIP exerts mitodepressive and clastogenic effects on V fuba root tip cells and should be used with caution, especially in in vivo investigations concerning inhibition of the activity of PAL and its products. We suppose that AIP at low concentrations does not exert clastogenic effects because different authors observed that concentrations in the range 20-30 pM do not have an inhibitory effect on the growth of examined plants. However, further research is required to elucidate the clastogenic effect of AIP 3. CONCLUSION The key function of PAL in phenylpropanoid metabolism makes this enzyme a challenging target for the development of powerful inhibitors specifically inter-
807
fering with phenylalanine deamination and thus blocking the formation compounds derived from cinnamic acid. AIP is a potent inhibitor of PAL which is often used in physiological studies. As shown in this paper, this compound at high concentrations inhibits growth of plants and has a mitodepressive and clastogenic effect in Viciafuba root tip cells. It is very important to know side-effects of commonly-used inhibitors and to use them in as low a concentration as possible. 4. METHODS 4.1. Plant material. Seedsof bean (Viciafaba L. ssp. minor var. Dino) were obtained from the Institute of Plant Breeding and Acclimatization (Bar&k, Poland). The seeds were germinated in Petri dishes on Whatman filter paper wetted with 10 cm3 distilled water (control) or AIP (25,50 or 100 pM) in darkness at 25 “C. Growth was measured in 4-d old seedlings. Dry weight was determined by placing freshly weighed seedlings in an oven at 80 “C for 24 h.
4.2. The cytogenetic assay. Experiments were performed according to the protocol of the International Programme of Chemical Safety (IPCS [8]). Seeds were soaked for 18 h in tap water and then germinated in moist Perlite at 22 “C. The tested chemicals were dissolved in l/150 M Sorensen’s phosphate buffer (pH 6.0), immediately before use. Seedlings with 1.5-3 cm long seminal roots were incubated for 2 h in darkness in aerated AIP solution at concentrations of 25, 50 and 100 uM. Maleic hydrazide (MH) (100 @I) was used as a positive control. This compound has been recommended by IPCS in cytogenetic assays because it is relatively stable, easily available and has low toxicity. As a negative control, we used the Sorensen’s buffer as above. The recovery period (26 h) included treatment with 0.05 % colchicine during the last 3 h. The meristems were fixed in a mixture of ethanol/ glacial acetic acid (3/l, v/v). The meristems were stained according to the Feulgen procedure and squashed preparations were made permanent by the dry-ice method. The slides were randomly coded before analyses. Four root tips were used for one experimental series. The mitotic activity and number of micronuclei in 1 000 cells from each slide were determined as well as the number of aberrations in 50 metaphase plates from one meristem. 4.3. Statistical analysis. Each assay was conducted in triplicate and experiments were repeated at least two times. SE was calculated between experiments. Acknowledgements. We thank Dr Jerzy Zen (Institute of Organic Chemistry, Biochemistry and Biotechnology, Technical University of Wroclaw, Poland) for providing us with AIP. vol. 36 (11) 1998
808
R. Osiecka, K.M. Janas
REFERENCES [I 1 Gaisser S.. Heide L.. Inhibition and regulation of shikonin biosynthesis in suspension cultures of Lithosprl-~UI~Z, Phytochemistry 41 (1995) 1065-l 072. [2] Giirlach J., Raesecke H.-R., Rentsch D., Regenass M., Roy P., Zala M., Keel C.H., Boller T., Amrhein N., Schmid J., Temporally distinct accumulation of transcripts encoding enzymes of the prechorismate pathway in elicitor-treated, cultured tomato cells. Proc. Nat1 Acad. Sci. USA 92 (1995) 3 166-3 170. [3] Janas K.M., Olechnowicz D.. 1-Amino-3-phenylpropylphosphonic acid. the inhibitor of L-phenylalanine ammonia-lyase activity in higher plants. Acta Biochim. Pol. 41 (1994) 191-193. [4] Janas K.M., Osiecka R., Effect of aminophosphonates on starch accumulation in Spirodela punctuta, Biol. Bull. Poznari (Suppl) (1995) 70. [5] Janas K.M., Filipiak A., Kowalik J., Mastalerz P.. Knypl J.S., I-Amino-2-phenylethylphosphonic acid: an inhibitor of L-phenylalanine ammonia-lyase in vitro. Acta Biochim. Pol. 32 (1985) 131-143. [6] Kafdrski P., Lejczak B., Biological activity of aminophosphonic acids, Phosphorus Sulfur Silicon 63 ( 1991 ) 193-215. [7] Kaiser I., Engelberth J., Groth B., Weiler E.W., Touchand methyl jasmonate-induced lignification in tendrils of Bryonia dioica Jacq., Bot. Acta 107 (1994) 24-29. [Sl Kanaya N., Gill B.S., Grover IS., Murin A., Osiecka R., Sandhu S.S., Andersson H.C., Viciafaba chromosomal assay, Mutat. Res. 3 IO (I 994) 23 l-247. [9] Mauch-Mani B., Slusarenko A.J., Production of salicylic acid precursors is a major function of phenylala-
Planr
Physiol.
Biochern.
nine ammonia-lyase in the rch~slance ol’ A~trhido/,.st.c to Pf~rnrw.cpot~tr ptrmsitiur. Plant Cell 8 (1996) 203-2 12. [ IO] Meuwly P., Miilder\ W.. Buchala A.. Mitraux J.-P., Local and systemic biosynthesis 01’ salicylic acid in infected cucumber plants, Plant Physiol. I OY I IO%\ 1107-I 114. [I I] Nakashima J., Takabe K.. Fujita M.. Saiki H.. lnhihition of phenylalanine ammonia-lyase activity cause5 the depression 01‘ lignin accumulation 01‘ \econdarq wall thickening in isolated Zimitr mesophyll cell5. Protoplasma lY6 (1997) 99--107. [ 121 Omura M.. Kazunori N.. Toshio H., Tomoya A., Variation in the chromosome number of cultured shoot tip of Vitis spp. by p-fluorophenylanine treatment. Japan. J. Breed. 37 (1987) 127-132. [13] Reuber S.. Leitsch J.. Krause G.H.. Weissenbijck G., Metabolic reduction of phenylpropanoid compounds in primary leaves of rye (Se&e cer-eale L.) leads to increased UV-B sensitivity of photosynthesis. Z. Naturforsch. 18~ ( 1993) 749.-756. [14] Ryser U.. Keller B., Ultrastructural localization of a bean glycine-rich protein in unlignified primary walls of protoxylem cells, Plant Cell 4 (1992) 773-783. [ 151 Sato Y.. Sugiyama M.. G6recki R.J., Fukuda H.. Komamine A.. Interrelationship between lignin deposition and the activities of peroxidase isoenzymes in differentiating tracheary elements of Zinniu. Analysis using L-a-aminooxy-P-phenylpropionic acid and 2aminoindan-2-phosphonic acid. Planta I89 ( 1993) 584-589. 1161 Swietlin’ska Z.. iuk J.. Cytotoxic effects of maleic hydrazide, Mutat. Res. 55 (1978) 15-30. [ 171 Zori J., Amrhein N., Inhibitors of phenylalanine ammonia-lyase: 2-aminoindan-2-phosphonic acid and related compound. Liebigs Ann. Chem. ( 1992) 625-628.
Mitodepressive and clastogenic effects of some aminophosphonates, inhibitors of phenylalanine ammonia-lyase. I. 2-Aminoindan-2-phosphonic acid Regina Osiecka’*, Krystyna M. Janas ’ Department of Plant Cytology and Cytochemistry, *Department of Plant Growth Regulation, University oflbdi, ul. Banacha 12/16, 90-237t6di, Poland. * Author to whorn correspondence should be addressed (fax +48 42 35 44 23; email [email protected]) (Received June 16, 1998; accepted July 28, 1998)
Abstract - The phosphonic analogue of L-phenylalanine, 2-aminoindan-2-phosphonic acid (AIP) decreased the growth of seedlings and the mitotic activity of root tip cells of bean (Viciafabu L. ssp. minor var. Dino) in a dose-dependent manner. AIP at a concentration of 100 pM induced micronuclei and chromosomal aberrations in Mcia root tip meristems. 0 Elsevier, Paris 2-Aminoindan-2:-phosphonic acid I chromosomal aberrations 1 maleic hydrazide I micronuclei I mitotic index I growth I viciu fuba AIP, 2-aminoindan-2-phosphonic acid / MH, maleic hydrazide / PAL, phenylalanine ammonia-lyase / PheP, I-amino-2phenylethylphosphonic acid / PhPP, 1-amino-3-phenylpropylphosphonic acid
1. INTRODUCTION Phenylalanine ammonia-lyase (PAL, EC 4.3.1 S) is a key enzyme which catalyses the deamination of phenylalanine to truns-cinnamic acid which is further converted to a large range of phenylpropanoid-derived secondary products such as coumarins, lignin, flavonoids etc. Inhibitors of PAL are useful tools in the study of these metabolic pathways as well as in the elucidation of the physiological roles of their products. Aminoalkanephosphonic acids are broadly defined as amino acid1 analogues in which the carboxylic group is replaced by a phosphonic or related group which has the same function (phosphonous, phosphinic, phosphine oxide, etc.). These structural analogues of amino acids usually bind as their carboxylic counterparts to the active sites of enzymes or other cell receptors. As inhibitors of metabolic processes, they are used as anticancer drugs, pesticides, etc. [6]. Most PAL inhibitors are derivatives of phenylalanine. Some of the phosphonic analogues of phenylalanine have been screened as putative inhibitors of PAL in vitro and in vivo, e.g. 2-aminoindan-2-phosphonic Plant Physiol.
Biochem.,
0981-9428/98/l
l/O
Elsevier,
Paris
acid (AIP), 1-amino-2-phenylethylphosphonic acid l-amino-3-phenylpropyl-phosphonic acid WW, (PhPP) [3,5, 171. AIP is a potent PAL inhibitor (Ki value of 0.08 yM, whereas the Km value for phenylalanine is 45 pM for buckwheat PAL). This compound effectively inhibits anthocyanin synthesis in buckwheat seedlings with an I,, of ca. 1.5 @I [17]. AIP blocks lignification [7, 141 and accumulation of phenylalanine-derived phenolic compounds in elicitor-treated cultured tomato cells [2]. However, different seedlings react with different sensitivity when treated with AIP. Usually 20-30 pM of AIP have been used [ 1,7]. Sometimes much higher concentrations of AIP up to 50-100 @I are needed [9-l 1, 14, 151. However, most experiments showed that this compound also decreased the growth of plants [I, 4, 131. Knowledge on the side-effects of enzyme inhibitors is very important if we want to use these compounds in physiological investigations. Because AIP is a PAL inhibitor which is often used in physiological studies, our first experiment was undertaken to examine the influence of this compound on the growth and mitotic activity in Vicia root tip cells and to determine the
806
R. Osiecka, K.M. Janas
potential clastogenic action by means of micronucleus and chromosomal aberration tests. 2. RESULTS AND DISCUSSION AIP decreased root elongation of Kcia jiba seedlings, determined after 4 days of cultivation in darkness, as a function of its concentration. Hypocotyl length increased at lower concentrations of AIP (25 @VI), but at 50 pM, it had no significant effect on the growth of hypocotyls (table Z). An inhibitory effect on hypocotyls was observed when seedlings were cultivated at the highest concentration of AIP. Fresh weight was reduced in both hypocotyls and roots (table Z). In roots of V fuba seedlings growing in the presence of AIP, an increase in dry mass from 70 mg in control roots to 76.5 mg and 74.5 mg in the presence of 25 pM and 50 pM AIP, respectively, was observed. In roots cultivated at the highest concentration of AIP (100 @VI), dry mass decreased. The growth retarding effect was accompanied by a decrease in root tip mitotic activity figure 1). The mitotic indices in meristems were 63 %, 54 % and 33 % of the negative control value for AIP at concentrations of 25, 50 and 100 pM, respectively @gure I). Sato et al. [15] and Nakashima et al. [l I] found that low concentrations of AIP did not inhibit lignification of tracheary elements in isolated Zinnia eleguns L. mesophyll cells. Therefore, they used AIP concentration as high as 100 pM in the culture medium. Hence. we also used this concentration in further experiments. Because maleic hydrazide (MH) decreases growth of roots, as does AIP, we also compared the activity of MH with that of AIP. MH has been used to retard the growth of several plants, e.g. grass, hedges, trees. It is known that MH in plants primarily acts on dividing cells. This compound’s interference with nucleic acid metabolism is the main reason for its inhibitory effects on plant cell division and growth. MH is known as a chromosome-
breaking agent (clastogcn). lirst described 111 195 I ] 161. MH-induced chromosomal aberrations are preferentially localized in heterochromatic segments. AIP at all tested concentrations decreased mitotic activity to a similar degree as MH. At low concentration (25 pM). its action is not as pronounced as that of MH (100 pM) (f&l/~ /). Tub10 II shows that AIP at a concentration of IO0 pM induces a significant increase in the frequency of chromosomal aberrations similar to MH. The frequency of chromosomal aberrations induced by AIP is &fold higher than that of spontaneous damages (negative control). but lower than that observed in the positive control (with MH) (table II). We observed chromosomal breaks (86 %) and NUpd type isochromatid breaks ( 16 %) (data not shown).
AIP induces four times more micronuclei in comparison to the negative control (Sorensen’s buffer) in Viciu root tip meristems (table II). The number of micronuclei induced by AIP is 13.2/l 000 cells. while in meristems treated with MH, the number of micronuclei is 17.4/l 000 cells in comparison to 3/l 000 cells in the negative control (ruble UJ.
Figure 1. Effect of AIP (25 FM, 50 FM and 100 PM) on mitotic ity of Vicia root tip cells. 0, Control;
Table I. The effect of 2-aminoindan-2-phosphonic grown
on Srensen’s
buffer
(control,
AIP (PM)
Length Roots
0
64.7 54.4 50.6 49.7
25 50 100
Plunt
Phvsiol.
Biochem
+ + + *
acid on growth of Vi& fuba seedlings. Growth was measured 0) or in the presence of various concentrations of AIP at 25 “C in darkness.
59.9 64.6 55.9 36.9
f 10.6 f 15.4 + 13.6 z!z 9.1
maleic
Roots I .2 I .o I.1 I .o
A f + +
0.30 0.05 0.06 0.1
Dry Hypocotyls 2.26 f 0.20 1.96 + 0.08 1.77 * 0.15
1.72 _t 0.15
hydrazide
in 4-d old seedlings
Fresh weight k)
of seedlings (mm) Hypocotyls
17.7 12.7 10.9 13.5
MH.
Roots 70.0 + 2.9 16.5 k 3.5 74.5 * 2.1 60.0 t 0.01
activ( 100 yM).
which
mass (mg) Hypocotyls 139+ 19 15222 139k 12 115 A 3.5
were
Clastogenicity of 2-aminoindan-2-phosphonic acid
Table II. Effect of AIP on frequency of chromosomal aberrations per cell and number of micronuclei (per 1 000 cells) in Vicia root tip cells. MH, Maleic hydrazide. Series
Control MH (100 PM) AIP (100 PM)
Number of micronuclei per 1 000 cells 3 17.4 13.2
Frequency of aberrations 0.005 0.045 0.04
These results show that AIP at the tested concentration (100 @vI) has a clastogenic effect manifested in the induction of chromosomal aberrations and micronuclei. It also has a mitodepressive effect, decreasing in a dose-dependent manner the mitotic activity of Vicia root tip cells. This may be the reason for the inhibition of the growth of Kcia seedlings. We suppose that the mitodepressive and clastogenic effects were not due to inh:ibition of PAL in vivo because another, but weak, inhibitor of PAL - D,L-PhPP (Ki value 3 @I for potato tuber PAL) - also exerts an effect similar to that of AIP, and a stronger inhibitor - D,L-PheP (Ki value 1.6 l&I for potato tubers) - did not affect micronuclei (in preparation). The way aminophosphonates act at the cellular level is still poorly understood. No data are available in the literature on the mutagenic activity of AIP or other aminophosphonates. p-Fluorophenylalanine, a known inhibitor of PAL, at concentrations of 25, 100 and 400 l&I, reduces the mitotic index, depending on the dose used, and inhibits the initial growth of cultured shoot tips of Vitis Zabruscana. Abnormal mitoses, including C-mitosis, lagging chromosomes, multipolar mitosis and micronuclei, have also been observed [ 121. Our results suggest that AIP exerts mitodepressive and clastogenic effects on V fuba root tip cells and should be used with caution, especially in in vivo investigations concerning inhibition of the activity of PAL and its products. We suppose that AIP at low concentrations does not exert clastogenic effects because different authors observed that concentrations in the range 20-30 pM do not have an inhibitory effect on the growth of examined plants. However, further research is required to elucidate the clastogenic effect of AIP 3. CONCLUSION The key function of PAL in phenylpropanoid metabolism makes this enzyme a challenging target for the development of powerful inhibitors specifically inter-
807
fering with phenylalanine deamination and thus blocking the formation compounds derived from cinnamic acid. AIP is a potent inhibitor of PAL which is often used in physiological studies. As shown in this paper, this compound at high concentrations inhibits growth of plants and has a mitodepressive and clastogenic effect in Viciafuba root tip cells. It is very important to know side-effects of commonly-used inhibitors and to use them in as low a concentration as possible. 4. METHODS 4.1. Plant material. Seedsof bean (Viciafaba L. ssp. minor var. Dino) were obtained from the Institute of Plant Breeding and Acclimatization (Bar&k, Poland). The seeds were germinated in Petri dishes on Whatman filter paper wetted with 10 cm3 distilled water (control) or AIP (25,50 or 100 pM) in darkness at 25 “C. Growth was measured in 4-d old seedlings. Dry weight was determined by placing freshly weighed seedlings in an oven at 80 “C for 24 h.
4.2. The cytogenetic assay. Experiments were performed according to the protocol of the International Programme of Chemical Safety (IPCS [8]). Seeds were soaked for 18 h in tap water and then germinated in moist Perlite at 22 “C. The tested chemicals were dissolved in l/150 M Sorensen’s phosphate buffer (pH 6.0), immediately before use. Seedlings with 1.5-3 cm long seminal roots were incubated for 2 h in darkness in aerated AIP solution at concentrations of 25, 50 and 100 uM. Maleic hydrazide (MH) (100 @I) was used as a positive control. This compound has been recommended by IPCS in cytogenetic assays because it is relatively stable, easily available and has low toxicity. As a negative control, we used the Sorensen’s buffer as above. The recovery period (26 h) included treatment with 0.05 % colchicine during the last 3 h. The meristems were fixed in a mixture of ethanol/ glacial acetic acid (3/l, v/v). The meristems were stained according to the Feulgen procedure and squashed preparations were made permanent by the dry-ice method. The slides were randomly coded before analyses. Four root tips were used for one experimental series. The mitotic activity and number of micronuclei in 1 000 cells from each slide were determined as well as the number of aberrations in 50 metaphase plates from one meristem. 4.3. Statistical analysis. Each assay was conducted in triplicate and experiments were repeated at least two times. SE was calculated between experiments. Acknowledgements. We thank Dr Jerzy Zen (Institute of Organic Chemistry, Biochemistry and Biotechnology, Technical University of Wroclaw, Poland) for providing us with AIP. vol. 36 (11) 1998
808
R. Osiecka, K.M. Janas
REFERENCES [I 1 Gaisser S.. Heide L.. Inhibition and regulation of shikonin biosynthesis in suspension cultures of Lithosprl-~UI~Z, Phytochemistry 41 (1995) 1065-l 072. [2] Giirlach J., Raesecke H.-R., Rentsch D., Regenass M., Roy P., Zala M., Keel C.H., Boller T., Amrhein N., Schmid J., Temporally distinct accumulation of transcripts encoding enzymes of the prechorismate pathway in elicitor-treated, cultured tomato cells. Proc. Nat1 Acad. Sci. USA 92 (1995) 3 166-3 170. [3] Janas K.M., Olechnowicz D.. 1-Amino-3-phenylpropylphosphonic acid. the inhibitor of L-phenylalanine ammonia-lyase activity in higher plants. Acta Biochim. Pol. 41 (1994) 191-193. [4] Janas K.M., Osiecka R., Effect of aminophosphonates on starch accumulation in Spirodela punctuta, Biol. Bull. Poznari (Suppl) (1995) 70. [5] Janas K.M., Filipiak A., Kowalik J., Mastalerz P.. Knypl J.S., I-Amino-2-phenylethylphosphonic acid: an inhibitor of L-phenylalanine ammonia-lyase in vitro. Acta Biochim. Pol. 32 (1985) 131-143. [6] Kafdrski P., Lejczak B., Biological activity of aminophosphonic acids, Phosphorus Sulfur Silicon 63 ( 1991 ) 193-215. [7] Kaiser I., Engelberth J., Groth B., Weiler E.W., Touchand methyl jasmonate-induced lignification in tendrils of Bryonia dioica Jacq., Bot. Acta 107 (1994) 24-29. [Sl Kanaya N., Gill B.S., Grover IS., Murin A., Osiecka R., Sandhu S.S., Andersson H.C., Viciafaba chromosomal assay, Mutat. Res. 3 IO (I 994) 23 l-247. [9] Mauch-Mani B., Slusarenko A.J., Production of salicylic acid precursors is a major function of phenylala-
Planr
Physiol.
Biochern.
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