Hairy roots of Lithospermum erythrorhizon, belladonna and carrot, which were induced by inoculation with Agrobacterium rhizogenes harboring the Ri plasmid, were cultured on a medium containing 0.1, 1, 10, 30 or 100μM W-7 or W-5, calmodulin antagonists. Growth rates of L. erythrorhizon and belladonna hairy roots cultured on all media containing W-7 were lower than that of the roots cultured without W-7. Growth of carrot hairy roots was inhibited by W-7 above 30μM. Inhibition rates of the root growth by high concentrations of W-5 were lower than those of the growth by the same concentrations of W-7. In the case ofthe development of lateral roots on hairy roots, 30 and 100μM W-7 or W-5 inhibited formation of lateral roots. The number of lateral roots formed by culturing on a medium containing W-7 was lower than that of the roots formed on the medium containing W-5. These results strictly suggest that calmodulin acts upon the growth of hairy roots and the development of lateral roots on hairy roots

Arashida  Miyuki

Kamada  Hiroshi

Kato  Ryoichi

Kodama  Masahiro

Suzuki  Takashi

Yamagata University Academic Repository

Bull. of Yamagata Univ., Nat. Sci., Vol.15, No.3, Feb. 2003Inhibitions of Growth and Lateral BranchDevelopment by Calmodulin Antagonists inHairy Roots of Lithospermum erythrorhizon,Atropa belladonna and Daucus carotaRyoichi Kato†, Miyuki Arashida†, Masahiro Kodama†,Hiroshi Kamada‡ and Takashi Suzuki††(Received March 29, 2002)AbstractHairy roots of Lithospermum erythrorhizon, belladonna and carrot,which were induced by inoculation with Agrobacterium rhizogenes har-boring the Ri plasmid, were cultured on a medium containing 0.1, 1,10, 30 or 100µM W-7 or W-5, calmodulin antagonists. Growth ratesof L. erythrorhizon and belladonna hairy roots cultured on all mediacontaining W-7 were lower than that of the roots cultured without W-7. Growth of carrot hairy roots was inhibited by W-7 above 30µM.Inhibition rates of the root growth by high concentrations of W-5 werelower than those of the growth by the same concentrations of W-7. Inthe case of the development of lateral roots on hairy roots, 30 and100µM W-7 or W-5 inhibited formation of lateral roots. The numberof lateral roots formed by culturing on a medium containing W-7 waslower than that of the roots formed on the medium containing W-5.These results strictly suggest that calmodulin acts upon the growthof hairy roots and the development of lateral roots on hairy roots.†Biology Laboratory, Faculty of Education, Yamagata University, Yamagata, 990–8560Japan‡Institute of Biological Sciences, University of Tsukuba, Tsukuba, Ibaragi, 305–8572Japan††Science Laboratory, Faculty of Education, Yamagata University, Yamagata, 990–8560Japan79Ryoichi Kato, Miyuki Arashida, Masahiro Kodama, Hiroshi Kamada and Takashi SuzukiAbbreviationsW-7, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide; W-5, N-(6-aminohexyl)-naphthalenesulfonamide.Key wordsAtropa belladonna—Calmodulin antagonist—Daucus carota—Hairyroots—Root growth and lateral branch development—Lithospermumerythrorhizon.1 IntroductionCalmodulin plays important roles in many physiological processes in plantcells as well as animal cells (Dieter 1984, Sun 2000, Chung et al. 2000,Lenartowska et al. 2001). Muto and Hirosawa (1987) reported that growth ofadventitious roots formed in stem cuttings of Tradescantia fluminensis couldbe inhibited by incubation in a solution containing a low concentration of acalmodulin antagonist: triﬂuoperazine or chlorpromazine, and they discussedinvolvement of calmodulin in the growth of the roots. These antagonists bindvery tightly to isolated calmodulin (Levin and Weiss 1976, 1977). However,an aﬃnity of the antagonists for cytoplasmic membrane is so high that theantagonists seriously damage cultured mammalian cells (Osborn and Weber1980). These reports suggest that using of triﬂuoperazine or chlorpromazinein in vivo experiments is unsuitable for detecting the action of calmodulin.It has been clearly demonstrated that the Ri plasmid present in Agrobac-terium rhizogenes causes the transformation of plant cells via the introduc-tion of T-DNA from the Ri plasmid into the genomic DNA of plant cells. Ithas also been demonstrated that the transformed plant cells give rise to mas-sive roots, the so-called hairy roots (White and Nester 1980, Tepfer 1984,Shanks and Morgan 1999, Giri and Narasu 2000). The transformed plantcells can continue to produce hairy roots even after Agrobacterium was elim-inated. Hairy roots grow vigorously in a phytohormone-free medium andprovide a useful material for studies on secondary metabolite production(Kamada et al. 1986, Bourgaud et al. 2001, Facchini 2001, Kim et al. 2002).But, there are very few physiological investigations into hairy root growth(Kato et al. 1989, Bonhomme et al. 2000, Tanaka et al. 2001) and lateralbranch development on hairy roots (Morgan and Shanks 2000, Balvanyos etal. 2001).In this report, we investigated the involvement of calmodulin in thegrowth of hairy roots and the development of lateral branches on the roots,80Hairy roots and calmodulinusing W-7 and W-5 as calmodulin antagonists. W-7 and W-5 can penetratecytoplasmic membrane and distribute mainly in cytoplasm (Hidaka et al.1981).2 Materials and MethodsPlant material—The procedures for the induction of hairy roots were de-scribed by Kato et al. (1989) and by Shimomura et al. (1991). Carrot seeds(Daucus carota L. cv. US-Harumakigosun) were sown on vermiculite at 25◦Cunder the continuous light (ca. 3 k lux). One-week-old seedlings were surface-sterilized with 1% sodium hypochlorite solution for 15min and rinsed threetimes with sterilized distilled water. Sterile belladonna (Atropa belladonnaL.) and Lithospermum erythrorhizon Sieb. et Zucc. plants were obtained byculturing the shoot tips of ﬁeld-grown plants. They were maintained by re-peated shoot culture on a Murashige and Skoog’s (MS) medium containing3% sucrose and 1% agar, at 25◦C under a 18-h light (ca. 4 k lux) / 6-h darkcondition. Carrot hypocotyls, belladonna and L. erythrorhizon internodeswere cut into 20-mm-long segments and placed with the basal part uppermoston the MS medium. The cut ends of these segments were inoculated by a nee-dle with Agrobacterium rhizogenes, which had been grown on YEB medium(Vervliet et al. 1974) containing 1.5% agar. The segments of belladonna andL. erythrorhizon were infected with A. rhizogenes strain 15834 and those ofcarrot were infected with strain 2659. Hairy roots appeared at the inocula-tion sites three weeks later. The growing root tips of the hairy roots werecut oﬀ and cultured on an MS medium containing 3% sucrose, 1% agar andan antibiotic (1mg/ml carbenicillin) at 25◦C under a 16-h light (ca. 3 k lux)/ 8-h dark condition, to eliminate the bacteria from the roots. The roottips were subcultured three more times at two-week intervals on a fresh MSmedium supplemented with the antibiotic. The root tips of belladonna andcarrot hairy roots were re-subcultured on an MS medium containing 3% su-crose and 0.2% Gellan Gum (Kelco, Division of Merck & Co., Inc., San Diego,U.S.A) without antibiotic at monthly intervals at 25◦C. The tips of L. ery-throrhizon hairy roots were re-subcultured at two-month intervals at 25◦C onthe same medium and later on a Root Culture (RC) medium [EMBO coursemanual 1982 as follows (mg/l): Ca(NO3)2 ·4H2O (288), KNO3 (80), KCl (65),MgSO4 ·7H2O (370), Na2SO4 ·10H2O (226.7), NaH2PO4 ·4H2O (21.5), H3BO3(1.5), ZnSO4 ·7H2O (2.65), KI (0.75), Na2MoO4 ·2H2O (0.25), CuSO4 ·5H2O(0.02), MnCl2 ·4H2O (6.0), FeCl3 (3.2), EDTA-2Na (7.4), thiamine-HCl (0.1),nicotinic acid (0.5), pyridoxin-HCl (0.1) and glycine (3.0)] containing 1.5%sucrose and 0.3% Gellan Gum (pH 4.9).81Ryoichi Kato, Miyuki Arashida, Masahiro Kodama, Hiroshi Kamada and Takashi SuzukiIncubation with calmodulin antagonists—Segments (length: 15mm) ofroot tips were cut from these hairy roots. Ten segments of belladonna andcarrot hairy roots were put side by side in a Petri dish (diameter: 94mm)on an MS medium containing 3% sucrose, 0.2% Gellan Gum and variousconcentrations (zero, 0.1, 1, 10, 30 and 100µM) of W-7 or W-5. Ten seg-ments of L. erythrorhizon hairy roots were put side by side in the same dishon a RC medium containing 1.5% sucrose, 0.3% Gellan Gum and variousconcentrations of W-7 or W-5. The dishes were covered with a double thick-ness of aluminum foil. They were incubated at 24◦C for 6, 8, 13 or 16 days.Growth of each hairy root was measured with a slide caliper. At the carrothairy roots, the number of lateral branches formed on the roots was visuallycounted.W-7 and W-5 were obtained from Seikagaku Corporation, Tokyo, Japan.3 ResultsEstablished cultures of hairy roots exhibited rapid growth with lateralroots on the hormone-free media. In order to conﬁrm that true transfor-mation had occurred, the presence of opines (mannopine and agropine, orcucumopine) was monitored by the method described earlier (Kamada et al.1986, Isogai et al. 1990), and both opines were detected (data not shown).Growth rates—Thirty segments of Lithospermum erythrorhizon hairy rootswere cultured on a RC medium containing zero, 0.1, 1, 10, 30 or 100µMW-7for 13 days. Growth rates of the hairy roots cultured on the all media con-taining W-7 were lower than that of the roots cultured without W-7 (zero),and then the rates of the roots became lower as W-7 concentrations increased(Fig. 1). Segments of L. erythrorhizon hairy roots were cultured for 13 dayson the medium containing W-5 in the same concentrations of W-7. Growthrates of hairy roots cultured on the media containing W-5 below 10µM wereequal to that of the roots cultured without W-5 (zero), but then the ratesof the roots cultured on the media containing W-5 above 30µM were lowerthan that of the roots cultured without W-5 (Fig. 1).We tried to determine whether the calmodulin antagonists inhibit thegrowth of hairy roots in other plant species. Segments of belladonna hairyroots were cultured on an MS medium containing zero, 0.1, 1, 10, 30 or100µM of W-7 or W-5 for 6 days. Growth of the roots was inhibited byW-7 above 0.1µM or by W-5 above 1µM (Fig. 2). Growth rates of the rootscultured on the medium containing W-7 were lower than those of the rootscultured on the medium containing W-5 at 10, 30 and 100µM antagonists,respectively. And then a diﬀerence between inhibition rates by W-7 and W-582Hairy roots and calmodulinFig. 1. Eﬀects of W-7 and W-5 on the growthof Lithospermum erythrorhizon hairy roots. Seg-ments (length: 15mm) of the tips of L. erythrorhi-zon hairy roots were cultured on an MS mediumcontaining zero, 0.1, 1, 10, 30 or 100µM W-7 (●)or W-5 (○), in the dark at 24◦C for 13 days. Eachpoint represents the average of measurements fromat least 30 segments of hairy roots, with the stan-dard error.Fig. 2. Eﬀects of W-7 and W-5 on the growth ofbelladonna hairy roots. Segments of the tips ofbelladonna hairy roots were cultured on an MSmedium containing zero, 0.1, 1, 10, 30 or 100µMW-7 (●) or W-5 (○), in the dark at 24◦C for 6days. Each point represents the average of mea-surements from at least 30 segments of hairy roots,with the standard error.became higher as antagonists concentrations increased (Fig. 2).Segments of carrot hairy roots were cultured on an MS medium containingzero, 0.1, 1, 10, 30 or 100µM of W-7 or W-5 for 8 days. Growth rates of theroots cultured on three media containing 0.1, 1 and 10µM antagonists wereequivalent to those of the roots cultured without antagonist (zero) (Fig. 3).Thirty micro-mole W-5 did not inhibit the root growth, but 30µM W-7 did(Fig. 3). Inhibition rate of the root growth by 100µM W-7 was higher thanthat of the growth by 100µM W-5 (Fig. 3).Number of lateral branches—The eﬀects of the calmodulin antagonists onthe development of lateral roots on hairy roots were investigated in the nextexperiments. Thirty segments of carrot hairy roots were cultured on an MSmedium containing zero, 0.1, 1, 10, 30 or 100µM of W-7 or W-5 for 16 days.The number of the lateral roots formed on the media containing antagonists83Ryoichi Kato, Miyuki Arashida, Masahiro Kodama, Hiroshi Kamada and Takashi SuzukiFig. 3. Eﬀects of W-7 and W-5 on the growth ofcarrot hairy roots. Segments of the tips of car-rot hairy roots were cultured on an MS mediumcontaining zero, 0.1, 1, 10, 30 or 100µM W-7 (●)or W-5 (○), in the dark at 24◦C for 8 days. Eachpoint represents the average of measurements fromat least 30 segments of hairy roots, with the stan-dard error.Fig. 4. Eﬀects of W-7 and W-5 on the developmentof lateral roots on carrot hairy roots. Segmentsof the tips of carrot hairy roots were cultured onan MS medium containing zero, 0.1, 1, 10, 30 or100µM W-7 (●) or W-5 (○), in the dark at 24◦Cfor 16 days. Each point represents the average ofmeasurements from at least 30 segments of hairyroots, with the standard error.below 10µM was equal to that of the roots formed on an antagonist-freemedium (zero), and then the number of lateral roots formed on antagonistsabove 30µM was lower than that of the roots formed on an antagonist-freemedium (Fig. 4). At 30 and 100µM the antagonists, the number of lateralroots induced on the medium containing W-7 was lower than that of theroots induced on the medium containing W-5 (Fig. 4).4 DiscussionCalmodulin regulates various physiological responses in animal and plantcells (Dieter 1984, Sun 2000, Chung et al. 2000, Lenartowska et al. 2001).It was shown that calmodulin involves in growth of adventitious roots in-duced in stem cuttings of Tradescantia fluminensis, by an incubation of theroots in a solution containing triﬂuoperazine or chlorpromazine (Muto andHirosawa 1987). These calmodulin antagonists, however, tightly combine84Hairy roots and calmodulinwith cytoplasmic membrane and damage the cells without direct inhibitionof the calmodulin action (Osborn and Weber 1980). Applications of theseantagonists to intact cells or tissues are unﬁt for a detection of the calmod-ulin function. Hairy roots, which are originated from transformation of plantcells by the Ri plasmid of Agrobacterium rhizogenes, have been induced fromsome plant materials to produce secondary metabolites (Kamada et al. 1986,Bourgaud et al. 2001, Facchini 2001, Kim et al. 2002). But, only a few studieshave been performed on the analyses of hairy roots growth (Kato et al. 1989,Bonhomme et al. 2000, Tanaka et al. 2001) and lateral branch developmenton hairy roots (Morgan and Shanks 2000, Balvanyos et al. 2001).We determined involvement of calmodulin in the growth of hairy rootsusing W-7 and W-5, which are able to penetrate cytoplasmic membrane(Hidaka et al. 1981). Segments of L. erythrorhizon, belladonna and carrothairy roots were cultured on media containing various concentrations of W-7. Growth rates of L. erythrorhizon and belladonna hairy roots culturedon the media containing W-7 were lower than those of the roots culturedwithout W-7 (Figs. 1 and 2). Growth of carrot hairy roots was inhibited byW-7 above 30µM (Fig. 3). Segments of those roots were cultured on mediacontaining W-5, a chlorine-deﬁcient analogue of W-7 that weakly interactswith calmodulin (Hidaka et al. 1981), at the same concentrations of W-7 tomake a close examination of the role of calmodulin in the growth of hairyroots. Inhibition rates of the root growth by high concentrations of W-5 werelower than those of the growth by the same concentrations of W-7 (Figs. 1, 2and 3). These results strictly suggest that calmodulin is concerned in hairyroot growth. To the best of our knowledge, this is the ﬁrst report provedexactly on involvement of calmodulin in the growth of plant roots.In order to determine whether the calmodulin antagonists inhibit devel-opment of lateral roots on hairy roots, segments of carrot hairy roots werecultured on an MS medium containing various concentrations of W-7 or W-5.At 30 and 100µM the antagonists, both W-7 and W-5 inhibited formation oflateral roots and the number of lateral roots formed on the medium contain-ing W-7 was lower than that of the roots formed on the medium containingW-5 (Fig. 4). These results show that calmodulin acts upon the developmentof lateral roots on hairy roots.The involvement of calmodulin in cell division in plant cells is suggestedby the following facts. (1) Young dividing and growing cells contained morecalmodulin than matured non-growing cells in pea seedlings (Muto and Miya-chi 1984). (2) Distribution of calmodulin related to that of tubulin in thecell cycle of onion and pea root meristematic cells (Wick et al. 1985). (3)Calmodulin was localizing at kinetochore microtubles in the mitotic appara-tus in Haemanthus endosperm cells (Vantard et al. 1885). Growth of hairy85Ryoichi Kato, Miyuki Arashida, Masahiro Kodama, Hiroshi Kamada and Takashi Suzukiroots consists of cell division and cell elongation. An important event inthe development of lateral roots on hairy roots is cell division, which causesprimordium formation of lateral roots. Inhibition rate of hairy root growthby each concentration of W-7 or W-5 shown in Figure 3 is similar to thatof formation of lateral roots of the roots by W-7 or W-5 shown in Figure 4.These results suggest that calmodulin plays an important role in cell di-vision in hairy roots. These may have some reference to the reports thatcalmodulin-binbing protein is involved in cell division in pea seedlings (Dayet al. 2000) and that expression of calmodulin mRNA is closely correlatedwith cell division in tobacco anthers (Chen et al. 1999).References[Balvanyos et al. 2001] Balvanyos, I., L. Kursinszki and E. Szoke, PlantGrowth Regul. 34: 339-345. 2001.[Bonhomme et al. 2000] Bonhomme, V., D. Laurain-Mattar and M. A. Flini-aux, J. Nat. Prod. 63: 1249-1252. 2000.[Bourgaud et al. 2001] Bourgaud, F., A. Gravot, S. Milesi and E. Gontier,Plant Sci. 161: 839-851. 2001.[Chen et al. 1999] Chen, S. R., Y. T. Lu and H. Y. Yang, Chinese Sci. Bull.44: 142-146. 1999.[Chung et al. 2000] Chung, W. S., S. H. Lee, J. C. Kim, W. D. Heo, M. C.Kim, C. Y. Park, H. C. Park, C. O. Lim, W. B. Kim and J. F. Harper,Plant Cell 12: 1393-1407. 2000.[Day et al. 2000] Day, I. S., C. Miller, M. Golovkin and A. S. N. Reddy, J.Biol. 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Inhibitions of Growth and Lateral Branch Development by Calmodulin Antagonists in Hairy Roots of Lithospermum erythrorhizon, Atropa belladonna and Daucus carota

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Inhibitions of Growth and Lateral Branch Development by Calmodulin Antagonists in Hairy Roots of Lithospermum erythrorhizon, Atropa belladonna and Daucus carota

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