Chen et al.

Inhibition of NAA-induced Adventitious Roots in Mung Bean Cuttings by Kinetin, Zeatin, Ethidium Bromide and Other DNA Intercalators

Jun Chen, Francis H. Witham and Charles W. Heuser

Department of Horticulture, The Pennsylvania State University, University Park, PA 16801

Correspondence should be addressd to Francis H. Witham Ph.D.
Email: frank_witham@agcs.cas.psu.edu

Submitted for publication: September 1995

Keywords: Vigna radiata, adventitious root growth, auxins, ethidium bromide, intercalating chemicals, cytokinins, inhibitors

Chemicals and Abbreviations: cycloheximide (CH); Indole-3-acetic acid (IAA); 6-methylpurine (6- MP); 1-Naphthaleneacetic Acid (NAA); 6-furfuryl aminopurine (Kinetin); 6-(4-hydroxy-3-methyl- trans-2-butenyl-amino) purine (Zeatin); 4-[2-(3,5-dimethyl-2-oxocyclohexyl)-2-hydroxyethyl]-2,6- piperidinedione (cyloheximide); 3,8-diamino-5-ethyl-6-phenyl-phenanthridine bromide (ethidium bromide or EB); 3,6-bis[Dimethylamino] acridinium chloride hemi-[zinc chloride] (acridine orange); 5,11-Dimethyl-6H-pyrido[4,3-b] carbazole (ellipticine); 3,6-diaminoacridine (proflavine)

Title Page Abstract Introduction Materials and Methods Results
Discussion Conclusions Acknowledgements References Table of Contents

Figure 1 Figure 2 Figure 3 Figure 4 Figure 5
Figure 6 Figure 7 Figure 8 Figure 9 Table I

ABSTRACT

Adventitious root formation on hypocotyls of cuttings of mung bean, Vigna radiata [ L.] R. Wilcz. cv. Berken was stimulated by NAA at 10 to 500 然. Following a l ml pulse of NAA (10 -4 M), roots were observed on cuttings between 60 and 72 h with maximum root numbers at 96 h. Auxin-induced rooting was inhibited by kinetin, zeatin, 6- methylpurine, cyloheximide and the intercalating chemicals, ethidium bromide, acridine orange, ellipticine and proflavine at 10 to 100 然. Kinetics of the inhibition indicated that 6-methylpurine, ethidium bromide and zeatin act similarly while cycloheximide inhibition is somewhat different. Inhibition of the NAA-induced root initiation by either ethidium bromide or zeatin was reversed by increasing concentrations of the synthetic auxin. Studies with space-filling models show that both ethidium and NAA can intercalate via their accomodation between base pairs of model DNA and with hydrogen bonding of ethidium at each phosphate oxygen of opposing DNA strands.

INTRODUCTION

Auxins are known to promote adventitious root formation on hypocotyls of mung bean cuttings (1, 2). Adventitious roots, initiated in phloem parenchyma cells exhibited histologically visible nuclear and nucleoli enlargement at 12 h followed by cell division between 20 and 24 h after NAA treatment (1). Well developed root primordia and root emergence were observed by 48 h and 72 h, respectively. Further, 6-methylpurine (6-MP), a suspected inhibitor of DNA dependent RNA synthesis, suppressed the nuclear and nucleolar enlargement prior to cell division and subsequent root formation up to 12 h after NAA treatment (3).

Although the timing of the incorporation of labelled thymidine and uridine into the cells has been correlated only with the wounding response following cutting, the uptake of these ribosides may represent the action of endogenous auxins released upon wounding (4). The inhibition of adventitious rooting by actinomycin-D, puromycin and 6-MP indicated that the action of of NAA with respect to adventitious root

As a continuation of past studies on the rooting of mung bean cuttings, the results presented herein describe the inhibition of NAA-induced adventitious root formation by several chemicals, including those known to be DNA-intercalating agents. The two acridine derivatives, 3,6-bis[Dimethylamino] acridinium chloride hemi-[zinc chloride] (termed Acridine orange) and 3,6-diaminoacridine (proflavine) and the alkaloid, 5,11-Dimethyl-6H-pyrido[4,3-b] carbazole (ellipticine), based on their intercalating properties and similar chemistry (5), were chosen in this study as likely inhibitors of rooting. Further, the molecule, 3,8-diamino-5-ethyl-6-phenyl-phenanthridine bromide (ethidium bromide or EB), a DNA intercalating agent (6,7,8) and inhibitor of DNA synthesis (9, 10) is known to bind preferentially to double-stranded polynucleotides (11) albeit on one ocassion had been shown to bind to t RNA (12).

The exact nature of the interactive groups during binding of ethidium via intercalation with DNA is not completely known, although weak binding occurs at relatively high concentrations of EB in which one molecule may be bound per nucleotide (11, 13). Also, evidence based on competitive binding in darkness between ethidium and mono-azido photoaffinity probes (produced by derivatization with azide at the 3 and/or 8 position of ethidium) provided strong evidence that the binding site of ethidium to DNA is non-covalent (14).

The noncovalent binding of phytohormones and some growth regulators in vivo is generally believed to be a characteristic feature of their association with a receptor(s). In fact, studies with Corey, Pauling, Koltun (CPK) space-filling models indicate that phytohormones and some growth regulators can bind, according to realistic chemical parameters, noncovalently and via intercalation to DNA (15). Much of the past evidence indicates, at least circumstantially, that auxin-induced rooting requires DNA replication, transcription and cell division. An important consideration is whether NAA binds to DNA in vivo as a necessary step in gene expression or some other cellular component leading to the initiation and development of adventitious roots in mung bean cuttings.

One of the initial goals of our studies was to determine if the intercalating chemicals, especially EB, with their unique properties and noncovalent binding to DNA might operate as inhibitors of adventitious root formation in hypocotyls of mung bean. As inhibitors with a known action on DNA, they would be important tools in understanding the action of NAA and other auxins in gene-mediated adventious root formation and other such responses. Therefore, time course inhibition patterns of EB and zeatin were compared with those of 6-MP, a putative inhibitor of RNA synthesis, and cycloheximide (CH), a suspected inhibitor of protein synthesis. The kinetics of zeatin and EB inhibition was also compared to the former compounds, since little is known about the action of zeatin and EB as inhibitors of root growth.

The interaction of EB and zeatin with NAA presumably involving a hypothetical receptor as a function of the rooting response was superficially evaluated on the basis of inhibition reversal by increasing concentrations of NAA . In view of past work (15 - 17), it was of interest also to construct CPK space-filling models of EB and NAA and their respective DNA complexes to detemine any similar binding features or obvious noncovalent binding sites between dinucleotides of double-stranded DNA. The results show for the first time that several intercalating agents operate as adventitious root initiation inhibitors. These chemicals, especially EB, may be useful in future molecular approaches to an understanding of this important process.

MATERIALS AND METHODS

Plant Material . Vigna radiata (L.) R. Wilcz. cv. Berken, mung bean seeds were surface-sterilized in 10% Clorox (v/v) for 10 min and rinsed in distilled water. The seeds were soaked in aerated distilled water for approximately 24 h and then sown 1 cm deep in perlite contained in trays (29 x 18 x 5 cm). During germination and development, the seedlings were grown 7 days, maintained at 25 C and constant lighting of 35 痠ol.s-1.m-2 and watered with tap water ever other day and on alternate days with a salt solution consisting of Ca (NO3)2.4 H2O (85 mM) and H3BO3 (1.6 mM).

Cuttings and incubation. Uniform cuttings of 7- day seedlings were produced by removing a small portion of the hypocotyls and attached roots exactly 3 cm below the cotyledonary node. Each cutting therefore consisted of a 3-cm hypocotyl, epicotyl, two primary leaves and the unexpanded trifoliate bud. The cuttings were initially placed in distilled water. Then ten cuttings were selected at random and placed in a 19 x 65 mm shell vial containing 1 ml of test solution. The initial test solution was absorbed within 2 h followed by the addition of sufficient distilled water to bring the level of liquid to the cotyledonary nodes of the cuttings in each vial. Additional distilled water was added each 24 h to maintain that level for the entire experimental time. The vials were arranged at random in the growth room under the same temperature and light conditions described for seedling development. After 120 h incubation, unless otherwise indicated, the number of roots per cutting was recorded.

Statistical methods. The value of each point on the graphs in the results section is based on the mean of 30 cuttings, unless otherwise stated. Statistical analyses of the root numbers, regression analyses, and graph construction of all the data points were performed with a Macintosh II computer, equipped with the statistical software package, Stat View II, Abacus Concepts, Inc.

RESULTS

NAA and adventitious root formation . Adventitious root formation was stimulated by NAA between 10 and 500 然, showing the optimum root number at 500 然 followed by inhibition at 1000 然 NAA and higher concentrations (Fig. 1).

Time course of root formation. A progressive increase in adventitious root number with time was observed in NAA-treated cuttings (Fig. 2). Observable rooting was evident between 60 and 72 h while maximum root number was observed at 96 h (and later) incubation time. In subsequent experiments, cuttings were incubated for 120 h as by this time the roots were longer and easier to count. The average number of roots found on NAA-treated cuttings at 120 h was approximately 20 to 25 more than the water controls.

Inhibition of NAA-induced rooting. The NAA-induced rooting of mung bean cuttings was inhibited by the cytokinins, kinetin and zeatin, the putative transcription and translation inhibitors, 6-MP and CH, respectively and the DNA intercalaters: proflavine, ellipticine, acridine orange and EB (Table I). Inhibition was observed at a concentration of 1 然 for all the inhibitors with more pronounced inhibition at the higher concentrations of 10, 50 and 100 然 . Kinetin and zeatin, appeared to be as effective as EB as inhibitors of the NAA-induced rooting of mung bean cuttings (Table I).

Zeatin inhibited both IAA- and NAA-induced rooting at concentrations of 0.1 to 50 然 (Fig. 3). The relatively higher levels of IAA (500 然) to NAA (100 然) and approximately equivalent rooting responses illustrates the effectiveness of NAA over that of the less active IAA. Also, the step-wise inhibition by zeatin within a low concentration range indicates an interaction consistent with the hormonal action of this chemical. Similarly, the inhibition curve showing the effect of EB illustrates the concentration dependent action of this compound on NAA-induced adventitious rooting (Fig. 4).

Although not seen in the higher concentration ranges (Table I), 6-MP stimulated rooting at low concentrations alone or in the presence of NAA (Fig. 5). Progressive inhibition of rooting, however, at relatively higher concentrations of 6-MP between 5 to 20 然 6-MP was apparent (Fig. 5).

Kinetics of the effects of the different inhibitors on rooting. Seedlings, treated with NAA only, exhibited progressive increases in root numbers with time (Fig. 6). In contrast, seedlings treated with NAA followed by cycloheximide 24 h later, showed complete inhibition of rooting. The time course of the response observed for the other compounds also added 24 h after NAA showed some inhibition, but unlike that of CH, the chemicals did not stop some initial root formation (early slope increases) and appeared to be responsible for progressive (6-MP treated cuttings) or abrupt (EB, zeatin treated cuttings) inhibition over time (Fig. 6).

Reversal of EB and zeatin inhibition of Rooting by in increasing concentrations of NAA. The inhibition of rooting at a given amount of EB was progressively reversed as concentrations of NAA (10, 20, 50, and 100 然) were increased (Fig. 7). In the absence of inhibitor, NAA stimulated slightly higher root numbers than the inhibitor containing solutions. However, at At each level of EB, increased concentrations of NAA overcome resulting in increased root numbers. The antagonistic nature of EB to NAA was evaluated by increasing concentrations of NAA with set amounts of EB . Results of experiments showing the interaction of the two substances are shown (Fig. 7) where the reciprocal of the rooting response is plotted against the reciprocal of the NAA concentration alone and in the presence of several concentrations of the inhibitor, EB. The fact that the resultant family of straight lines developed by regression analysis about the appropriate points exhibit a common intercept at the ordinate, indicates competitive inhibition between NAA and EB. However, for various reasons discussed later, this data is by no means conclusive.

Similar Lineweaver-Burk (18) plot analyses were used to evaluate the interaction of zeatin and NAA (Fig. 8). With the exception of the highest concentration of zeatin (8然), the slopes of the lines shown for the lower concentrations of zeatin (1, 2, 4 然) exhibit separate intercepts on the y axis. Extrapolation of this family of lines indicates individual intercepts at negative values along the abscissa. These results therefore, suggest that the inhibition of rooting by zeatin is uncompetitive and, while not ruling out the interaction of zeatin with DNA, the phytohormone inhibition appears to be different than that of ethidium.

Studies with space-filling models. A hypothetical ethidium-DNA complex is best illustrated with CPK space-filling models in which ethidium (Fig. 9 A) is intercalated (inserted) between the base-paired double-stranded dinucleotide 5'-dTdA- 3' and complimentary sequence, 3'-dAdT-5 ' (Fig.9 B). Interestingly, the internuclear distance of the ethidium molecule between positions 3 and 8 (locations of the primary aryl amino groups of ethidium) approximates the distance between the phosphate oxygens of the two DNA strands (at given DNA conformations). Therefore, the ethidium was sandwiched between base pairs so that the planar aromatic surfaces were parallel to the surfaces of the base pairs on each side with each aryl amino group at positions 3 and 8 hydrogen bonded to the phosphate oxygen of the complimentary strands ' (Fig. 9 B). The positioning of the intercalated ethidium model between base pairs is similar to that previously suggested (6 - 8) except that the amino groups are not free within the complex, but hydrogen bonded to the phosphate oxygens ' (Fig. 9B).

The 5-ethyl and 6-phenyl substituents projected above the base pairs and the major groove impose no steric hindrance to the complex formation ' (Fig. 9 B), although they might seemingly influence uncoiling (19), DNA replication and ultimately RNA synthesis. While other possible relationships of the ligand to the Base-pair cavities are possible (ie. hydrogen bonding to the deoxyribose ring oxygens, etc.), we only present one illustration based on reasonable chemical and physical parameters.

A similar CPK model illustrating a hypothetical interaction of NAA and DNA shows that NAA exhibits one hydroxyl group ' (Fig. 9 C), which under the appropriate conditions, may bind to a phosphate oxygen between base pairs via hydrogen bonding ' (Fig. 9 D ). In this instance, NAA is bound between 5'-dTdA-3' and its complimentary strand.

DISCUSSION AND CONCLUSIONS

The dose-response curve of NAA-induced adventitious root formation, characterized typically by a concentration-dependent linear response progressing to an optimum with subsequent leveling off at several higher concentrations (Fig. 1), implicates NAA with a regulatory site associated with the rooting response. The general relationship of the rooting response to NAA concentration is typical of other dose-response curves for various plant growth regulators and phytohormones. Further, the time course of NAA -induced rooting (Fig. 2) is consistent with past observations relating to time of root initiation, development of root primordia, and the emergence of adventitious roots (1).

The inhibition of NAA-induced adventitious root formation of mung bean cuttings by the DNA intercalaters, acridine orange, proflavine, the alkaloid, ellipticine and EB (Table I) is the first account of adventitious root inhibition by these intercalating chemicals. Ethidium, along with the acridines and derivatives, is known to bind via intercalation with DNA (7, 12) . In fact, countless studies dealing with the isolation and purification of DNA and southern blot analyses show that ethidium effectively binds to DNA with the resulting DNA-ethidium complex (s) being detected as a highlighted fluorescent band (s) on the appropriate electrophoretic gel (20). Interestingly, kinetin and zeatin dramatically inhibited NAA-induced rooting of mung bean cuttings as effectively as the intercalating chemicals.

The separate inhibition curves illustrating the concentration-dependent action of zeatin (Fig. 3), EB ' (Fig. 4) and 6-MP ' (Fig. 5) show that inhibitor activity of each chemical simply is not due to a single threshold dose, but is proportionally related to concentration. Also, the stimulation of adventitious rooting by very low concentrations of 6-MP may reflect a direct promotive effect of the molecule per se or an active demethylated purine derivative (s) of 6-MP. Additionally, it appears that EB may promote adventitious rooting at low concentrations (Fig. 4) This oberservation in itself is worth further study.

The kinetics of inhibition showed that the cuttings incubated in NAA for the entire incubation period exhibited progressive increases in root numbers with time (Fig. 6). When incubated in NAA, followed 24 h after by an inhibitory level of 6-MP, there was initially slight inhibition (first 24 hours after 6-MP addition) which was followed by complete inhibition (abrupt slope change). These results indicate the presence of a preformed essential biochemical, possibly mRNA, at the time of 6-MP addition which is subsequently depleted with further synthesis inhibited by 6-MP. Similarly, zeatin and EB, exhibit the same pattern of inhibition as 6-MP. Cycloheximide, on the other hand, inhibited root number increases immediately after addition. This pattern is suggestive of direct suppression of protein synthesis and eventhough it has been reported that CH may inhibit other cellular processes (21), the type of inhibition relating to the kinetics indicates that the action of CH at least is different than that of EB, zeatin or 6-MP. At increasing concentrations, NAA reversed either the EB ' (Fig. 7) or zeatin ' (Fig. 8) inhibition of adventitious root development . This reversal of EB or zeatin inhibition by NAA (Fig. 7) indicates that each inhibitor may dissociate from a receptor, and in view of the EB intercalating activity, a receptor possibly at the level of DNA. The noncovalent nature of ethidium-DNA complexes (14) is a priori consistent with such interactions. In addition, past studies with CPK space-filling models show that Zeatin can interact by intercalation with DNA in a noncovalent manner (15) and eventhough zeatin appears to operate in an uncompetitive fashion to NAA ' (Fig. 8), this finding is consistent with the general idea that zeatin inhibits elongation directly rather than root initiation.

We recognize that the application of Lineweaver-Burk kinetics to a biological response such as rooting, which probably involves several or more catenary events, is not sufficient to conclude whether a given cellular site is operative in the NAA-induced rooting response and ethidium inhibitory action ' (Fig. 7). In fact, the analysis of the response at more than three or four separate auxin levels at a given concentration of inhibitor and the inclusion of more than three coordinates in the construction of ' Figure 7, was not feasible due to the curvilinear nature of the response at higher growth regulator concentrations. The same problem appears to be encountered by others in attempts to apply Lineweaver-Burk kinetics to chemical-biological response systems (22). Nevertheless, the implication that ethidium, a known DNA intercalating chemical, generally binds to genomic DNA at or close to a site that may be influenced by NAA is indeed intriguing and poses some interesting questions for further experimentation with the intercalators described here.

The studies with the CPK space-filling models show that it is chemically feasible for ethidium ' (Fig. 9 B) as well as NAA ' (Fig.9 D) to interact with DNA via intercalation. In the case of ethidium a plausible explanation of the observed suppressed rates of diazotization of intercalated aminoacridine derivatives, when complexed with DNA, is that the van der Walls contour of both the upper and lower base pairs of the DNA may shelter and restrict the reactivity of the amino groups of the intercalated chemicals in their orientation close to the deoxyribose oxygens (8). However, Hydrogen bond formation to the phosphate oxygens, through rotation of the amino nitrogens as illustrated here ' (Fig.9B), would be effective also in protecting the amino groups in the complex. We have shown, therefore, another model which illustrates the features of EB intercalation between base pair cavities of DNA in which the EB binds noncovalently to one or two phosphate oxygens via hydrogen bonding (Fig. 9). In this regard, the CPK space-filing models show precise chemical interactions and when constructed according to realistic chemical and physical parameters they provide insights that may be more informative about molecular interactions than many laboratorty experiments.

In consideration of the interactions of models of NAA and EB with DNA, the earlier studies of Sen and Das (23), demonstrating the direct binding of IAA to DNA from a variety of organisms, will have to be investigated with IAA and purified DNA from mung bean cuttings. Also, The use of intercalating chemicals and appropriate cDNA probes in future studies of the putative interaction of auxins with DNA, either free or as ligands of recepter-carrier molecules, should provide additional resolution of auxin molecular activity and the hormonally activated gene expression that appears to be associated with the rooting response in mung bean.

ACKNOWLEDGEMENTS:

We are grateful to Xiaosong Zhang for her able technical assistance. Also, support from the Department of Horticulture and the Agricultural Experiment Station, The Pennsylvania State University is gratefully acknowledged.

REFERENCES

  1. Blazich, F. A. and C. W. Heuser.(l979). A histological study of adventitious root initiation in mung bean cuttings. J. Amer. Soc. Hort. Sci. 104: 63-67.

  2. Hess, C. E. (l964). Naturally occurring substances which stimulate root initiation, p. 517-527. In: J. P. Nitsch ( ed.). Regulateurs Naturels de la Croissance Vegetale, C.N.R.S., Paris.

  3. Blazich, F. A. and C.W. Heuser. (l981). Effects of selected putative inhibitors of ribonucleic acid or protein synthesis on adventitious root formation in mung bean cuttings. J. Amer. Soc. Hort. Sci. 106: 8-11.

  4. Tripepi, R. R., C.W. Heuser, and J. C. Shannon. ( l983). Incorporation of tritiated thymidine and uridine into adventitious-root initial cells of Vigna radiata. J. Amer. Soc. Hort. Sci. l08: 469-474.

  5. Bauer, W. and J. Vinograd. (l968). The interaction of closed circular DNA with intercalative dyes. I. The super helix density of SV40 DNA in the presence and absence of dye. J. Mol. Biol. 33: 141-171. MEDLINE

  6. Lerman, L. S. (1961). Structural considerations in the interaction of DNA and acridines. J. Mol. Biol. 3: 18-30.

  7. Lerman, L. S. (l963). The structure of the DNA-acridine complex. Proc. Nat. Acad. Sci., Wash. 49: 94-102.

  8. Lerman, L. S. (l964). Amino group reactivity in DNA- aminoacricine complexes. J. Mol. Biol. 10: 367-380.

  9. Newton, B. A. (l957). The mode of action of phenanthridines: the effect of ethidium bromide on cell division and nucleic acid synthesis. J. Gen. Microbiol., 17: 718-730.

  10. Tomchick, R. and H. G. Mandel. (l964). Biochemical effects of ethidium bromide in micro-organisms. J. Gen. Microbiol. 36: 225-236.

  11. Le Pecq, J. B. and C. Paoletti. (l967). A fluorescent complex between ethidium bromide and nucleic acids. Physical-chemical characterization. J. Mol. Biol. 27: 87- 106. MEDLINE

  12. Bittman, R. (1969). Studies of the binding of ethidium bromide to transfer ribonucleic acid: Absorption, fluorescence, ultracentrifugation and kinetic investigations. J. Mol. Biol. 46: 251-268. MEDLINE

  13. Waring, M.J. (l965). Complex formation between ethidium bromide and nucleic acids. J. Mol. Biol. 13: 269-282. MEDLINE

  14. Yielding, L. K. (1989). Studies of the binding and biological actions of ethidium, p. 241-259. In: P.E. Nielsen (ed.). Photochemical Probes in Biochemistry Kluwer Academic Publishers, Dordrecht (Netherlands); Boston (USA).

  15. Witham, F. H., L. B. Hendry, and O. L. Chapman. (l978). Chirality and stereochemical recognition in DNA-phytohormone interactions: A model approach. Origins of Life 9: 7-15.

  16. Hendry, L. B., F. H. Witham, and O. L. Chapman. (1977). Gene regulation: the involvement of stereochemical recognition in DNA-small molecule interactions. Perspect. Biol. and Med. 21: 120-130. MEDLINE

  17. Witham, F. H. and L. B. Hendry. (l992). Computer modeling of gibberellin- DNA binding. J. Theor. Biol. 155: 55-67.

  18. Lineweaver, H. and D. Burk. (l934). The determination of enzyme dissociation constants. J. Am. Chem. Soc. 56: 658-666.

  19. Wang, J.C. (1974). The degree of unwinding of the DNA Helix by ethidium. I. Titration of twisted PM2 DNA molecules in alkaline cesium chloride density gradients. J. Mol. Biol. 89: 783-801. MEDLINE

  20. Le Pecq, J. B. (l971). Use of ethidium bromide for separation and determination of nucleic acids of various conformational forms and measurement of their associated enzymes, p. 41-86. In: Methods of Biochemical Analysis, D.Glick (ed.). Interscience Publishers.

  21. MacDonald, I. R. and R.J. Ellis. (l969). Does cycloheximide inhibit protein synthesis specifically in plant tissues? Nature 222: 791-792.

  22. Koshimizu, K. and H. Iwamura. (1988). Cytokinins, p.153-199. In: N. Takahashi (ed.). Chemistry of Plant Hormones. CRC Press Inc. Boca Raton, Fl.

  23. Sen, S. P. & Das, J. (1982). Probable site(s) of plant hormone action. - In Recent Developments in Plant Sciences. S.M. Sircar Mem. Vol. on behalf of the Bot. Soc. of Bengal (Sen, S. P. ed), pp. 61-78. Today and Tomorrow's Print. & Publ., New Delhi. ISBN 0880652411.

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