- Email: [email protected]
Induction of antheridium differentiation in the fern Anemia phyllitidis L. Sw. by halogenated base analogues
Abteilung Allgemeine Botanik, Vniversitat VIm, Federal Republic of Germany
Induction of Antheridium Differentiation in the Fern Anemia phyllitidis L. Sw. by Halogenated Base Analogues H. SCHRAUDOLF With 4 figures Received June 12, 1979 . Accepted August 1, 1979
Summary The halogenated deoxynucleosides 5-bromo-2-deoxyuridine, 5-iodo-2-deoxyuridine and 5bromo-2-deoxycytidine substitute, though with less efficiency, for antheridiogens or gibberellins in the antheridium induction in gametophytes of Anemia phyllitidis. This effect is enhanced by cultivation of the prothalli under low light intensities. In contrast to antheridiogens and gibberellins, these halogenated deoxynucleosides are unable to cause dark-germination of Anemia spores. 5-Bromouracil and 5-bromouridine, but also 2-aminopurine, proved to be inactive regarding both antheridium induction and dark-germination.
Key words: Fern, gametophyte, antheridium, differentiation, bromodeoxyuridine, bromodeoxycytidine.
Introduction Differentiation of the antheridium in gametophytes of Anemia phyllitidis is induced by the diterpenoid pheromons antheridiogen AAnl and AAn2 (NAF, 1959; NAKANISHI et aI., 1971; ENDO et aI., 1972; SCHRAUDOLF, 1972) while gibberellins are able to substitute for these native compounds (SCHRAUDOLF, 1962, 1966 a; VOELLER, 1964). Though considerable attempts have been made to unravel the molecular basis of antheridium induction, conclusive data on the biochemical mechanisms underlying the process are however lacking. Since hormonal control of cell differentiation is not blocked by inhibitors of protein and nucleic acid synthesis, 5-bromo-deoxyuridine (BUdR) has a striking influence on the manifestation of antheridium pattern (SCHRAUDOLF, 1967 a, 1977 a), or chloroplast structure (SCHRAUDOLF and SONKA, 1979). Sporadic formation of antheridia could be observed during the studies on analogue treated Anemia prothalli. The substitution of antheridiogen by a base analogue can give rise to new points in our understanding of the causal events of hormonal regulation in antheridium formation especially because differentiation of male Z. Pflanzenphysiol. Bd. 96. S. 67-75. 1980.
68
H. SCHRAUDOLF
gametangia was also observed in prothalli of Polyp odium crassifolium treated with BUdR under non inductive light conditions. The present study has been undertaken mainly to examine and quantify the earlier observations so as to improve the experimental parameters in antheridium induction by base analogues. Material and Methods Spores of Anemia phyllitidis L. Sw. were harvested from plants grown in the green house of the University of Ulm. The standard growth conditions and culture media were published elsewhere (SCHRAUDOLF, 1966 a). Red light obtained from red fluorescent tubes (colour 15; Phillips-TL) combined with two layers of plexiglass no. 501 (ROHM and HAAS) gave an irradiance at the level of the spores of about 1.0 X 10-4 joule cm-2. Low intensity irradiation rates of 1.1 X 10-4 joule cm-2 have been obtained below 2.25 0/0 grey plexiglass. (ROHM and HAAS) in white light field (Phillips TL G7/32 Warmton). For the quantification of antheridium differentiation at least 200 prothallia stained with aceto carmine have been evaluated microscopically.
Results
When spores of Anemia are germinated and grown in the presence of high concentrations of BUdR (2 X 10-3 M), the developing gametophytes are only sporadically induced to form antheridia. The simultaneous reduction of light intensity, however, remarkably increases the effect of this base-analogue. Transfer of spores previously kept in red light for 48 h into either continuous dark (D) or white light of low intensity (WWL) causes the differentiation of antheridia after only a few vegetative cell divisions (Tab. 1). The same effect is observed with samples kept under red light throughout (R). Under the same conditions, 5-bromouridine (BrU) Table 1: Induction of antheridia in prothalli of Anemia phyllitidis grown and afterwords cultured in red light (R) in darkness (D) or white light of low intensity (WWL).
Ofo antheridia-bearing prothalli R Control
WWL
D
0
0
BUdR 2 X 10-3 M 10-3 M 5X10-4 M
50,0 32.5 25.4
14.8 14.8 13.0
BrU 2 X 10-3 M
0
0
0
BU 2X10- 3 M
0
0
0
Z. Pjlanzenphysiol. Bd. 96. S. 67-75. 1980.
0 8.0 4.2 1.0
Induction of antheridium differentiation
69
and 5-bromouracil (BU) are completely inactive, as well as 2-aminopurine, a base-analogue that causes like BUdR growth inhibition and severe aberrations in the antheridium-patterns of hormon-treated prothalli (SCHRAUDOLF, 1977 a). On the other hand, 5-bromodeoxycytidine (BCdR) and with lower activity also 5-iododeoxyuridine (IUdR) are able to induce premature antheridium differentiation (Tab. 2). In the first instance, it can be postulated that only those base-analogues which are capable of causing specific base-transitions in the DNA can serve as active substituents for the native antheridiogen. Table 2: Effect of different halogenated deoxynucleosides on antheridium differentiation in Anemia phyllitidis. 0/0
antheridia-bearing prothalli
R Control
WWL
D
0
0
0
BUdR 5 X 10-4 10-4 M 5 X 10-5 M 1O-5 M
53.4 28.5 21.0 16.7
27.5 18.5 19.7 15.2
10.5 1.2 0 0
BCdR 5 X 10-4 M 10-4 M 5 X 10-5 M 10-5 M
6.4 29.5 41.2 40.0
9.3 13.6 19.4 11.3
6.8 5.7 5.4 4.3
IUdR 5 X 10-4 M 10-4 M 5 X 10-:; M 10-5 M
11.5 9.8 3.4 0
5.1 1.2 0 0
1.0 1.2 1.0 0
Difficulties in the quantification of the inductive effects by the above chemicals arise from the simultaneous disturbance of the normal antheridium patterns (SCHRAUDOLF, 1967 a, 1977 a). The formation of teratological structures which frequently are no longer identifiable as sexual organs (Fig. 1) certainly represent one of the important reasons for the decreasing percentage of antheridia-bearing prothalli counted in samples which had been treated with high BCdR concentrations (Tab. 2). Under those conditions also two-dimensionally growing prothalli can be observed occasionally even after continuous exposure of the samples to red light (Fig. 2). Ethyl acetate extracts from acidified growth media of prothalli which already had differentiated antheridia after BUdR-treatment proved to be inactive in bioassays. The possibility of premature synthesis of antheridiogens as a prerequisite of the observed effects of the base-analogues can thus be excluded. This view is confirmed
z. Pjlanzenphysiol. Bd. 96. S. 67-75. 1980.
70
H.
SCHRAUDOLF
Fig. 1: Beginning of pattern simplification in an antheridium (green antheridium [Dopp, 1959]) induced by 10-4 M BCdR in continuous red light. Acetocarmine.
Fig. 2: 2-dimensional growth of Anemia prothalli in continuous red light induced by BCdR (5 X 10-4 M). Acetocarmine. Z. Pflanzenphysiol. Bd. 96. S. 67-75. 1980.
Induction of antheridium differentiation
71
by the observation that cyclic-Y,S'-AMP which sensibilizes prothalli against the natural antheridiogen (SCHRAUDOLF, 1977 b) had even an inhibitory effect in the BUdR-induced antheridio-genesis (Tab. 3). Table 3: Mutual effects of BUdR and cycl.-3,S-AMP on antheridium induction of Anemia phyllitidis.
Ofo antheridia-bearing prothalli
R Control
WWL
0
0
0
0
0
0
20.0 15.0 1.0
17.0 13.8 0
14.1 1.0
11.8
c-3',S-AMP
(S X 10-4 M)
BUdR
10-3 M 10-4 M 10-5 M
c-3',S-AMP (5 X 10-4 M)
2.0 0.8 0
+
BUdR
10-3 M 10-4 M 10-5 M
D
o
6.9
o
o
o o
With increasing age of the prothalli, specific areas of the gametophytes lose their sensitivity towards antheridiogens and also towards gibberellins, which can substitute for this native hormone. Short-time plasmolysis, however, can restore the sensibility of these cells (SCHRAUDOLF, 1966 b). Regenerated prothalli obtained from plasmolyzed cells redifferentiate sexual organs after hormone application. Identical effects can be observed with BUdR as the triggering signal (Fig. 3). These results indicate a far-reaching conformity of the various processes involved in the hormonal as well as in the BUdR-caused effects on gametangium differentiation. In some species of the Polypodiaceae (sensu Wettstein), antheridium differentiation in young gametophytes is controlled by the phytochrome system (SCHRAUDOLF, 1967 b). In these ferns, premature development of gametangia occurs under dark or far-red conditions whereas exposure to red or white light causes only vegetative growth. Also in the latter system, however, the incorporation of BUdR in the growth medium initiates differentiation of antheridia. Spores of Polyp odium crassifolium kept on 10-5 M BUdR show premature formation of gametangia under non-inductive light conditions (Fig. 4). While antheridiogens and gibberellins are able to induce germination of Anemia spores in darkness, a similar situation is brought about by none of the base analogues active in antheridium induction. Z. PJlanzenphysiol. Bd. 96. S. 67-75. 1980.
72
H.
SCHRAUDOLF
. 20um
Fig. 3: Induction of antheridia by 5 X 10-4 M BUdR in regenerates of plasmolyzed Anemia prothalli Acetocarmine.
,
20um
.
Fig. 4: Antheridium induction by BUdR (5 X 10- 5 M)
folium , cultured in continuous red light. Acetocarmine. Z. Pflanzenphysiol. Bd. 96. S. 67-75. 1980.
In
ProthaIIi of Polypodium crassi-
Induction of antheridium differentiation
73
Discussion The thymidine analogue BUdR blocks cell differentiation not only in isolated animal (RUTTER et al., 1973; LANGEN, 1975; WEINTRAUB, 1975) and plant tissues (MEINS, 1976; BEZDEK et al., 1977; VYSKOT et al., 1977) but also in intact plants (QUILLOT, 1971; HESS, 1966). However in some cases, induction of specific differentiation by BUdR has already been reported as in the case of neuroplastoma cells (SCHUBERT and JACOB, 1970; SIMATOV and SACHS, 1975). An accelerated rate of floral initiation under non inductive light conditions in Arabidopsis thaliana (HIRONO and REDEl, 1966; BROWN, 1970; REDEl, 1975) and premature rhizogenesis in mung bean cuttings (ANZAI et al., 1971) are the only induction effects reported· in higher plants. Although general interest has been evinced in the action of this inhibitor in both animal and plant tissues, the substitution of bormonal signal in the regulation of differentiation and development by BUdR has not been reported so far. The role of BUdR in cell differentiation has not been clearly established. Further, the complex effect of the analogue in antheridium differentiation of Anemia on one hand, and the disturbance of the antheridium pattern itself on the other, makes the interpretation of the process more cumbersome. Since light is not essential for the induction of antheridium by BUdR, an increased radiation-sensitivity of the analogue-substituted DNA can be excluded as a factor involved in antheridium formation. Nevertheless, the incorporation into specific positions of Anemia DNA is necessary to anticipate this effect. Furthermore, application of a broad spectrum of growth inhibitors including 2-aminopurine which, like BUdR, gives rise to base transitions during DNA replication and thereafter to specific deformation in antheridium pattern (SCHRAUDOLF, 1977 b), fails to induce male gametangia. The incorporation of BUdR into the total (Koop, 1973) and chloroplastic DNA (SONKA and SCHRAUDOLF, 1979) has been reported in the gametophyte of Anemia. The high efficiency of BCdR in this reaction is noteworthy. CULLEN and BICK (1978) observed an increased deoxycytidine deaminase activity in a BUdR-treated hamster cell line and discussed a cascade like effect caused by BCdR, whereby a low level of BCdU is converted into BUdR. This again results in BUdR substitution of DNA, enhancing deoxycytidine deaminase activity. Such a cascade can offer an explanation to the activity of BCdR in induction as well as in pattern deformation of the antheridia. However, the experiments that are currently being carried out in our laboratory indicate that the inhibition of ribonucleotide reductase-catalyzed reduction of cytidine diphosphate by BUdR triphosphate (MEUTH and GREEN, 1974; KAUFMAN and DAVIDSON, 1978) participates in the effects attributed to BUdR. At least deoxycytidine is able to reverse the BUdR effects on pattern deformation of antheridia as well as a reduction in growth rate of the pro thalli caused by this analogue.
z. Pflanzenphysiol. Ed. 96. S. 67-75. 1980.
74
H. SCHRAUDOLF
In Onoclea sensibilis, reduced light intensity to near darkness leads to a gradial decay of a block in antheridium formation which at least gives rise to spontaneous gametangium differentiation without the interaction of an additional chemical signal, if the correlated suspension of cell divisions is overcome by sucrose added to the medium (NAF et aI., 1974). On the other hand, in Anemia gametophytes, the reduced light intensity even up to darkness alone can never cause spontaneous antheridium formation. Since cell divisions continue, though at a reduced rate, in all the light conditions used in the present study, a congruity between the effects initiated by sucrose and those of BUdR is not probable. This view is further strengthened by the observation that plasmolysis or meristem excision alone are unable to cause spontaneous antheridium differentiation in Anemia (SCHRAUDOLF, 1966 b), while in the case of the gametophytes of Onoclea these treatments are sufficient to induce male gametangia (NAF, 1961, 1962). Thus Anemia needs the presence of antheridiogen for this induction, a factor for which gibberellins or BUdR can substitute. As a consequence of our fragmentary knowledge of the molecular events which finally lead to the differentiation of antheridia, all statements on the possible effects of base-analogues must remain highly speculative. We thus have started to analyze the in vitro-effects of halogenated uri dine nucleosideson various enzymes involved in nucleotide-turnover. Acknowledgements V.
My thanks are due to Mrs. GUHA and Mr. Russ for skilful technical assistence and to Dr. J. PHILIP, Calicut, for checking the English version of the manuscript.
References ANZAI, T., H. SHIBAOKA, and M. SIMO-KORIYAMA: Plant Cell Physiol. 12,695 (1971). BEZDEK, M., B. VYSKOT, 1. TKADLECEK, and Z. KARPFEL: Biochem. Physiol. Pflanz. 171, 537
(1977).
BROWN, J. A. M.: The role of competitive analogues of uri dine in the induction of floral morphogenesis. In: BERNIER, G. (Ed.): Cellular and molecular aspects of floral induction, 117-138. Longman, London, 1970. CULLEN, B. R. and M. D. BICK: Biochim. Biophys. Acta 517, 158 (1978). Dopp, W.: Ber. Deut. Botan. Ges. 72, 11 (1959). ENDO, M., K. NAKANISHI, U. NAF, W. McKEON, and R. WALKER: Physiol. Plant. 26, 183
(1972). GUILLOT, A.: Planta 97, 269 (1971). HESS, D.: Z. Pflanzenphysiol. 54, 356 (1966). HIRaNO, Y. and G. P. REDEl, Planta 71, 107 (1966). KAUFMAN, E. R. and R. 1. DAVIDSON: Somatic Cell Ge:Jet. 4, 587 (1978). Koop, H. U.: Protoplasma 77, 343 (1973). LANGEN, P.: Antimetabolites of Nucleic Acid Metabolism, 54-56 and 263-267. Gordon and Breachi, New York, London, Paris 1975. MEINS, F. jr.: Planta 129, 239 (1976).
Z. PJlanzenphysiol. Bd. 96. S. 67-75. 1980.
Induction of antheridium differentiation
75
MEUTH, M. and H. GREEN: Cell 2, 109 (1974). NAF, U.: Nature 184, 798 (1958). - Nature 189, 900 (1961). - Ann. Rev. Plant Physiol. 13, 507 (1962). NAF, U.,]. SULLIVAN, and M. CUMMINS: Development. BioI. 40, 355 (1974). NAKANISHI, K., M. ENDO, and U. NAF:]. Am. Chern. Soc. 93, 5579 (1971). REDEl, G. P.: Genetic Mechanisms in Differentiation and Development. In: L. LEDOUX (Ed.): Genetic Manipulations with Plant Material, 183-209. Plenum Press, New York, London, 1975. RUTTER, W.]., R. L. PICTET, and P. W. MOURS: Ann. Rev. Biochem. 42, 601 (1973). SCHRAUDOLF, H.: BioI. Zentr. 81, 731 (1962). - Planta 68, 335 (1966 a). - BioI. Zentr. 85, 349 (1966 b). - Planta 74, 123 (1967 a). - Planta 76, 37 (1967b). - Z. Pflanzenphysiol. 66, 189 (1972). - Experientia 33, 1161 (1977 a). - Z. Pflanzenphysiol. 84, 49 (1977 b). SCHRAUDOLF, H. and]. SONKA: Europ.]. Cell BioI. 19, 135 (1979). SCHUBERT, D. and F. JACOB: Proc. Nat!. Acad. Sci. U.S. 67, 247 (1970). SIMANTOV, R. and L. SACHS: Dev. BioI. 45, 382 (1975). SONKA, ]. and H. SCHRAUDOLF: Z. Naturforsch. 34 c, 449 (1979). VYSKOT, G., Z. KARPFEL, and M. BEZDEK: Planta 137, 247 (1977). WEINTRAUB, H.: The Organization of Red Cell Differentiation. In: ]. REINERT and H. HOLTZER (Ed.): Cell Cycle and Cell Differentiation, 27-42. Springer-Verlag, Berlin, Heidelberg, New York, 1975.
H. SCHRAUDOLF, Universitat Ulm, Abteilung Allgemeine Botanik (BioI. II), Oberer Eselsberg, D-7900 Ulm.
Z. Pflanzenphysiol. Bd. 96. S. 67-75. 1980.
Induction of Antheridium Differentiation in the Fern Anemia phyllitidis L. Sw. by Halogenated Base Analogues H. SCHRAUDOLF With 4 figures Received June 12, 1979 . Accepted August 1, 1979
Summary The halogenated deoxynucleosides 5-bromo-2-deoxyuridine, 5-iodo-2-deoxyuridine and 5bromo-2-deoxycytidine substitute, though with less efficiency, for antheridiogens or gibberellins in the antheridium induction in gametophytes of Anemia phyllitidis. This effect is enhanced by cultivation of the prothalli under low light intensities. In contrast to antheridiogens and gibberellins, these halogenated deoxynucleosides are unable to cause dark-germination of Anemia spores. 5-Bromouracil and 5-bromouridine, but also 2-aminopurine, proved to be inactive regarding both antheridium induction and dark-germination.
Key words: Fern, gametophyte, antheridium, differentiation, bromodeoxyuridine, bromodeoxycytidine.
Introduction Differentiation of the antheridium in gametophytes of Anemia phyllitidis is induced by the diterpenoid pheromons antheridiogen AAnl and AAn2 (NAF, 1959; NAKANISHI et aI., 1971; ENDO et aI., 1972; SCHRAUDOLF, 1972) while gibberellins are able to substitute for these native compounds (SCHRAUDOLF, 1962, 1966 a; VOELLER, 1964). Though considerable attempts have been made to unravel the molecular basis of antheridium induction, conclusive data on the biochemical mechanisms underlying the process are however lacking. Since hormonal control of cell differentiation is not blocked by inhibitors of protein and nucleic acid synthesis, 5-bromo-deoxyuridine (BUdR) has a striking influence on the manifestation of antheridium pattern (SCHRAUDOLF, 1967 a, 1977 a), or chloroplast structure (SCHRAUDOLF and SONKA, 1979). Sporadic formation of antheridia could be observed during the studies on analogue treated Anemia prothalli. The substitution of antheridiogen by a base analogue can give rise to new points in our understanding of the causal events of hormonal regulation in antheridium formation especially because differentiation of male Z. Pflanzenphysiol. Bd. 96. S. 67-75. 1980.
68
H. SCHRAUDOLF
gametangia was also observed in prothalli of Polyp odium crassifolium treated with BUdR under non inductive light conditions. The present study has been undertaken mainly to examine and quantify the earlier observations so as to improve the experimental parameters in antheridium induction by base analogues. Material and Methods Spores of Anemia phyllitidis L. Sw. were harvested from plants grown in the green house of the University of Ulm. The standard growth conditions and culture media were published elsewhere (SCHRAUDOLF, 1966 a). Red light obtained from red fluorescent tubes (colour 15; Phillips-TL) combined with two layers of plexiglass no. 501 (ROHM and HAAS) gave an irradiance at the level of the spores of about 1.0 X 10-4 joule cm-2. Low intensity irradiation rates of 1.1 X 10-4 joule cm-2 have been obtained below 2.25 0/0 grey plexiglass. (ROHM and HAAS) in white light field (Phillips TL G7/32 Warmton). For the quantification of antheridium differentiation at least 200 prothallia stained with aceto carmine have been evaluated microscopically.
Results
When spores of Anemia are germinated and grown in the presence of high concentrations of BUdR (2 X 10-3 M), the developing gametophytes are only sporadically induced to form antheridia. The simultaneous reduction of light intensity, however, remarkably increases the effect of this base-analogue. Transfer of spores previously kept in red light for 48 h into either continuous dark (D) or white light of low intensity (WWL) causes the differentiation of antheridia after only a few vegetative cell divisions (Tab. 1). The same effect is observed with samples kept under red light throughout (R). Under the same conditions, 5-bromouridine (BrU) Table 1: Induction of antheridia in prothalli of Anemia phyllitidis grown and afterwords cultured in red light (R) in darkness (D) or white light of low intensity (WWL).
Ofo antheridia-bearing prothalli R Control
WWL
D
0
0
BUdR 2 X 10-3 M 10-3 M 5X10-4 M
50,0 32.5 25.4
14.8 14.8 13.0
BrU 2 X 10-3 M
0
0
0
BU 2X10- 3 M
0
0
0
Z. Pjlanzenphysiol. Bd. 96. S. 67-75. 1980.
0 8.0 4.2 1.0
Induction of antheridium differentiation
69
and 5-bromouracil (BU) are completely inactive, as well as 2-aminopurine, a base-analogue that causes like BUdR growth inhibition and severe aberrations in the antheridium-patterns of hormon-treated prothalli (SCHRAUDOLF, 1977 a). On the other hand, 5-bromodeoxycytidine (BCdR) and with lower activity also 5-iododeoxyuridine (IUdR) are able to induce premature antheridium differentiation (Tab. 2). In the first instance, it can be postulated that only those base-analogues which are capable of causing specific base-transitions in the DNA can serve as active substituents for the native antheridiogen. Table 2: Effect of different halogenated deoxynucleosides on antheridium differentiation in Anemia phyllitidis. 0/0
antheridia-bearing prothalli
R Control
WWL
D
0
0
0
BUdR 5 X 10-4 10-4 M 5 X 10-5 M 1O-5 M
53.4 28.5 21.0 16.7
27.5 18.5 19.7 15.2
10.5 1.2 0 0
BCdR 5 X 10-4 M 10-4 M 5 X 10-5 M 10-5 M
6.4 29.5 41.2 40.0
9.3 13.6 19.4 11.3
6.8 5.7 5.4 4.3
IUdR 5 X 10-4 M 10-4 M 5 X 10-:; M 10-5 M
11.5 9.8 3.4 0
5.1 1.2 0 0
1.0 1.2 1.0 0
Difficulties in the quantification of the inductive effects by the above chemicals arise from the simultaneous disturbance of the normal antheridium patterns (SCHRAUDOLF, 1967 a, 1977 a). The formation of teratological structures which frequently are no longer identifiable as sexual organs (Fig. 1) certainly represent one of the important reasons for the decreasing percentage of antheridia-bearing prothalli counted in samples which had been treated with high BCdR concentrations (Tab. 2). Under those conditions also two-dimensionally growing prothalli can be observed occasionally even after continuous exposure of the samples to red light (Fig. 2). Ethyl acetate extracts from acidified growth media of prothalli which already had differentiated antheridia after BUdR-treatment proved to be inactive in bioassays. The possibility of premature synthesis of antheridiogens as a prerequisite of the observed effects of the base-analogues can thus be excluded. This view is confirmed
z. Pjlanzenphysiol. Bd. 96. S. 67-75. 1980.
70
H.
SCHRAUDOLF
Fig. 1: Beginning of pattern simplification in an antheridium (green antheridium [Dopp, 1959]) induced by 10-4 M BCdR in continuous red light. Acetocarmine.
Fig. 2: 2-dimensional growth of Anemia prothalli in continuous red light induced by BCdR (5 X 10-4 M). Acetocarmine. Z. Pflanzenphysiol. Bd. 96. S. 67-75. 1980.
Induction of antheridium differentiation
71
by the observation that cyclic-Y,S'-AMP which sensibilizes prothalli against the natural antheridiogen (SCHRAUDOLF, 1977 b) had even an inhibitory effect in the BUdR-induced antheridio-genesis (Tab. 3). Table 3: Mutual effects of BUdR and cycl.-3,S-AMP on antheridium induction of Anemia phyllitidis.
Ofo antheridia-bearing prothalli
R Control
WWL
0
0
0
0
0
0
20.0 15.0 1.0
17.0 13.8 0
14.1 1.0
11.8
c-3',S-AMP
(S X 10-4 M)
BUdR
10-3 M 10-4 M 10-5 M
c-3',S-AMP (5 X 10-4 M)
2.0 0.8 0
+
BUdR
10-3 M 10-4 M 10-5 M
D
o
6.9
o
o
o o
With increasing age of the prothalli, specific areas of the gametophytes lose their sensitivity towards antheridiogens and also towards gibberellins, which can substitute for this native hormone. Short-time plasmolysis, however, can restore the sensibility of these cells (SCHRAUDOLF, 1966 b). Regenerated prothalli obtained from plasmolyzed cells redifferentiate sexual organs after hormone application. Identical effects can be observed with BUdR as the triggering signal (Fig. 3). These results indicate a far-reaching conformity of the various processes involved in the hormonal as well as in the BUdR-caused effects on gametangium differentiation. In some species of the Polypodiaceae (sensu Wettstein), antheridium differentiation in young gametophytes is controlled by the phytochrome system (SCHRAUDOLF, 1967 b). In these ferns, premature development of gametangia occurs under dark or far-red conditions whereas exposure to red or white light causes only vegetative growth. Also in the latter system, however, the incorporation of BUdR in the growth medium initiates differentiation of antheridia. Spores of Polyp odium crassifolium kept on 10-5 M BUdR show premature formation of gametangia under non-inductive light conditions (Fig. 4). While antheridiogens and gibberellins are able to induce germination of Anemia spores in darkness, a similar situation is brought about by none of the base analogues active in antheridium induction. Z. PJlanzenphysiol. Bd. 96. S. 67-75. 1980.
72
H.
SCHRAUDOLF
. 20um
Fig. 3: Induction of antheridia by 5 X 10-4 M BUdR in regenerates of plasmolyzed Anemia prothalli Acetocarmine.
,
20um
.
Fig. 4: Antheridium induction by BUdR (5 X 10- 5 M)
folium , cultured in continuous red light. Acetocarmine. Z. Pflanzenphysiol. Bd. 96. S. 67-75. 1980.
In
ProthaIIi of Polypodium crassi-
Induction of antheridium differentiation
73
Discussion The thymidine analogue BUdR blocks cell differentiation not only in isolated animal (RUTTER et al., 1973; LANGEN, 1975; WEINTRAUB, 1975) and plant tissues (MEINS, 1976; BEZDEK et al., 1977; VYSKOT et al., 1977) but also in intact plants (QUILLOT, 1971; HESS, 1966). However in some cases, induction of specific differentiation by BUdR has already been reported as in the case of neuroplastoma cells (SCHUBERT and JACOB, 1970; SIMATOV and SACHS, 1975). An accelerated rate of floral initiation under non inductive light conditions in Arabidopsis thaliana (HIRONO and REDEl, 1966; BROWN, 1970; REDEl, 1975) and premature rhizogenesis in mung bean cuttings (ANZAI et al., 1971) are the only induction effects reported· in higher plants. Although general interest has been evinced in the action of this inhibitor in both animal and plant tissues, the substitution of bormonal signal in the regulation of differentiation and development by BUdR has not been reported so far. The role of BUdR in cell differentiation has not been clearly established. Further, the complex effect of the analogue in antheridium differentiation of Anemia on one hand, and the disturbance of the antheridium pattern itself on the other, makes the interpretation of the process more cumbersome. Since light is not essential for the induction of antheridium by BUdR, an increased radiation-sensitivity of the analogue-substituted DNA can be excluded as a factor involved in antheridium formation. Nevertheless, the incorporation into specific positions of Anemia DNA is necessary to anticipate this effect. Furthermore, application of a broad spectrum of growth inhibitors including 2-aminopurine which, like BUdR, gives rise to base transitions during DNA replication and thereafter to specific deformation in antheridium pattern (SCHRAUDOLF, 1977 b), fails to induce male gametangia. The incorporation of BUdR into the total (Koop, 1973) and chloroplastic DNA (SONKA and SCHRAUDOLF, 1979) has been reported in the gametophyte of Anemia. The high efficiency of BCdR in this reaction is noteworthy. CULLEN and BICK (1978) observed an increased deoxycytidine deaminase activity in a BUdR-treated hamster cell line and discussed a cascade like effect caused by BCdR, whereby a low level of BCdU is converted into BUdR. This again results in BUdR substitution of DNA, enhancing deoxycytidine deaminase activity. Such a cascade can offer an explanation to the activity of BCdR in induction as well as in pattern deformation of the antheridia. However, the experiments that are currently being carried out in our laboratory indicate that the inhibition of ribonucleotide reductase-catalyzed reduction of cytidine diphosphate by BUdR triphosphate (MEUTH and GREEN, 1974; KAUFMAN and DAVIDSON, 1978) participates in the effects attributed to BUdR. At least deoxycytidine is able to reverse the BUdR effects on pattern deformation of antheridia as well as a reduction in growth rate of the pro thalli caused by this analogue.
z. Pflanzenphysiol. Ed. 96. S. 67-75. 1980.
74
H. SCHRAUDOLF
In Onoclea sensibilis, reduced light intensity to near darkness leads to a gradial decay of a block in antheridium formation which at least gives rise to spontaneous gametangium differentiation without the interaction of an additional chemical signal, if the correlated suspension of cell divisions is overcome by sucrose added to the medium (NAF et aI., 1974). On the other hand, in Anemia gametophytes, the reduced light intensity even up to darkness alone can never cause spontaneous antheridium formation. Since cell divisions continue, though at a reduced rate, in all the light conditions used in the present study, a congruity between the effects initiated by sucrose and those of BUdR is not probable. This view is further strengthened by the observation that plasmolysis or meristem excision alone are unable to cause spontaneous antheridium differentiation in Anemia (SCHRAUDOLF, 1966 b), while in the case of the gametophytes of Onoclea these treatments are sufficient to induce male gametangia (NAF, 1961, 1962). Thus Anemia needs the presence of antheridiogen for this induction, a factor for which gibberellins or BUdR can substitute. As a consequence of our fragmentary knowledge of the molecular events which finally lead to the differentiation of antheridia, all statements on the possible effects of base-analogues must remain highly speculative. We thus have started to analyze the in vitro-effects of halogenated uri dine nucleosideson various enzymes involved in nucleotide-turnover. Acknowledgements V.
My thanks are due to Mrs. GUHA and Mr. Russ for skilful technical assistence and to Dr. J. PHILIP, Calicut, for checking the English version of the manuscript.
References ANZAI, T., H. SHIBAOKA, and M. SIMO-KORIYAMA: Plant Cell Physiol. 12,695 (1971). BEZDEK, M., B. VYSKOT, 1. TKADLECEK, and Z. KARPFEL: Biochem. Physiol. Pflanz. 171, 537
(1977).
BROWN, J. A. M.: The role of competitive analogues of uri dine in the induction of floral morphogenesis. In: BERNIER, G. (Ed.): Cellular and molecular aspects of floral induction, 117-138. Longman, London, 1970. CULLEN, B. R. and M. D. BICK: Biochim. Biophys. Acta 517, 158 (1978). Dopp, W.: Ber. Deut. Botan. Ges. 72, 11 (1959). ENDO, M., K. NAKANISHI, U. NAF, W. McKEON, and R. WALKER: Physiol. Plant. 26, 183
(1972). GUILLOT, A.: Planta 97, 269 (1971). HESS, D.: Z. Pflanzenphysiol. 54, 356 (1966). HIRaNO, Y. and G. P. REDEl, Planta 71, 107 (1966). KAUFMAN, E. R. and R. 1. DAVIDSON: Somatic Cell Ge:Jet. 4, 587 (1978). Koop, H. U.: Protoplasma 77, 343 (1973). LANGEN, P.: Antimetabolites of Nucleic Acid Metabolism, 54-56 and 263-267. Gordon and Breachi, New York, London, Paris 1975. MEINS, F. jr.: Planta 129, 239 (1976).
Z. PJlanzenphysiol. Bd. 96. S. 67-75. 1980.
Induction of antheridium differentiation
75
MEUTH, M. and H. GREEN: Cell 2, 109 (1974). NAF, U.: Nature 184, 798 (1958). - Nature 189, 900 (1961). - Ann. Rev. Plant Physiol. 13, 507 (1962). NAF, U.,]. SULLIVAN, and M. CUMMINS: Development. BioI. 40, 355 (1974). NAKANISHI, K., M. ENDO, and U. NAF:]. Am. Chern. Soc. 93, 5579 (1971). REDEl, G. P.: Genetic Mechanisms in Differentiation and Development. In: L. LEDOUX (Ed.): Genetic Manipulations with Plant Material, 183-209. Plenum Press, New York, London, 1975. RUTTER, W.]., R. L. PICTET, and P. W. MOURS: Ann. Rev. Biochem. 42, 601 (1973). SCHRAUDOLF, H.: BioI. Zentr. 81, 731 (1962). - Planta 68, 335 (1966 a). - BioI. Zentr. 85, 349 (1966 b). - Planta 74, 123 (1967 a). - Planta 76, 37 (1967b). - Z. Pflanzenphysiol. 66, 189 (1972). - Experientia 33, 1161 (1977 a). - Z. Pflanzenphysiol. 84, 49 (1977 b). SCHRAUDOLF, H. and]. SONKA: Europ.]. Cell BioI. 19, 135 (1979). SCHUBERT, D. and F. JACOB: Proc. Nat!. Acad. Sci. U.S. 67, 247 (1970). SIMANTOV, R. and L. SACHS: Dev. BioI. 45, 382 (1975). SONKA, ]. and H. SCHRAUDOLF: Z. Naturforsch. 34 c, 449 (1979). VYSKOT, G., Z. KARPFEL, and M. BEZDEK: Planta 137, 247 (1977). WEINTRAUB, H.: The Organization of Red Cell Differentiation. In: ]. REINERT and H. HOLTZER (Ed.): Cell Cycle and Cell Differentiation, 27-42. Springer-Verlag, Berlin, Heidelberg, New York, 1975.
H. SCHRAUDOLF, Universitat Ulm, Abteilung Allgemeine Botanik (BioI. II), Oberer Eselsberg, D-7900 Ulm.
Z. Pflanzenphysiol. Bd. 96. S. 67-75. 1980.