Phototropism in Hypocotyls of Radish. VII. Involvement of the Growth Inhibitors, Raphanusol A and B in Phototropism of Radish Hypocotyls

,.PlantPhysiol. Vol. 135.pp. 110-113(1989}

Phototropism in Hypocotyls of Radish. VII. Involvement of the Growth Inhibitors, Raphanusol A and B in Phototropism of Radish Hypocotyls KOJI HASEGAWA

* and SHIGENORI TOGO

Department of Biology, College of Liberal Arts, Kagoshima University, Korimoto 1-21-30, Kagoshima, 890, Japan Received March 6,1989 . Accepted May 11, 1989

Summary Using a physicochemical determination, the distribution of the endogenous growth inhibitors, raphanusol A and B, was measured at the lighted and shaded sides of etiolated radish (Raphanus sativus var. hortensis f. gigantissimus Makino) hypocotyls 60 min after the start of phototropic stimulation. The phototropic curvature was still developing then in the first positive, indifferent response, and the second positive phototropic curvatures induced by unilateral broad blue light (half band width 43 nm, Amax 448 nm). The contents of raphanusol A and B increased at the lighted side in both the first and second positive curvature; only a slight increase was seen in the region of indifference, whereas their contents in the shaded side in either phototropic curvature were similar to those in the dark control. The differential distribution of raphanusol A and B in the hypocotyls is closely correlated with growth suppression at the lighted side in the first and second positive phototropic curvatures. Unilateral applications of raphanusol A and B caused the hypocotyls to bend towards the site of application because of more growth inhibition at the treated side than at the opposite one.

Key words: Raphanus sativus, growth inhibitor, phototropism, raphanusol. Abbreviation: IAA

=

indole-3-acetic acid.

Introduction We have previously isolated three neutral growth inhibitors from light-grown radish seedlings and identified them by spectrometric analyses as 1-/3, 4-di-0-(4-hydroxy-3,S-dimethoxycinnamoyl) gentiobiose (Hasegawa and Miyamoto 1980, Hase and Hasegawa 1982), 1-/3-0-(4-hydroxy-3,S-dimethoxycinnamoyl)-D-glucose (Hasegawa and Hase 1981) and 3-methoxy-4-methylthio-2-piperithione (Hasegawa et a1. 1982). For these compounds the names raphanusol A and B (Fig. 1), and raphanusanin were proposed, respectively. The contents in the radish seedlings increased greatly in relation to the exposure to red light, but decreased or maintained their initial level in the dark (Hasegawa and Miyamoto 1980, Hasegawa and Hase 1981, Hasegawa et al. 1982). Thus, we

* To whom reprint requests should be sent. © 1989 by Gustav Fischer Verlag, Stuttgart

concluded that the inhibitors might playa role in the phytochrome-controlled light inhibition of hypocotyl growth of the radish seedlings. Recently, it was demonstrated that of the raphanusanin growth inhibitors, the cis- and trans-compounds (Hasegawa et al. 1986) are not only involved in light inhibition of hypocotyl growth but also in phototropism of the hypocotyl; cisand trans-raphanusanin content increased in the lighted side during both the first and second positive curvatures (Noguchi et aI. 1986, Noguchi and Hasegawa 1987, Hasegawa et al. 1987, Sakoda et al. 1988). Their contents turned out to be closely correlated: on the one hand with the light intensity at either side of the phototropically stimulated hypocotyls and, on the other hand, with the growth inhibition at those sides (Noguchi and Hasegawa 1987, Bruinsma et al. 1989). Furthermore, their unilateral application suppressed the growth of the hypocotyl at the applied side more than at the op-

Raphanusols in phototropism of radish hypocotyl

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posite one, causing the hypocotyl to bend towards the site of application (Noguchi et al. 1986). However, whether or not the raphanusols A and B are also involved in the cryptochrome-controlled phototropism of the radish hypocotyl has not yet been reported. In this paper we describe the relationship between the distribution of raphanusol A and B in the hypocotyls and the flank growth rates of these hypocotyls during developing phototropism, as well as the effect of unilateral application of raphanusols A and B on the growth rates of the treated and opposite sides.

111

Determination of raphanusol A and B Per experiment, 10 phototropically stimulated hypocotyls were harvested 60 min after the start of illumination. The hypocotyl sections from 0 to 2 cm below the hook of the seedlings were excised and bisected into the lighted and shaded sides with a razor under dim green light. The bisected hypocotyls were immediately fixed in 8 ml of cold acetone (O°C) and stored at - 20°C until analysis of raphanusol A and B. Samples were ground with pieces of dry ice in a mortar and extracted with 50 volumes of 70 % cold acetone for 30 min at O°C. The extract was filtered through Toyo No.1 filter paper and the residue rinsed with 30 volumes of cold acetone. The filtrate was evaporated to an aqueous solution in vacuo at 35°C. After evaporation, Xo volume of K-phosphate buffer (pH 7.8, 1 M) was added to the aqueous residue and the solution was partitioned three times against an equal volume of ethyl acetate. The neutral fraction was dried over anhydrous Na2S04 and then evaporated to dryness in vacuo at 35°<;:. The crude material was taken up in 10 ml of 40 % methanol in water, brought on a CIS Sep-pak cartridge column (Waters) pretreated with 40 % methanol in water, and eluted with 10 ml of 40 % methanol in water. The combined eluate was evaporated to dryness in vacuo at 35°C and finally separated by HPLC (Wakopak CIS, Wako Pure Chemical Industries, Japan, water: methanol = 3:2, vlv, 1.2ml·min- l , 333nm detector, Amax of raphanusol A and B: 333 nm). Retention times of raphanusol A and B were 7.5 and 2.5 min, respectively. After the area of the peaks was determined, the amounts of endogenous raphanusol A and B were calculated from standard curves. Known amounts of genuine raphanusol A and B were added to the other half of each sample during extraction in order to determine losses during the purification procedures. Overall recovery was about 70 %, all data were corrected on this basis. The experiments were repeated three times.

Materials and Methods Plant material Sakurajima radish (Raphanus sativus var. hortensis f. gigantissimus Makino) seeds were germinated on double layers of moistened filter paper in a tray in the dark at 25°C. Three days later, uniform seedlings were transplanted into trays filled with moist vermiculite under a photo morphogenetically inactive intensity (0.03 !Lmol . m - 2. S - I) of dim green light and kept in the dark at 25°C for one more day.

Phototropic stimulation Four-d-old, etiolated seedlings (hypocotyl length about 4 em) were unilaterally illuminated with broad blue light (half band width 43nm, Amax 448nm) for 30s, 5 min or 50 min. The blue light was obtained from a blue fluorescent lamp (National High Light S, National Corp., Japan) through a blue acrylic plate (Kyowalite PG, Kyowa Gas Chemical Industry Corp., Japan) attached to a 3-cm wide, horizontal slit of a non-reflecting black box. The illumination was given over the whole of the seedlings. Incident energy was 0.46 !Lmol· m - 2. S -I at the plant level. At least twenty seedlings were used per experiment. The hypocotyl curvature and the growth rates at the lighted and shaded sides of the hypocotyls were directly measured using photographs 60 and 90 min after the start of illumination. The maximal curvature was observed to be reached by 90 min. To measure the growth rates at the lighted and shaded sides and the dark control of the hypocotyls, ion exchange resin beads, Amberlite XAD-2 smeared with lanolin, were positioned on the lighted and shaded sides from 0 to 2 cm below the hook of 10 seedlings under dim green light. The distances between the beads were measured on the photographs using a map-measuring device.

Unilateral application of raphanusol A and B Attempts to induce curvature by unilateral application of raphanusol A and B were done using uniform, 4-d-old, etiolated radish seedlings. One, 0.3 or O!Lg of genuine raphanusol A or B in 1 mg lanolin were unilaterally applied along the length from 0 to 2 cm below the hook of 6 seedlings. Simultaneously, ion exchange resin beads were positioned on the smeared and opposite sides from 0 to 2 cm below the hook. Seedlings with or without raphanusol A or B were incubated in the dark at 25°C. Photographs were taken 2 h after the start of incubation. The bending response and the distances between the upper and lower beads on the applied and opposite sides were measured on the photographs. The experiments were repeated three times.

Results Table 1 shows the bending degrees and the growth rates at the lighted and shaded sides of hypocotyls subjected to the three different fluences. Maximal curvature was observed at 90 min. Illumination at 14 /Lmol . m - 2 of unilateral blue light produces the so-called first positive phototropic curvature, at 138 /Lmol· m -2 the indifferent phototropic response, and at 1380 /Lmol· m -2 the second positive phototropic curvature, respectively. The growth rates at the shaded sides were almost equal to those in darkness at any phototropic curvature. On the contrary, the growth rates at the lighted sides were suppressed. The pattern of bending and growth inhibition is very similar to the previously reported data (Sakoda et

112

KOJI HASEGAWA and SHIGENORI TOGO

Table 1: The bending degrees and growth rates of the dark control and unilaterally illuminated radish hypocotyls. Elongation was determined 60 min after the start of phototropic stimulation. Each value is the mean of 10 observations ± SE. Curvature (0) Phototropism (fluence) 60 min 90 min Dark control l±1 0±1 First positive (14 I'mol· m -2) 7±2 15±3 Indifferent (138I'mol· m -2) HI 8±1 Second positive (1380 I'mol . m - 2) 17±2 3l±2

30

Elongation increment (mm) Lighted side Shaded side 0.95±0.05 0.98±0.04 0.70±0.06

0.9l±0.04

0.89±0.07

1.00±0.07

0.32±0.10

0.99±0.11

Inhibitor Plain lanolin Raphanusol A (0.3 I'g) (1.0 I'g) Raphanusol B (0.3 I'g) (1.0 I'g)

Curvature (degree) 0.8± 1.0 5.0±2.8 8.8±0.8 6.8± 1.2 11.5± 1.6

Elongation increment (mm) Applied side Opposite side 1.25 ± 0.15 1.29 ± 0.17 1.04±0.05 1.1l± O. 13 0.62±0.04 0.89±0.11 0.89±0.16 1.06±0.17 0.60±0.11 0.89±0.04

induced the hypocotyl to bend towards the site of application. The magnitude of the bending was proportional to the concentrations used. Raphanusol A is at least as active as raphanusol B on a molar basis. The treatments suppressed the growth rates at the treated sides at both concentrations. The growth rates at the opposite sides were only slightly suppressed by the treatments at the higher concentration, and hardly so at the lower one.

Raphonuso I B

Raphanuso I A

Table 2: Effects of raphanusol A and B, applied to one side of etiolated radish hypocotyls, on hypocotyl curvature and the growth rates of applied and opposite sides. The curvature and growth rates were measured 2 h after unilateral applications of 1.0, 0.3 or 0 I'g inhibitor per seedling. Average values of 6 seedlings.

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Fig. 2: Lateral distribution of raphanusol A and B in the lighted (0) and shaded (.) sides of radish hypocotyls 60 min after the start of phototropic stimulation. Bars indicate SE. al. 1988); the measure of phototropic curvature depends on the magnitude of growth suppression at the lighted sides. The contents of endogenous raphanusol A and B in the lighted and shaded sides of the hypocotyls at the first positive, the indifferent response and the second positive phototropic curvature are shown in Fig. 2. The plant materials were harvested 60 min after onset of illumination, at which time the curvature is still developing. The content in the shaded sides was almost equal to that in the dark control at any phototropic curvature. On the contrary, the contents of both raphanusol A and B were considerably increased at the first and, particularly, the second positive response, whereas they were equal in both sides at the indifferent response. Whether or not unilateral application of exogenous raphanusol A or B on the hypocotyls, resulting in unequal distribution, causes differential flank growth of the hypocotyls was also determined (Table 2). Plain lanolin was not effective. Unilateral applications of exogenous raphanusol A or B

Evidence that phototropism of higher plants is caused by light-induced, local accumulation of growth inhibitors in the presence of an unchanged, even distribution of auxin has recently been provided by many researchers. In green sunflower hypocotyls, it has been reported that a lateral redistribution of indole-3-acetic acid (IAA) was not shown in unilaterally illuminated hypocotyls (Bruinsma et al. 1975, Feyerabend and Weiler 1988); on the contrary, neutral growth inhibitors, including cis-xanthoxin, accumulated at the lighted side of the hypocotyls during phototropic curvature (Thompson and Bruinsma 1977, Franssen and Bruinsma 1981, Shen-Miller et al. 1982, Hasegawa et al. 1983). In etiolated and de-etiolated radish hypocotyls, the first positive, the indifferent response and the second positive phototropic curvatures induced by unilateral blue light were shown to be caused by the lateral distribution of growth inhibitors, cis- and trans-raphanusanins, in the hypocotyls (Hasegawa et al. 1986, Noguchi et al. 1986, Noguchi and Hasegawa 1987, Hasegawa et al. 1987, Sakoda et al. 1988, Bruinsma and Hasegawa 1989, Bruinsma et al. 1989). IAA was evenly distributed over the lighted and shaded sides in the first and second positive phototropic curvatures; no net exchange of IAA between the peripheral and central cell layers was observed during these curvatures (Sakoda and Hasegawa 1989). In oat coleoptiles, it has been shown that both extractable and diffusible IAA were evenly distributed over the lighted and shaded sides of the coleoptiles during phototropic curvature and that growth inhibitors increased in the lighted side (Hasegawa and Sakoda 1988, Bruinsma and Hasegawa 1989, Bruinsma et al. 1989, Hasegawa et al. 1989). From the results of the present study (Tables 1 and 2, and Fig. 2), it is concluded that the lateral distribution of endogenous raphanusol A and B in the phototropically stimulated

Raphanusols in phototropism of radish hypocotyl

hypocotyls is closely correlated with the differential growth rates of the hypocotyls, and that raphanusol A and B play an important role not only in the phytochrome-mediated light inhibition but also in the cryptochrome-mediated phototropism of the radish hypocotyls. Thus, we conclude that phototropism in radish hypocotyls may be regulated by a !ateral gradient of raphanusols as well as of raphanusanins. We propose that the growth inhibitors, raphanusol A and B in addition to raphanusanins act as phototropism-regulating substances in the radish hypocotyl.

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HASEGAWA, K. and T. HASE: Raphanusol B: a growth inhibitor of light-grown radish seedlings. Plant Cell Physio!. 22, 303-306 (1981). fuSEGAWA, K., E. KNEGT, and J. BRUINSMA: Caprolactam, a lightpromoted growth inhibitor in sunflower seedlings. Phytochemistry 22,2611-2612 (1983). HASEGAWA, K. and K. MIYAMOTO: Raphanusol A: a new growth inhibitor of light-grown radish seedlings. Plant Cell Physio!. 21, 363-366 (1980).

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HASEGAWA, K., H. NOGUCHI, T. IWAGAWA, and T. fuSE: Phototropism in hypocotyls of radish. 1. Isolation and identification of growth inhibitors, cis- and trans-raphanusanins and rap hanusamide, involved in phototropism of radish hypocotyls. Plant Physio!. 81, 976-979 (1986). fuSEGAWA, K., H. NOGUCHI, C. TANOUE, S. SANDO, M. TAKADA, M. SAKODA, and T. fuSHIMOTO: Phototropism in hypocotyls of radish. IV. Flank growth and lateral distribution of cis- and transraphanusanins in the first positive phototropic curvature. Plant Physio!. 85, 379-382 (1987). HASEGAWA, K. and M. SAKODA: Distribution of endogenous indole-3acetic acid and growth inhibitor(s) in phototropically responding oat coleoptiles. Plant Cell Physio!. 29, 1159-1164 (1988). fuSEGAWA, K., M. SAKODA, and J. BRUINSMA: Revision of the theory of phototropism in plants: a new interpretation of a classical experiment. Planta (in press). HASEGAWA, K., S. SHIIHARA, T. IWAGAWA, and T. fuSE: Isolation and identification of a new growth inhibitor, raphanusanin from radish seedlings and its role in light inhibition of hypocotyl growth. Plant Physio!' 70,626-628 (1982). NOGUCHI, H. and K. HASEGAWA: Phototropism in hypocotyls of radish. Ill. Influence of unilateral or bilateral illumination of various light intensities on phototropism and distribution of cis- and trans-raphanusanins and raphanusamide. Plant Physio!. 83, 672-675 (1987). NOGUCHI, H., K. NISHITANI, J. BRUINSMA, and K. fuSEGAWA: Phototropism in hypocotyls of radish. II. Role of cis- and transraphanusanins, and raphanusamide in phototropism of radish hypocotyls. Plant Physio!. 81, 980-983 (1986). SAKODA, M. and K. HASEGAWA: Phototropism in hypocotyls of radish. VI. No exchange of endogenous indole-3-acetic acid between peripheral and central cell layers during first and second positive phototropic curvatures. Physio!. Plant. (in press). SAKODA, M., T. MATSUOKA, S. SANDO, and K. HASEGAWA: Phototropism in hypocotyls of radish. V. Lateral distribution of cisand trans-raphanusanins and raphanusamide in various phototropisms induced by unilateral broad blue light. J. Plant Physio!. 133, 110-112 (1988). SHEN-MILLER, J., E. KNEGT, E. VERMEER, and J. BRUINSMA: Purification and lability of cis-xanthoxin, and its occurrence in phototropically stimulated hypocotyls of Helianthus annuus L. Z. Pflanzenphysio!. 108,289-294 (1982). THOMPSON, J. M. and J. BRUINSMA: Xanthoxin: a growth inhibitor in light-grown sunflower seedlings, Helianthus annuus L. J. Exp. Bot. 28,804-810 (1977).