Growth and Gravireaction of Maize Roots Treated with a Phytotropin

Growth and Gravireaction of Maize Roots Treated with a Phytotropin ART E.

GmssLER*), PAUL-EMILE PILET1) and GERARD F. KATEKAR*)

*) CSIRO, Division of Plant Industry, GPO Box 1600, Canberra City, ACT 2601, Australia 1)

Institute of Plant Biology and Physiology, University of Lausanne, B£timent de Biologie, CH-1015 Lausanne, Switzerland

Received June 25, 1984 · Accepted December 4, 1984

Summary Effeas of the phy!otropin 1-(2'-carboxyphenyl)-3-phenylpropane-1,3-dione (CPD) on growth and gravireaC!ion of intaCI roots and apical root segments of Zea mays L. cv LG-11 were analysed. It is concluded that the compound aC!s through sites of aaion in the extension zone and/or the root cap. The nature of the effects is consistent with the proposal that phy!otropins may inhibit the gravitropic response by interfering in some way with the movement or aaion of the growth substance(s) emanating from the root cap.

Key words: Zea mays L., CPD, elongation, elongation zone, gravireaction, growth substance, phytotropin, root cap.

Introduction

Tropic responses in plants can be affected by a wide variety of synthetic compounds, and structure - activity relationships have been developed for some of them (Mentzer et al., 1950; Jones et al., 1954a,b; Schneider, 1970; Brown et al., 1973; Geissler et al., 1975). These relationships have been reassessed and it was found that many compounds had common chemical and similar physiological properties (Katekar, 1976). From this it was conceived that there exists a class of synthetic chemicals which could be provisionally described in biological and chemical terms, although their mode of action and relationship to the plant functions they affect are unknown. Compounds which embody this tentatively defined concept were termed phytotropins because of their profound effect on the tropic responses of plants (Katekar and Geissler, 1980). It has been shown that phytotropins can interact at specific binding sites in maize coleoptiles, and it was concluded that they may achieve their physiological effects by acting through receptors (Katekar et al., 1981). One of the processes which phytotropins affect and thus would appear to involve their receptors, is the root gravitropic response (Katekar et al., 1981; Geissler and Katekar, 1982; Larkin et al., 1982). It was therefore considered that an analysis of the Abbreviations: CPD

=

1-(2'-carboxyphenyl)-3-phenylpropane-1,3-dione.

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26

Au E. GEissLER, PAUL-EMILE PILET and GERARD F. KATEKAR

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Fig. 1: Growth {increase over initial length in mm ± twice standard error), after 6 h. Intact roots, treated with CPD for 2h. Roots placed horizontally, in the dark {A) or light (B). CPD: 10- 8 -10- 4 M. C = Control. Data is the mean of 6 experiments, each with 40±5 roots. Initial length of roots 15±4mm. way the gravitropic response is affected by the compounds may assist in the understanding of the response itself, as well as in the elucidation of the function of phytotropin receptors. The effect of a phytotropin on growth and gravireaction of maize roots was therefore assessed. CPD was chosen, since it is one of the most active members of the class (Katekar and Geissler, 1977 a, b) and does not interfere with the normal development of the root cap (Geissler and Katekar, 1982).

Materials and Methods In general, the methods previously described by Pilet {1977) and Ney and Pilet {1980}, were followed. Seeds of Zea mays L. cv. LG-11 were soaked for 24h. Selected seeds were then positioned between filter papers and germination took place in the dark {20 ± 1 °C) for 48 h. The technique allowed roots to grow vertically and when the primary root reached a length of 15±4mm, intact roots were mounted either horizontally {with the embryo uppermost) or vertically in plastic frames {Pilet, 1977). Apical root segments {10.0±0.2mm) were mounted horizontally with the cut surface in contact with moist filter papers. The frames were placed in plastic boxes in which a humid atmosphere was maintained {90±5%). Growth and gravireaction took place in either the dark or in light {white fluorescent tubes, Philips 8W/33, 260 p. W em- 2) at 20 ± 1 °C. All manipulations and treatments were carried out under green safety lights. Root growth and curvatures were simultaneously recorded by means of shadow photographs. Recordings were made 6 h after treatment, or at hourly intervals over this period. CPD, which was dissolved in 3-3-dimethyl glutaric acid, NaOH buffer {pH 6.0), was applied in various ways.

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Treatment of maize roots with phytotropin

27

1. In some assays the entire roots of the seedlings were immersed for 2 h in beakers containing

the compound at several concentrations. The roots were subsequently quickly blotted and mounted horizontally on the frames as described above and either kept in darkness or placed in light. Root growth and curvature were measured after 6 h, or as in the time-course assays at hourly intervals. Where apical root segments were used, the segments were excised after the 2 h treatment period. 2. In other assays a 5 ,J droplet containing CPD was applied for 2 h to the basal cut surface of vertically placed apical root segments, before they were horizontally mounted. They were either kept in darkness or exposed to light, and measurements were taken after 6 h. Alternatively a 5 ,J droplet containing CPD was applied for 2 h to the tip of intact vertically placed roots, before segments were excised. Intact roots and apical segments were subsequently horizontally mounted and kept in the dark or placed in light. Measurements were taken after

6h.

3. In the final series of assays, entire roots were treated for 2 h by immersion and were subsequently half decapitated (Pilet, 1977). The roots were mounted, either vertically or horizontally and placed in light. Curvature was measured after 6 h. Control roots and apical root segments were treated in a similar way with 3-3-dimethyl glutaric acid, NaOH buffer (pH 6.0), which does not affect root growth or gravireaction. Chemicals: CPD was obtained by synthesis (Brown et al., 1973).

Results and Discussion The results obtained with intact control roots show that growth was more inhibited in light than in dark (Fig. 1 A, B). These results agree with other data that growth of roots of serveral plant species is inhibited more in light than in dark, and that this inhibition is dependent on the perception of light by the root cap (Wilkins and Wain, 1974). In some maize cultivars, it has been concluded that white light can influence the level of growth substances in roots (Pilet, 1975, 1979; Wilkins and Wain, 1975; Suzuki et al., 1979; Pilet and Rivier, 1980). When intact roots were treated with CPO, growth was inhibited at 10- 6 M and 10- 4 M both in light and in darkness (Fig. 1A, B). It would appear that growth inhibition due to CPD in intact roots is in addition to inhibition caused by light, with growth of light grown roots being almost completely inhibited at 1Q- 4 M (Fig. 1 B). The results on gravireaction show a greater response in intact control roots exposed to light (Fig. 2 B) than in control roots grown in darkness (Fig. 2 A) and they agree with previous data (Scott and Wilkins, 1969; Pilet, 1979; Geissler and Katekar, 1982). CPD inhibited gravireaction at all concentrations, both in light (Fig. 2 B) and in darkness (Fig. 2 A). The presence of light is therefore not an essential requirement for CPD to exert its gravitropic effects. The time course analyses of the rate of growth and gravireaction in intact roots are shown in Figs. 3 and 4. The rate of growth in dark grown control roots was fairly constant during the 6 h period of the experiment (Fig. 3 A). CPD inhibited the growth rate during the first 4 h of the experiment, but between 4 and 6 h the growth rate was greater than that of controls (Fig. 3 A). Essentially similar results were obtained with light exposed treated roots (Fig. 3 B). The data on gravireaction show that the rate of curvature in dark grown control roots was extremely rapid during the first

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ART E. GEISSLER, PAUL-fum.E PILET and GERARD F. KATEKAR

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Fig.2: Gravireaction (curvature in degrees ± twice standard error}, after 6h. Intact roots, treated with CPD for 2 h. Roots placed horizontally in the dark (A} or light (B). CPD: 10- 8 -10- 4 M. C = Control. Data is the mean of 6 experiments, each with 40±5 roots. Initial length of roots 15 ±4 mm.

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Fig. 3: Growth (in mm ± twice standard error}, 0-6 h measurements. Intact roots, treated with CPD for 2h. Roots placed horizontally in the dark (A} or light (B). Control (a}; CPD: 10- 6 M (b). Data is the mean of 6 experiments, each with 40±5 roots.

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Treatment of maize roots with phytotropin

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Fig.4: Gravireaction (curvature in degrees ± twice standard error), 0-6 h measurements. Intact roots, treated with CPD for 2 h. Roots placed horizontally in the dark (A) or light (B). Control (a); CPD: 10- 6 M (b). Data is the mean of 6 experiments, each with 40±5 roots.

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Table 1: Growth (mm) and Gravireaction (degrees) of apical maize root segments maintained in the dark or in the light for 6 h in a horizontal position following a 2 h treatment with a 5 ,.J. buffered droplet with or without CPD. Application was to the basal cut section of vertically placed root segments prior to gravi-stimulation. Treatment

Concentration

Growth-darkness

Control 10- 6 M 10- 4 M

Means 1.46 1.15 0.81

Student's t values 0.64°8 1.47ns

Growth-light

Control 10- 6 M 10- 4 M

1.19 1.01 0.67

0.53°8 1.72ns

Gravireaction-darkness

Control 1o- 6 M 10- 4 M

25.3 11.7 3.8

2.49* 4.49**

Gravireaction-light

Control 10- 6 M 10- 4 M

46.1 30.7 4.2

2.13ns 6.78***

Data are mean values (mm or degrees) for 5 experiments each with 30±4 root segments for each treatment. ns = not significant (P > 0.05) * = significant at 5% level (P < 0.05) ** = significant at 1% level (P < 0.01) *** =significant at 0.1% level (P <0.001)

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Fig.5: Growth (in mm ±twice standard error), 0-6h measurements. Apical root segment; 2h treatment of intact roots (by immersion) with CPD. Segments placed horizontally in the dark (A) or light (B). Control (a); CPD: 10- 6 M (b). Data is the mean of 5 experiments, each with 35±5 segments.

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Fig. 6: Gravireaction (curvature in degrees ± twice standard error), 0-6h measurements. Apical root segments; 2 h treatment of intact roots (by immersion) with CPD. Segments placed horizontally in the dark (A) or light (B). Control (a); CPD: 10- 6 M (b). Data is the mean of 5 experiments, each with 35±5 segments.

2 h, after which it was considerably less (Fig. 4 A). In comparison the rate of curvature was less in dark grown, CPD treated roots (Fig. 4 A). Similar results were obtained with light exposed roots (Fig. 4 B). The recovery of growth rate (Fig. 3) would indicate that the action of the compound is reversible with respect to root growth. It ]. Plant PbysioL VoL 119. pp. 25-34 (1985}

Treatment of maize roots with phytotropin

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Table2: Growth (mm) and Gravireaction (degrees) of intact primary maize roots and apical maize root segments maintained in the dark or in light for 6 h in a horizontal position, following a 2 h treatment with a 5 JLl buffered droplet with or without CPD. Application was to the tip of intact and vertically placed roots before roots and root segments were gravistimulated. Treatment

Concentration

Means

Growth-darknessintact root

Control 10- 6 M 10- 4 M

4.8 4.0 3.7

0.87ns 1.41ns

Growth-darknesssegments

Control 10- 6 M 10- 4 M

1.52 1.45 1.09

o.uns 0.87ns

Growth-lightintact roots

Control 10- 6 M 10- 4 M

3.1 3.7 1.5

-0.94ns 2.83*

Growth-lightsegments

Control 10- 6 M 10- 4 M

1.24 1.42 0.95

-0.37ns 0.77ns

Gravireaction-darkness intact roots

Control 10- 6 M 10- 4 M

31.4 26.2 8.9

0.87ns 4.70**

Gravireaction-darkness segments

Control 10- 6 M 10- 4 M

28.2 17.0 0.8

2.46* 6.58***

Gravireaction-light intact roots

Control 10- 6 M 10- 4 M

63.5 38.6 12.7

3.56** 8.04***

Gravireaction-light segments

Control 10- 6 M 10- 4 M

44.1 35.9 1.1

Student's t values

1.52ns 10.49***

Data are mean values (mm or degrees) for 5 experiments each with 30±4 roots or segments for each treatment. Explanation of t values as for Table 1 Initial length of intact roots: 15 ±4mm Initial length of root segments: 10.0±0.2 mm Initial angle of intact roots: 1. 9 ± 1.5 degrees Initial angle of root segments: -1.3 ±0.8 degrees

can be noted that there is no corresponding increase in the rate of curvature [i.e. gravitropic response (Fig. 4)]. However, inhibition of the gravitropic response is also reversible, because complete removal of phytotropin will allow roots to respond normally to gravity (Geissler and Katekar, 1982). Reversibility of the gravitropic response is also observed in the root segment experiments (Fig. 6). Apical root segments were used to determine the possible site of action of CPD. When the compound was applied to the cut surface of the segments ]. Plant Physiol. Vol. 119. pp. 25-34 (1985}

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ART E. GEISSLER, PAUL-EMILE PILET and GERARD F. KATEKAR

Table 3: Curvature (degrees) of intact maize roots maintained in light for 6 h in a vertical or horizontal position following a 2 h treatment by immersion in a buffered solution with or without CPD. Roots are fixed vertically (a) or horizontally; (b) remaining half of root tip lowermost; (c) remaining half of root tip uppermost. Arrows indicate direction of curvature. Treatment

Concentration

Means

Student's t values

a)

Control 1o- 6 M

34.5 10.3

3.97**

Control 1o- 6 M

58.2 15.8

5.33**

31.3 4.5

4.48**

b)

J

c)

Data are the means (degrees) for 4 experiments each with 30±4 roots for each orientation. Explanation oft values as for Table 1.

gravireaction was in most instances significantly inhibited while growth was not affected (Table 1). Similar results were obtained when CPD was applied to the root tip of apical segments (Table2). Finally the rate of growth and gravireaction in apical root segments was also affected by CPD (Figs. 5 and 6). Since these results are essentially similar to those obtained with intact roots (Figs. 3 and 4; Table 2), it is concluded that whatever the effect on the remainder of the plant, CPD can affect growth and gravireaction by acting in the root tip and/or elongation zone. It can also be concluded that CPD moves acropetally to its site of action since the gravitropic response was in most instances significantly affected when CPD was applied to the basal cut section of root segments (Table 1). Whether the compound could move basipetally has still to be determined because the effects observed when CPD was applied to the root tip of segments (Table2) could have been due to its action in this specific region rather than the elongation zone. It has been concluded that phytotropins can alter the root gravitropic response through their effect on the action or distribution of growth substances present in the plant (Geissler and Katekar, 1982). The above evidence is consistent with this. Possible ways that CPD could affect the gravitropic response, therefore, include a direct ]. Plant Physiol. Vol. 119. pp. 25-34 (1985}

Treatment of maize roots with phytotropin

33

or indirect inhibitory effect in the elongation zone, alteration of the distribution of growth substances in the root cap or elongation zone, or alteration of the flow of growth regulator(s} (i.e. the cap-inhibitor} from the root cap to the elongation zone which control the response Gackson and Barlow, 1981}. The latter two possibilities would be in accord with the known auxin transport inhibitory properties of the phytotropins (Katekar and Geissler, 1980}. A direct inhibitory effect alone would seem unlikely, because many powerful root growth inhibitors are known which do not prevent the gravitropic response (Katekar and Geissler, 1980; Jackson and Barlow, 1981}. Further, the results here show that the response can be abolished at differing amounts of inhibition (Table2}, and also when the rate of growth is variable (Fig. 3 and 4}. An indirect inhibitory action would imply that the compound may act by interfering in some way with the action of the growth substances which cause inhibition. Since the cap inhibitor causes inhibition, interference with the action of this substance is a plausible possibility. An asymmetric distribution of the cap-inhibitor can be achieved by surgical means. Thus, a root that has been half decapitated will curve towards the side which is still intact, such curvature being a response to the action of regulators from the root cap (Pilet, 1975, 1977}. Treatment with CPD reduced this curvature, regardless of the orientation of the roots with respect to gravity (Table3}. CPD can therefore affect the gravitational response by means independent of any ability it may have to prevent the asymmetric distribution of the cap-inhibitor. Prevention of flow of the capinhibitor to the root tip, of itself, cannot explain all observations, because it is known to have a net inhibitory effect, and preventing its flow (e.g. by removal of the root cap) causes an increase, not a decrease in growth (Pilet, 1972} and such an increase is not observed here. However, the two effects (inhibitory and gravitational} may be independent. It is postulated that phytotropins can inhibit the root gravitropic response by preventing the flow of growth substances from the root cap, by interfering with the action of those substances in the elongation zone in some way, or both these mechanisms. Phytotropins can do this independently of any effect they may have on the distribution of growth substances in the root cap or elongation zone, and of any effect they may have in the remainder of the plant. Acknowledgements We wish to thank Mrs Cl Grandchamp for her skilful assistance.

References BROWN, B. T., 0. JoHANsEN, G. F. KATEKAR, and W. H. F. SASSE: The effect on root geotropism of certain orthocarboxyphenylpropanones. Pestic. Sci. 4, 473-483 (1973). GEISSLER, A. E. and G. F. KATEKAR: Phytotropism V: The effect of 1-(2'-carboxyphenyl)-3-phenylpropane-1,3-dione on the root and root cap of Zea mays L. J. Exp. Bot. 33, 952-965 (1982).

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GEISSLER, A. E., J. L. HuPPATZ, and G. F. KATEKAR: Effect of substituted pyrazoles and related compounds on geotropism in cress seedlings. Pestic. Sci. 6, 441-450 (1975). jACKSON, M. B. and P. W. BARLow: Root geotropism and the role of growth regulators from the cap: a re-examination. Plant Cell and Environment. 4, 107-123 (1981). JoNEs, R. L., T. P. METCALFE, and W. A. SEXTON: The relationship between the constitution and the effect of chemical compounds on plant growth. Derivatives and analogues of 2-benzoylbenzoic acid. J. Sci. Fd. Agric. 5, 32-38 (1954 a). - - - The relationship between the constitution and the effect of chemical compounds on plant growth. Some derivatives of fluorene. J. Sci. Fd. Agric. 5, 44-47 (1954 b). KATEKAR, G. F.: Inhibitors of the root geotropic response in plants: a correlation of molecular structures. Phytochemistry 15, 1421-1423 (1976). KATEKAR, G. F. and A. E. GEISSLER: Auxin transport inhibitors II. 2-(1-pyrenoyl) benzoic acid, a potent inhibitor of polar auxin transport. Aust. J. Plant Physiol. 4, 321-325 (1977 a). - - Auxin transport inhibitors III. Chemical requirements of a class of auxin transport inhibitors. Plant Physiol. 60, 826-829 (1977b). - - Auxin transport inhibitors IV. Evidence of a common mode of action for a proposed class of auxin transport inhibitors: the phytotropins. Plant Physiol. 66, 1190-1195 (1980). KATEKAR, G. F., J. F. NAVE, and A. E. GEISSLER: Phytotropins III. Naphthylphthalamic acid binding sites on maize coleoptile membranes as possible receptor sites for phytotropin action. Plant Physiol. 68, 1460-1464 (1981). LARKIN, P. J., W. R. ScowcROFT, A. E. GEISSLER, and G. F. KATEKAR: Phytotropins IV. Effect of phytotropins on cultured plant cells and protoplasts. Aust. J. Plant Physiol. 9, 297-307 (1982). MENTZER, C., D. MoLHO et H. PACHECO: Relations, entre la structure chimique ell'inhibition des tropismes chez les vegetaux. Bull. Ste. Chim. Biol. 32, 572-582 (1950). NEY, D. and P. E. PILET: Importance of the caryopsis in root growth and georeaction. Physiol. Plant. 50, 166-168 (1980). PILET, P. E.: Root cap and root growth. Planta 106, 169-171 (1972). - Abscisic acid as a root growth inhibitor: physiological analyses. Planta 122, 299-302 (1975). - Growth inhibitors in growing and geostimulated maize roots: In: P. E. PILET (Ed.), Plant Growth Regulation, 115-128. Springer, Berlin, Heidelberg, New York, 1977. - Kinetics of the light-induced georeactivity of maize roots. Planta 145, 403-404 (1979). PILET, P. E. and L. RlviER: Light and dark georeaction of maize roots: Effect and endogenous level of abscisic acid. Pl. Sc. Lett. 18,201-206 (1980). SCHNEIDER, G.: Morphactins: physiology and performance. Ann. Rev. Plant Physiol. 21, 499-536 (1970). ScoTT, T. K. and M. B. WILKINS: Auxin transport in roots IV. Effect of light on IAA transport and geotropic responsiveness in Zea roots. Planta 87, 249-258 (1969). SuzuKI, T., N. KoNDo, and T. FUJII: Distribution of growth regulators in relation to the light induced geotropic responsiveness in Zea roots. Planta 145, 323-329 (1979). WILKINS, H. and R. L. WAIN: The root cap and control of root elongation in Zea mays L. seedlings exposed to white light. Planta 121, 1-8 (1974). - - The role of the root cap in the response of the primary roots of Zea mays L. seedlings to white light and gravity. Planta 123, 217-222 (1975).

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