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Novel Physiological Properties of Two Cytokinin Antagonists
J
• JOURNAL OF • PLANT PHYSIOLOGY
Plant Physiol. Vol. 156. pp. 623-627 (2000)
http://www.urbanfischer.de/journals/jpp
© 2000 URBAN &FISCHER Verlag
Novel Physiological Properties of Two Cytokinin Antagonists M. Somleva h
,
V. Kapchina-Toteva2, V. Alexieva 1, I. Sergiev 1 and E. Karanov 1
1
Institute of Plant Physiology, Bulgarian Academy of Sciences, Acad. G. Bonchev Str., BI. 21, 1113 Sofia, Bulgaria
2
Faculty of Biology, University of Sofia, 8 D. Tzankov Boul., 1421 Sofia, Bulgaria
Received August 10, 1999 . Accepted November 15, 1999
Summary
The promotive effect of two cytokinin antagonists 2-chloro-4-cyclobutyl-amino-6-ethylamino-l,3,5triazine and N-(4"pyridyl)-O-(4-chlorophenyl) carbamate on somatic embryos production directly or through callus in leaf explants of the grass Dactylis glomerata L. (orchardgrass) was established. The data for the endogenous contents of hydrogen peroxide and lipid peroxidation products during the earliest stages of somatic embryogenesis suggest that anticytokinins induce some alterations in the production of active oxygen species. Both compounds activated the defence enzymes 5 days after culture initiation. The triazine derivative enhanced catalase activity in more differentiated leaf segments while the phenyl ureatype anticytokinin had the same effect on peroxidase activity. Additional activation of catalase and slight reduction of peroxidase activity were determined in lO-day-old cultures. During induction of somatic embryogenesis in Dactylis leaf explants the main difference in the physiological action of the cytokinin antagonists tested was related to lAA-oxidase activity. A possible role of the anti cytokinin-induced changes in cell oxidative status on the explant embryogenic response is discussed.
Key words: Dactylis glomerata L., anticytokinins, catalase, hydrogen peroxide, malondialdehyde, fAA-oxidase activity, peroxidase, somatic embryogenesis. Abbreviatiom: ACK 1 = 2-chloro-4-cyclobutyl-amino-6-ethylamino-l,3,5-triazine; ACK2 dyl)-O-(4-chlorophenyl) carbamate; MDA malondialdehyde.
=
Introduction
The diversity of the processes in plant growth and development that can be affected by cytokinins has been well documented. Processes in which cytokinins are involved include cell division, branching of shoots, leaf senescence, chloroplast development and nutrient metabolism. On the other hand, the control role of cytokinins in a broad range of oxidative processes has also been reported (Musgrave, 1994; Gidrol et al., 1994; Deikman and Hammer, 1995). These roles for cytokinins are derived from numerous studies using exogenous application of these phytohormones and by the correlation of their endogenous levels with various developmental processes * Correspondence.
= N-(4-pyri-
(Binns, 1994; Schmulling et al., 1997). However, little is known about the mode of action of cytokinins. Application of cytokinin antagonists provides a new approach for investigations in this field. These compounds competitively inhibit cytokinin action in typical physiological reactions. Seven structural classes of cytokinin antagonists (anticytokinins) have been reported (Sergiev, 1999). Introduction of plant cells and tissues in in vitro cultures is associated with complex metabolic changes including alterations in the oxygen balance. Active oxygen species may affect the morphogenic response of the explants (De Marco and Roubelakis-Angelakis, 1996). Several tissue culture experiments upheld the concept that organization occurs in a more oxidizing environment than is needed for cell proliferation only. 0176-1617/00/156/623 $ 12.00/0
624
M. Somleva, V. Kapchina-Toteva, V. Alexieva, I. Sergiev and E. Karanov
Somatic embryogenesis systems developed for Dactylis glomerata L. (orchardgrass) are among the most advanced currently existing for the Poaceae (Conger et al., 1983). In vitro cultures of Dactylis leaf segments are a suitable system for comparative studies on somatic embryo formation directly from mesophyll cells and through callus. The mechanism{s) by which cytokinins regulate this process is not understood. Previous studies on Dactylis embryogenic cultures showed that exogenous cytokinins suppressed somatic embryogenesis in leaf explants (Wenck et al., 1988) while anticytokinins applied during the whole culture period stimulated somatic embryo formation (Somleva et al., 1995). The cytokinin antagonists 2-chloro-4-cyclobutyl-amino-6-ethylamino-l, 3, 5-triazine and N-{4-pyridyl)-O-{4-chlorophenyl) carbamate, structural analogues of purine and phenyl urea-type cytokinins, respectively, caused a significant reduction of the endogenous cytokinin levels during the earliest stages of somatic embryogenesis, which resulted in promotion of the explant embryogenic capacity (Somleva et al., 1999 b). In order to evaluate some novel physiological properties of these cytokinin antagonists we investigated their effects on the endogenous contents of hydrogen peroxide and products of lipid peroxidation as well as catalase, peroxidase, and IAA-oxidase activities during induction of direct and indirect somatic embryogenesis in Dactylis leaf explants.
Materials and Methods
Plant material Developing leaves from Dactylis glomerata L. (orchardgrass), the highly embryogenic genorype Embryogen-P (Conger and Hanning, 1991), were used as an explant source. After surface sterilization leaf halves from the basal parts of the innermost two leaves were cut transversely into 6 segments (3 mm long) and plated in the same order onto SH medium (Schenk and Hildebrandt, 1972), solidified with 0.8 % agar. The medium was supplemented with 30 J.LmollL 3,6-dichloro-o-anisic acid (dicamba) for induction of callus and embryo formation. Culture conditions were a temperature of 25 'c, in the dark.
Evaluation ofanticytokinin effects on the early explant development Filter sterilized 2-chloro-4-cyclobutyl-amino-6-ethylamino-l, 3, 5triazine (ACK 1) and N-(4-pyridyl)-O-(4-chlorophenyl) carbamate (ACK 2) were added to the Rrecooled auxin-containing SH medium at final concentrations 10-7 mollL and 10- 5 mollL, respectively. These concentrations were the most effective for stimulation of somatic embryogenesis (Somleva et al., 1995). Our previous studies showed that 5 and 10 days were the shortest periods for induction of embryogenic callus production in the younger leaf parts (1-4 segments) and direct somatic embryogenesis in the more differentiated 5-6 segments, respectively (Somleva and Karanov, 1996). The explants cultured for 5 and 10 days were transferred onto SH medium without any plant growth regulators for further development of the induced embryos and plant regeneration. The explant embryogenic response was estimated as I} the number of the explants with somatic embryos as a percentage of the total number of the plated leaf segments and ii) the number of somatic embryos produced through callus from 1-4 segments 5 and 10 days after culture initiation and directly formed somatic embryos from 5-6 segments after a lO-daylong culture period. Callus dry weight was also determined. Ex-
plants cultured for 5 and 10 days as well as the initial (non-cultured) explants were sampled for biochemical analyses.
Hydrogen peroxide determination The endogenous level of H 20 2 was determined spectrophotometrically (Iv = 390 nm) after incubation of leaf extracts with 1 moll L Kl. Hydrogen peroxide content was calculated using a standard curve.
Estimation oflipid peroxidation The level of lipid peroxidation in leaf tissues was measuted in terms of the thiobarbituric acid reaction product, malondialdehyde (MDA), whose content was determined by the procedure described by Dhindsa et al. (1981). The concentration ofMDA was calculated using its extinction coefficient of (155 mmollLt l . cm -I.
Enzyme assays Catalase (EC 1.11.1.6.) activity was determined according to the method of Brennann and Frenkel (1977). Guaiacol peroxidase (EC 1.11.1.7) activity was measured spectrophotometrically by monitoring the formation of tetraguaiacol (E = 26.6 mmollL -I . cm -I) from guaiacol at A.7o by the method of Dias and Costa (1983) with slight modifications. lAA-oxidase activity was evaluated spectrophotometrically according to the modified technique of Bohnsack and Albert (1977). Residual lAA was determined against a standard curve. Enzyme activities are presented as follows: catalase - H 2 0 2 destroyed [mmol. g-I FW· min-I]; peroxidase - guaiacol oxidized [J.Lmol . g-I FW. min-I]; lAA-oxidase activity - lAA degraded [mg. g-I FW. h -I]. Total soluble protein content was determined by Bradford's method (1976) using bovine serum albumin as a standard. The data presented are from four experiments, in 5-8 replications each. Statistical analyses were performed using the F-test (a significant at p
Anticytokinin effects on in vitro development oforchardgrass leaf explants In general, the compounds tested did not influence considerably the efficiency of somatic embryogenesis during culture initiation. Reduction in the number of the explants producing somatic embryos through callus after a 1O-day-Iong culture period was determined (Fig. 1 A). However, a significant increase in somatic embryo production was observed. The effect of the triazine-type antagonist was greater than that of the carbamate derivate. The first compound promoted the direct embryo formation as well (Fig. 1 B). The presence of both cytokinin antagonists in the medium slightly stimulated callus growth after the first culture period (136 % and 122 % of the control for triazine- and phenylurea-type compounds, respectively) but strong inhibition was observed 10 days after culture initiation (ca. 50 % of the control value).
Changes in the levels ofhydrogen peroxide and products of lipid peroxidation Both compounds caused a significant decrease in hydrogen peroxide content in the explants after the first culture period
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mation. A significant increase in the enzyme activity was measured in the 5th and 6 th segments cultured in the presence of the carbamate compound (Fig. 3 B). Culture of Dactylis leaf explants on the anticytokinin-containing medium for 5 days resulted in a sharp enhancement of IAA-oxidase activity, being more significant in the presence of the carbamate anticytokinin (Fig. 3 C). This compound did not show different effects depending on the explant age after this culture period. IAA-oxidase activity was decreased in all of the segments 10 days after culture initiation in the presence of the triazinetype antagonist. After the same period the carbamate anticytokinin significantly reduced IAA-oxidase activity in the younger 1-4 segments.
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Discussion
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number of somatic embtyos formed indirectly (1-4 segments) and directly (5-6 segments) from leaf explants cultured in the presence of auxin for 30 days was 31 and 18 per segment, respectively. (a significantly different at p
while elevated endogenous concentrations of HzO z were detected in lO-day-Iong cultures. The effects of the carbamate anticytokinin both on the reduction and on the accumulation of hydrogen peroxide were greater than those of the triazine compound (Fig. 2). MDA content was insignificantly altered except for its reduction (ca. 70 % of the control value) in leaf explants cultured in the presence of the triazine-type compound.
It has been shown that the application of cytokinin antagonists to in vitro cultures of leaf explants from Dactylis glomerata L. stimulated mainly indirect somatic embryogenesis (Somleva et al., 1995). The data presented here show that the presence of cytokinin antagonists in the culture medium during the first 5 days of culture initiation results in promotion of embryogenic callus growth. Probably the activated cell division increased the number of explant cells capable of acquiring embryogenic competence. This suggestion is in agreement with the data for the effects of anticytokinins on the explant embryogenic response. Both compounds did not induce considerable changes in the efficiency of somatic embryogenesis in Dactylis leaf explanrs compared with the control cultures while a significant increase in the number of somatic embryos formed through callus was observed. A sharp decrease of HzO z content was detected in the explants cultured in the presence of anticytokinins for 5 days. Because the activities of HzO z metabolizing enzymes peroxi-
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Fig. 2: Changes in the endogenous H 20 Z content in Dactylis leaf explants during induction of somatic embryogenesis in the presence of anticytokinins. The initial explants contained 33.827Ilmol . g -1 FW and 25.867Ilmol· g-1 FW in 1-4 segments and 5-6 segments, respectively. (a - significantly different at p
626
M. Somleva, V. Kapchina-Toteva, V. Alexieva, I. Sergiev and E. Karanov
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Fig. 3: Anticytokinin effects on catalase (A), peroxidase (B), and lAA-oxidase activity (C). The enzyme activities in non-cultured explants were 3.879 and 4.057 units· g-l FW for catalase; 20.759 and 30.787 units· g-l FW for peroxidase; 6.431 and 6.823 units. g-l FW for IAA-oxidase activity in 1-4 segments and 5-6 segments, respectively. (a - significantly different at p
Changes in the enzyme activities determined 5 days after culture initiation also reflected a slight cytokinin action of the compounds tested. In cultured Zinnia cells the expression of peroxidase was inhibited by an anticytokinin treatment (Church et al., 1988). The compounds used in our system showed a similar action after the second culture period while increased catalase activity was detected at the same time . Probably the detoxification of H 2 0 2 is due rather to its degradation by catalase than the utilization by peroxidase. Our previous results demonstrated opposite changes in the activities of these defence enzymes during induction of somatic embryogenesis in the presence only of auxin (Somleva et al., 1999 a), which again confirms the influence of both cytokinin antagonists on processes and/or enzymes involved in the oxidative status of plant cells. Since the main difference in the physiological effects of the cytokinin antagonists tested is related to IAA-oxidase activity we could suggest that both compounds influence the explant embryogenic response also through modulation of the endogenous active auxin pools. This mode of action could be a possible explanation for the different effects of both compounds on direct somatic embryogenesis as well. An anticytokinin is a compound that is believed to bind to a cytokinin receptor competitively with cytokinins and thus inhibits their biological effects (Brault et al., 1997). Taken together, the data for callus growth as well as changes in H 20 2 content and defence enzyme activities after the first culture period indicate a slight cytokinin activity of the compounds applied. However, both anticytokinins reduce the endogenous cytokinin levels in 5-day-old cultures (Somleva et al., 1999 a, b), which probably promotes the expression of the explant embryogenic capacity during the subsequent culture. Most probably the changes in the cell oxidative status induced by cytokinin antagonists additionally favored this process. It has been shown that higher antioxidant levels were detrimental to completion of somatic embryogenesis in wild carrot cell suspensions (Earnsnaw and Johnson, 1987). We can assume that the antagonistic action of the compounds applied for 5 days after culture initiation results rather in blocking of cytokinin biosynthesis than in alterations of the physiological action of these phytohormones. Some changes in the biological effects of cytokinins during the earliest stages of somatic embryogenesis were established in Dactylis leaf explants cultured for 10 days in the presence of cytokinin antagonists. The results presented here suggest that these compounds act as cytokinin antagonists or agonists during the different stages of such a complex and multistage morphogenic process as plant somatic embryogenesis. Acknowledgements
dase and catalase were not significantly changed in these cultures a possible influence of the cytokinin antagonists on some cellular electron transfer reactions could be suggested. The results for the changes in MDA content in Dactylis explants during induction of somatic embryogenesis support the suggestion that anticytokinins might induce some alterations in the production of active oxygen species without provoking oxidative stress.
We are grateful compounds.
to
Pro£ H. Iwamura for kindly providing of the
References
Binns, A.: Cytokinin accumulation and action: biochemical, genetic and molecular approaches. Anu. Rev. Plant Physiol. Plant Mol. BioI. 45, 173-196 (1994).
Anticytokinins and Somatic Embryogenesis Induction in Dactylis Bohnsack, C. w., and L. S. Albert: Early effects of boron deficiency on indole acetic-acid oxidase in squash root tips. Plant Physiol. 59, 1047-1050 (1977). Bradford, M. M.: A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein-dye binding. Anal. Biochem. 72,248-254 (1976). Brault, M., R. Maldiney and E. Miginiac: Cytokinin-binding proteins. Physiol. Plant. 100,520-527 (1997). Brennann, T., and C. Frenkel: Involvement of hydrogen peroxide in the regulation of senescence in pear. Plant Physiol. 59, 411-416 (1977). Church, D. L., and A. W. Galston: 4-Coumarate: coenzyme A ligase and isoperoxidase expression in Zinnia mesophyll cells induced to differentiate into tracheary elements. Plant Physiol. 88, 679682 (1988). Conger, B. v., and G. E. Hanning: Registration of Embryogen-P orchardgrass germ plasm with high capaciry for somatic embryogenesis from in vitro culture. Crop Sci. 31, 855 (1991). Conger, B. v., G. E. Hanning, D. J. Gray and J. K. McDaniel: Direct embryogenesis from mesophyll cells of orchardgrass. Science 221,850-851 (1983). De Marco, A., and K. A. Roubelakis-Angelakis: The complexiry of enzymic control of hydrogen peroxide concentration may affect the regeneration potential of plant protoplasts. Plant Physiol. 110, 137-145 (1996). Deikman, J., and P. E. Hammer: Induction of anthocyanin accumulation by cytokinins in Arabidopsis thaliana. Plant Physiol. 108, 47-57 (1995). Dhindsa, R. S., P. Plumb-Dhindsa and T. A. Thorpe: Leaf senescence correlated with increased levels of membrane permeabiliry and lipid peroxidation, and decreased levels of superoxide dismutase and catalase. J. Exp. Bot. 32, 93-101 (1981). Dias, M., and M. Costa: Effect of low salt concentrations on nitrate reductase and peroxidase of sugarbeet leaves. J. Exp. Bot. 34, 537-543 (1983).
627
Earnsnaw, B. A., and M. A. Johnson: Control of wild carrot somatic embryo development by antioxidants. Plant Physiol. 85, 273276 (1987). Gidrol, X., W. S. Lin, N. Degousee, S. F. Yip and A. Kush: Accumulation of reactive oxygen species and oxidation of cytokinin in germinating soybean seeds. Eur. J. Biochem. 224, 21-28 (1994). Musgrave, M. E.: Cytokinins and oxidative processes. In: Mok, D. W. S. and M. C. Mok (eds.): Cytokinins: Chemistry, Activiry and Function, pp. 167-178. CRC Press, Boca Raton, Ann Arbor, London, Tokyo (1994). Schenk, R. U., and A. C. Hildebrandt: Medium and techniques for induction and growth of monocoryledonous and dicoryledonous plant cell cultures. Can. J. Bot. 50, 199-204 (1972). Schmulling, T., S. Schafer and G. Romanov: Cytokinins as regulators of gene expression. Physiol. Plant. 100,505-519 (1997). Sergiev, I.: Cytokinin antagonists: structure - activiry relationships and physiological properties. Ph D Thesis, Bulgarian Academy of Sciences, Sofia (1999). Somleva, M., V. Alexieva, I. Sergiev and E. Karanov: Alterations in the activities of some hydrogen peroxide scavenging enzymes during induction of somatic embryogenesis in leaf explants from Dactylis glomerata L. C. R. d'Acad. Bulg. Sci. 52 (7) (1999 a) (in press). Somleva, M., T. Genkov, Y. Ivanova and E. Karanov: Endogenous cytokinin levels during induction of somatic embryogenesis in Dactylis glomerata leaf explants in the presence of cytokinin antagonists. Plant Cell Tiss. Org. Cult. (1999 b) (in press). Somleva, M., V. Kapchina, V. Alexieva and E. Golovinsky: Anticytokinin effects on in vitro response of embryogenic and non-embryogenic genotypes of Dactylis glomerata L. Plant Growth Regul. 16, 109-112 (1995). Somleva, M., and E. Karanov: Induction of direct and indirect somatic embryogenesis in Dactylis glomerata leaf explants: cell cycle kinetic studies. Acta Bot. Neer. 45(4), 576 (1996). Wenck, A. R., B. V. Conger, R. N. Trigiano and C. E. Sams: Inhibition of somatic embryogenesis in orchardgrass by endogenous cytokinins. Plant Physiol. 88,990-992 (1988).
• JOURNAL OF • PLANT PHYSIOLOGY
Plant Physiol. Vol. 156. pp. 623-627 (2000)
http://www.urbanfischer.de/journals/jpp
© 2000 URBAN &FISCHER Verlag
Novel Physiological Properties of Two Cytokinin Antagonists M. Somleva h
,
V. Kapchina-Toteva2, V. Alexieva 1, I. Sergiev 1 and E. Karanov 1
1
Institute of Plant Physiology, Bulgarian Academy of Sciences, Acad. G. Bonchev Str., BI. 21, 1113 Sofia, Bulgaria
2
Faculty of Biology, University of Sofia, 8 D. Tzankov Boul., 1421 Sofia, Bulgaria
Received August 10, 1999 . Accepted November 15, 1999
Summary
The promotive effect of two cytokinin antagonists 2-chloro-4-cyclobutyl-amino-6-ethylamino-l,3,5triazine and N-(4"pyridyl)-O-(4-chlorophenyl) carbamate on somatic embryos production directly or through callus in leaf explants of the grass Dactylis glomerata L. (orchardgrass) was established. The data for the endogenous contents of hydrogen peroxide and lipid peroxidation products during the earliest stages of somatic embryogenesis suggest that anticytokinins induce some alterations in the production of active oxygen species. Both compounds activated the defence enzymes 5 days after culture initiation. The triazine derivative enhanced catalase activity in more differentiated leaf segments while the phenyl ureatype anticytokinin had the same effect on peroxidase activity. Additional activation of catalase and slight reduction of peroxidase activity were determined in lO-day-old cultures. During induction of somatic embryogenesis in Dactylis leaf explants the main difference in the physiological action of the cytokinin antagonists tested was related to lAA-oxidase activity. A possible role of the anti cytokinin-induced changes in cell oxidative status on the explant embryogenic response is discussed.
Key words: Dactylis glomerata L., anticytokinins, catalase, hydrogen peroxide, malondialdehyde, fAA-oxidase activity, peroxidase, somatic embryogenesis. Abbreviatiom: ACK 1 = 2-chloro-4-cyclobutyl-amino-6-ethylamino-l,3,5-triazine; ACK2 dyl)-O-(4-chlorophenyl) carbamate; MDA malondialdehyde.
=
Introduction
The diversity of the processes in plant growth and development that can be affected by cytokinins has been well documented. Processes in which cytokinins are involved include cell division, branching of shoots, leaf senescence, chloroplast development and nutrient metabolism. On the other hand, the control role of cytokinins in a broad range of oxidative processes has also been reported (Musgrave, 1994; Gidrol et al., 1994; Deikman and Hammer, 1995). These roles for cytokinins are derived from numerous studies using exogenous application of these phytohormones and by the correlation of their endogenous levels with various developmental processes * Correspondence.
= N-(4-pyri-
(Binns, 1994; Schmulling et al., 1997). However, little is known about the mode of action of cytokinins. Application of cytokinin antagonists provides a new approach for investigations in this field. These compounds competitively inhibit cytokinin action in typical physiological reactions. Seven structural classes of cytokinin antagonists (anticytokinins) have been reported (Sergiev, 1999). Introduction of plant cells and tissues in in vitro cultures is associated with complex metabolic changes including alterations in the oxygen balance. Active oxygen species may affect the morphogenic response of the explants (De Marco and Roubelakis-Angelakis, 1996). Several tissue culture experiments upheld the concept that organization occurs in a more oxidizing environment than is needed for cell proliferation only. 0176-1617/00/156/623 $ 12.00/0
624
M. Somleva, V. Kapchina-Toteva, V. Alexieva, I. Sergiev and E. Karanov
Somatic embryogenesis systems developed for Dactylis glomerata L. (orchardgrass) are among the most advanced currently existing for the Poaceae (Conger et al., 1983). In vitro cultures of Dactylis leaf segments are a suitable system for comparative studies on somatic embryo formation directly from mesophyll cells and through callus. The mechanism{s) by which cytokinins regulate this process is not understood. Previous studies on Dactylis embryogenic cultures showed that exogenous cytokinins suppressed somatic embryogenesis in leaf explants (Wenck et al., 1988) while anticytokinins applied during the whole culture period stimulated somatic embryo formation (Somleva et al., 1995). The cytokinin antagonists 2-chloro-4-cyclobutyl-amino-6-ethylamino-l, 3, 5-triazine and N-{4-pyridyl)-O-{4-chlorophenyl) carbamate, structural analogues of purine and phenyl urea-type cytokinins, respectively, caused a significant reduction of the endogenous cytokinin levels during the earliest stages of somatic embryogenesis, which resulted in promotion of the explant embryogenic capacity (Somleva et al., 1999 b). In order to evaluate some novel physiological properties of these cytokinin antagonists we investigated their effects on the endogenous contents of hydrogen peroxide and products of lipid peroxidation as well as catalase, peroxidase, and IAA-oxidase activities during induction of direct and indirect somatic embryogenesis in Dactylis leaf explants.
Materials and Methods
Plant material Developing leaves from Dactylis glomerata L. (orchardgrass), the highly embryogenic genorype Embryogen-P (Conger and Hanning, 1991), were used as an explant source. After surface sterilization leaf halves from the basal parts of the innermost two leaves were cut transversely into 6 segments (3 mm long) and plated in the same order onto SH medium (Schenk and Hildebrandt, 1972), solidified with 0.8 % agar. The medium was supplemented with 30 J.LmollL 3,6-dichloro-o-anisic acid (dicamba) for induction of callus and embryo formation. Culture conditions were a temperature of 25 'c, in the dark.
Evaluation ofanticytokinin effects on the early explant development Filter sterilized 2-chloro-4-cyclobutyl-amino-6-ethylamino-l, 3, 5triazine (ACK 1) and N-(4-pyridyl)-O-(4-chlorophenyl) carbamate (ACK 2) were added to the Rrecooled auxin-containing SH medium at final concentrations 10-7 mollL and 10- 5 mollL, respectively. These concentrations were the most effective for stimulation of somatic embryogenesis (Somleva et al., 1995). Our previous studies showed that 5 and 10 days were the shortest periods for induction of embryogenic callus production in the younger leaf parts (1-4 segments) and direct somatic embryogenesis in the more differentiated 5-6 segments, respectively (Somleva and Karanov, 1996). The explants cultured for 5 and 10 days were transferred onto SH medium without any plant growth regulators for further development of the induced embryos and plant regeneration. The explant embryogenic response was estimated as I} the number of the explants with somatic embryos as a percentage of the total number of the plated leaf segments and ii) the number of somatic embryos produced through callus from 1-4 segments 5 and 10 days after culture initiation and directly formed somatic embryos from 5-6 segments after a lO-daylong culture period. Callus dry weight was also determined. Ex-
plants cultured for 5 and 10 days as well as the initial (non-cultured) explants were sampled for biochemical analyses.
Hydrogen peroxide determination The endogenous level of H 20 2 was determined spectrophotometrically (Iv = 390 nm) after incubation of leaf extracts with 1 moll L Kl. Hydrogen peroxide content was calculated using a standard curve.
Estimation oflipid peroxidation The level of lipid peroxidation in leaf tissues was measuted in terms of the thiobarbituric acid reaction product, malondialdehyde (MDA), whose content was determined by the procedure described by Dhindsa et al. (1981). The concentration ofMDA was calculated using its extinction coefficient of (155 mmollLt l . cm -I.
Enzyme assays Catalase (EC 1.11.1.6.) activity was determined according to the method of Brennann and Frenkel (1977). Guaiacol peroxidase (EC 1.11.1.7) activity was measured spectrophotometrically by monitoring the formation of tetraguaiacol (E = 26.6 mmollL -I . cm -I) from guaiacol at A.7o by the method of Dias and Costa (1983) with slight modifications. lAA-oxidase activity was evaluated spectrophotometrically according to the modified technique of Bohnsack and Albert (1977). Residual lAA was determined against a standard curve. Enzyme activities are presented as follows: catalase - H 2 0 2 destroyed [mmol. g-I FW· min-I]; peroxidase - guaiacol oxidized [J.Lmol . g-I FW. min-I]; lAA-oxidase activity - lAA degraded [mg. g-I FW. h -I]. Total soluble protein content was determined by Bradford's method (1976) using bovine serum albumin as a standard. The data presented are from four experiments, in 5-8 replications each. Statistical analyses were performed using the F-test (a significant at p
Anticytokinin effects on in vitro development oforchardgrass leaf explants In general, the compounds tested did not influence considerably the efficiency of somatic embryogenesis during culture initiation. Reduction in the number of the explants producing somatic embryos through callus after a 1O-day-Iong culture period was determined (Fig. 1 A). However, a significant increase in somatic embryo production was observed. The effect of the triazine-type antagonist was greater than that of the carbamate derivate. The first compound promoted the direct embryo formation as well (Fig. 1 B). The presence of both cytokinin antagonists in the medium slightly stimulated callus growth after the first culture period (136 % and 122 % of the control for triazine- and phenylurea-type compounds, respectively) but strong inhibition was observed 10 days after culture initiation (ca. 50 % of the control value).
Changes in the levels ofhydrogen peroxide and products of lipid peroxidation Both compounds caused a significant decrease in hydrogen peroxide content in the explants after the first culture period
625
Anticytokinins and Somatic Embryogenesis Induction in Dactylis
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number of somatic embtyos formed indirectly (1-4 segments) and directly (5-6 segments) from leaf explants cultured in the presence of auxin for 30 days was 31 and 18 per segment, respectively. (a significantly different at p
while elevated endogenous concentrations of HzO z were detected in lO-day-Iong cultures. The effects of the carbamate anticytokinin both on the reduction and on the accumulation of hydrogen peroxide were greater than those of the triazine compound (Fig. 2). MDA content was insignificantly altered except for its reduction (ca. 70 % of the control value) in leaf explants cultured in the presence of the triazine-type compound.
It has been shown that the application of cytokinin antagonists to in vitro cultures of leaf explants from Dactylis glomerata L. stimulated mainly indirect somatic embryogenesis (Somleva et al., 1995). The data presented here show that the presence of cytokinin antagonists in the culture medium during the first 5 days of culture initiation results in promotion of embryogenic callus growth. Probably the activated cell division increased the number of explant cells capable of acquiring embryogenic competence. This suggestion is in agreement with the data for the effects of anticytokinins on the explant embryogenic response. Both compounds did not induce considerable changes in the efficiency of somatic embryogenesis in Dactylis leaf explanrs compared with the control cultures while a significant increase in the number of somatic embryos formed through callus was observed. A sharp decrease of HzO z content was detected in the explants cultured in the presence of anticytokinins for 5 days. Because the activities of HzO z metabolizing enzymes peroxi-
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Fig. 2: Changes in the endogenous H 20 Z content in Dactylis leaf explants during induction of somatic embryogenesis in the presence of anticytokinins. The initial explants contained 33.827Ilmol . g -1 FW and 25.867Ilmol· g-1 FW in 1-4 segments and 5-6 segments, respectively. (a - significantly different at p
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Fig. 3: Anticytokinin effects on catalase (A), peroxidase (B), and lAA-oxidase activity (C). The enzyme activities in non-cultured explants were 3.879 and 4.057 units· g-l FW for catalase; 20.759 and 30.787 units· g-l FW for peroxidase; 6.431 and 6.823 units. g-l FW for IAA-oxidase activity in 1-4 segments and 5-6 segments, respectively. (a - significantly different at p
Changes in the enzyme activities determined 5 days after culture initiation also reflected a slight cytokinin action of the compounds tested. In cultured Zinnia cells the expression of peroxidase was inhibited by an anticytokinin treatment (Church et al., 1988). The compounds used in our system showed a similar action after the second culture period while increased catalase activity was detected at the same time . Probably the detoxification of H 2 0 2 is due rather to its degradation by catalase than the utilization by peroxidase. Our previous results demonstrated opposite changes in the activities of these defence enzymes during induction of somatic embryogenesis in the presence only of auxin (Somleva et al., 1999 a), which again confirms the influence of both cytokinin antagonists on processes and/or enzymes involved in the oxidative status of plant cells. Since the main difference in the physiological effects of the cytokinin antagonists tested is related to IAA-oxidase activity we could suggest that both compounds influence the explant embryogenic response also through modulation of the endogenous active auxin pools. This mode of action could be a possible explanation for the different effects of both compounds on direct somatic embryogenesis as well. An anticytokinin is a compound that is believed to bind to a cytokinin receptor competitively with cytokinins and thus inhibits their biological effects (Brault et al., 1997). Taken together, the data for callus growth as well as changes in H 20 2 content and defence enzyme activities after the first culture period indicate a slight cytokinin activity of the compounds applied. However, both anticytokinins reduce the endogenous cytokinin levels in 5-day-old cultures (Somleva et al., 1999 a, b), which probably promotes the expression of the explant embryogenic capacity during the subsequent culture. Most probably the changes in the cell oxidative status induced by cytokinin antagonists additionally favored this process. It has been shown that higher antioxidant levels were detrimental to completion of somatic embryogenesis in wild carrot cell suspensions (Earnsnaw and Johnson, 1987). We can assume that the antagonistic action of the compounds applied for 5 days after culture initiation results rather in blocking of cytokinin biosynthesis than in alterations of the physiological action of these phytohormones. Some changes in the biological effects of cytokinins during the earliest stages of somatic embryogenesis were established in Dactylis leaf explants cultured for 10 days in the presence of cytokinin antagonists. The results presented here suggest that these compounds act as cytokinin antagonists or agonists during the different stages of such a complex and multistage morphogenic process as plant somatic embryogenesis. Acknowledgements
dase and catalase were not significantly changed in these cultures a possible influence of the cytokinin antagonists on some cellular electron transfer reactions could be suggested. The results for the changes in MDA content in Dactylis explants during induction of somatic embryogenesis support the suggestion that anticytokinins might induce some alterations in the production of active oxygen species without provoking oxidative stress.
We are grateful compounds.
to
Pro£ H. Iwamura for kindly providing of the
References
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