The effect of ethylene on auxin-induced growth of excised rice coleoptile segments

Elongation growth induced by exogenous auxin of apical coleoptilesegments of etiolated rice seedlings was promoted by ethylene.In the absence of exogenous auxin, growth promotion was notobserved. The highest promotion by ethylene was obtained at10^–6 M of indole-3-acetic acid, a suboptimal concentrationfor auxin-induced elongation. Level of ethylene which achievedthe effect was less than 1 µl per liter of an incubationatmosphere. ¹Present address: The Ocean Research Institute, University ofTokyo, Nakano, Tokyo, Japan (Received May 27, 1970; )     

Involvement of Calcium in Adventitious Bud Initiation in Torenia Stem Segments

In Torenia stem segments cultured in vitro, active meristematicdivisions are induced in the epidermis by treatment with cytokinin,resulting in the formation of adventitious buds. Applicationof the calcium ionophore A23187 [GenBank] was found to induce meristematicdivisions in the absence of cytokinin. The induction by A23187 [GenBank] was inhibited by simultaneous addition of auxin, but not byanti-cytokinin. A two hour pre-treatment with A23187 [GenBank] was alsoeffective, but only when it was applied to the explants justafter their excision from mother plants. The A23187 [GenBank] -inducedmeristematic zones developed into dome-shaped structures, butnot into complete adventitious buds. Complete elimination ofcalcium from the culture medium caused 50% inhibition of A23187 [GenBank] -and/or cytokinin-induced initiation of meristematic divisions.When the explants were preincubated with EGTA and then culturedon a Ca-free medium containing EGTA, cytokinin failed to inducebud initiation. Similar inhibition was also obtained by lanthanum,a calcium antagonist, by verapamil, a calcium channel inhibitor,and by trifluoperazine and chlorpromazine, calmodulin inhibitors.These results support the idea that adventitious bud initiationinduced by cytokinin in Torenia stem segments may be mediated,at least partially, by an increase in the level of intracellularCa²⁺. ¹Bioscience Research Center, Mitsui Petrochemical IndustriesLtd., Waki-cho, Kuga-gun, Yamaguchi 740, Japan. (Received May 9, 1985; Accepted October 5, 1985)     

THE RELATIONSHIP OF GIBBERELLIN AND AUXIN IN PLANT GROWTH

No synergism was found between IAA and gibberellin in the Avenucurvature test and this bioassay thus measures changes in diffusibleauxin resulting from gibberellin treatment and not a synergisticaction of the gibberellin on the curvature response to auxin.Gibberellin treatment causes an increase in diffusible auxinfrom the stem apex of dwarf pea (Pisum sativum L. var. LittleMarvel) 24 to 48 hours before the elongation response in thestem. The increase in diffusible auxin in the stem apex of Centaureacyanus L. var. Blue Boy occurs four to six days before the boltingresponse to gibberellin treatment under short days. The stemtissues of both the dwarf pea and Centaurea show an elongationresponse to IAA when the IAA is applied in a manner simulatingthe stem apex. Thus the growth of the dwarf pea and the boltingof Centaurea brought about by treatment with gibberellin aredependent on an increase in diffusible auxin. ¹Present address: Biological Institute, College of General Education,University of Tokyo, Komaba, Meguro, Tokyo.     

THE MECHANISM OF GIEBERELLIN ACTION IN THE DWARF PEA

The increase in diffusible auxin in the dwarf pea followingtreatment with gibherellin has been shown not to involve theinhibition of IAA oxidase, the rate of basipetal auxin transport,decrease in growth inhibitor, the formation of a complex betweengibberellin and auxin, or the enzymatic conversion of tryptophanto ether-soluble auxin. The tryptophan conversion system fromplants treated with gibberellin formed four times more water-solubleauxin than did the enzyme preparation from control plants. Thusthe increase in water-soluble auxin appears to be the majorcause of the higher auxin level in plants treated with gibberellin. ¹Present address: Biological Institute, College of General Education,University of Tokyo, Komaba, Meguro, Tokyo.     

Effect of osmotic shock on auxin-induced cell extension, cell wall changes and acidification in Avena coleoptile segments

The effect of plasma membrane alteration caused by osmotic shockof different strengths on the auxin-induced responses of Avenacoleoptile cells was observed. Osmotic shock brought about by0.5–0.7 M mannitol solution for 10 or 30 min, followedby phosphate-buffer (1 mM, pH 6.0) treatment for 10 min at 4?Ccaused no significant inhibition of auxin-induced cell extension.The osmotic shock did not affect auxin-induced cell wall looseningrepresented by stress-relaxation time and a decrease in thenoncellulosic glucose level of the cell wall. The shock causedonly a temporary inhibition of transmembrane potential and noinhibition of oxygen consumption. However, it inhibited auxin-stimulatedH⁺ secretion which was reversed by 0.1 mM CaCl₂. We concludedthat the Osmotic shock may partly modify the plasma membranerelated to the hydrogen ion pump which interacts with auxin,but this modification which is reflected little by the transmembranepotential and cellular metabolism, is not closely related toauxin-induced cell wall loosening and thus cell extension inAvena coleoptiles. ³ Present address: Department of Botany, Faculty of Science,University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113, Japan (Received February 17, 1978; )     

Senescence of Rice Leaves XXIV. Involvement of Calcium and Calmodulin in the Regulation of Senescence

Effects of compounds that influenced calcium uptake and calmodulininhibitors on the senescence of detached rice leaves were examined.Chelators, ethyleneglycol-bis-(ß-aminoethyl ether)-N,N,N',N'-tetraaceticacid (EGTA) and l,2-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraaceticacid (BAPTA), significantly promoted senescence of detachedrice leaves in the dark and light. The effect of EGTA can bereversed by treating detached rice leaves with calcium. Verapamil,a calcium channel blocker, and lanthanum chloride, a calciumantagonist, promoted dark-induced, and suppressed BA- and light-retardedsenescence of detached rice leaves. Calcium ionophore A23187 [GenBank] and ruthenium red, believed to raise cytosolic level of Ca²⁺,were quite effective in retarding dark-induced and ABA-promotedsenescence of detached rice leaves. Calmodulin inhibitors, W-7,compound 48/80, chlorpromazine and trifluoperazine, significantlypromoted dark-induced, and suppressed BA- and light-retardedsenescence of detached rice leaves. It is concluded that cytosoliclevel of Ca²⁺ may regulate senescence of detached rice leavesthrough a calmodulin-dependent mechanism. (Received June 13, 1990; Accepted August 3, 1990)     

Auxin — Calcium Interaction in Adventitious Root Formation in the Hypocotyl Explants of Sunflower (Helianthus annuus L.)

Auxin-calcium interaction has been studied to understand their involvement in adventitious root initiation from the hypocotyl explants of sunflower (Helianthus annuus L.). When hypocotyl explants were cultured on MS medium (containing calcium), 1 mg l^-1 IAA was found to be optimal for root induction. However, the hypocotyl explants washed in EGTA (10_-5M) solution for the removal of extracellular calcium, when cultured on medium containing IAA and calcium, exhibited enhanced rooting response. When EGTA-washed explants were cultured on the medium supplemented with lanthanum chloride (10^-6 and 10^-5M), it resulted in the inhibition of the rooting response and this inhibitory effect could be alleviated by the simultaneous addition of IAA. Similar observations have been made by using calcium channel blockers, verapamil and TMB-8, and also a calmodulin inhibitor, trifluoperazine. A net influx of extracellular calcium in the differentiating cells is thus presumed to accompany the auxin-induced response. These results have been discussed in light of initial lack of polarity in the decapitated hypocotyl segments subjected to auxin treatment.     

Functional Correlation between Endogenous Phytohormone Levels and Hormone Autotrophy of Transformed and Habituated Soybean Cell Lines

We analysed the time course of the endogenous free IAA and cytokininlevels in hormone requiring and hormone autotrophic (both transformedand untransformed) Glycine max. L. Merr. cv. Mandarin tissuecultures. The auxin habituated line showed an enhanced endogenous IAAlevel, whereas the IAA as well as the cytokinin concentrationsin the cytokinin habituated line differed not significantlyfrom the non-habituated hormone requiring soybean callus. It were only the auxin habituated cells that could be inducedto fully habituated cells, from which a pale and a green typewas isolated. The phytohormone autotrophic growth of the paletype was sustained by enhanced IAA levels, whereas the greentype was characterised by elevated cytokinin concentrations. These results on the phytohormone content of partially and fullyhabituated soybean calli were compared with soybean crown galllines and discussed in view of the positive effect of exogenouslyapplied cytokinins on the endogenous IAA levels. ³Recipient of an Instituut voor Wetenschappelijk Onderzoek inNijverheid en Landbouw (I.W.O.N.L.) grant. ⁴Senior Research Associate Nationaal Fonds voor wetenschappelijkOnderzoek (N.F.W.O.). (Received March 25, 1988; Accepted July 7, 1988)     

THE ROLE OF AUXIN IN THE PHOTOPERIODIC TUBERIZATION IN BEGONIA EVANSIANA ANDR.

The formation of aerial tubers in Begonia plants, an SD response,was inhibited by IAA, NAA and IBA applied to their leaves duringthe dark periods. The effectiveness of IAA differed accordingto the time of application during the dark periods, and themost sensitive time varied with daylengths employed. In order to inhibit the tuberization under optimal photoperiods(8-hr SDs), IAA had to be applied during the first 2 days orso of the SDs. Under non-optimal photoperiods, however, IAAwas effective even when applied somewhat later. The auxin activity of leaf extracts from the plants subjectedto 8-hr SDs decreased during the first 2 or 3 days to a minimum,and then increased until finally began to decrease, again; undernon-optimal photoperiods, the minimum of auxin activity wasattained more slowly. The paper-chromatographic study suggestedthat the change in auxin activity was mainly due to the changein IAA content. The number of SDs making the auxin content minimal agreed withthe minimum number of SDs required for tuberization. On the basis of the above results, the part played by endogenousauxin in photoperiodic induction is discussed. ¹Present address: Institute for Agricultural Research, TôhokuUniversity, Sendai. ²Present address: Biological Institute, Yamaguchi University,Yamaguchi.     

Mechanism of cytokinin action on auxin-induced ethylene production

The stimulative effect of cytokinin on ethylene production hasbeen examined in etiolated mungbean hypocotyl segments. Therate of auxin-induced production linearly increased with timein a certain range of exogenous IAA concentration. The rateof induced production doubled with a 10-fold increase in exogenousIAA concentration or addition of benzyladenine at 5 µM.Benzyladenine did not suppress inactivation of the induced ethyleneproducing system. Although the free IAA level within the tissueswas slightly increased by benzyladenine accompanied by a decreasein IAAsp formation, the increased free IAA level did not accountfor the doubling of the production rate. When the tissues werepreincubated with benzyladenine alone followed by postincubationwith auxin, the rate of induced production in pretreated tissueswas significantly higher than that in untreated or buffer-treatedtissues supplied with auxin and benzyladenine simultaneouslyin the post-incubation medium. Formation and disappearance ofthe cytokinin effect were temperature dependent. The rate ofendogenous production was constant over an experimental periodand benzyladenine simply enhanced the rate several-fold aftera lag period. Kinetics of the cytokinin stimulation was notthe inductive type. Based on these observations, a possiblemechanism of the stimulative effect of cytokinin was discussed. ¹This research was partly supported by grants from the Ministryof Education of Japan (C-856043 and C-956037) and the AsahiPress. (Received May 7, 1975; )     

Electric field-directed cell shape changes, displacement, and cytoskeletal reorganization are calcium dependent

C3H/10T1/2 mouse embryo fibroblasts were stimulated by a steady electric field ranging up to 10 V/cm. Some cells elongated and aligned perpendicular to the field direction. A preferential positional shift toward the cathode was observed which was inhibited by the calcium channel blocker D-600 and the calmodulin antagonist trifluoperazine. Rhodaminephalloidin labeling of actin filaments revealed a field-induced disorganization of the stress fiber pattern, which was reduced when stimulation was conducted in calcium-depleted buffer or in buffer containing calcium antagonist CoCl2, calcium channel blocker D-600, or calmodulin antagonist trifluoperazine. Treatment with calcium ionophore A23187 had similar effects, except that the presence of D-600 did not reduce the stress fiber disruption. The calcium-sensitive photoprotein aequorin was used to monitor changes in intracellular-free calcium. Electric stimulation caused an increase of calcium to the micromolar range. This increase was inhibited by calcium-depleted buffer or by CoCl2, and was reduced by D-600. A calcium-dependent mechanism is proposed to explain the observed field-directed cell shape changes, preferential orientation, and displacement.     

Growth and cell wall changes in rice coleoptiles growing under different conditions II. Auxin-induced growth in coleoptile segments

The effect of auxin on growth, mechanical properties of thecell wall and cell wall sugar composition in rice coleoptilesegments excised at different ages from seedlings growing underdifferent conditions were investigated. Auxin markedly inducedgrowth only in segments excised from coleoptiles in the fastgrowth phase with a high content of non cellulosic glucose intheir cell walls. The response to auxin decreased with coleoptileage, accompanying a decrease in the amount of the noncellulosicglucose in the cell wall, suggesting a correlation between noncellulosicglucose content and growth capacity in response to auxin. Goodcorrelation among auxin-induced growth, auxin-induced decreasein the T_o value and auxin-induced decrease in the noncellulosicglucose content of the cell wall also was found. ¹ Present address: Departamento Fisiologia Vegetal, Facultadde Ciencias, Universidad de Salamanca, Salamanca, Spain. (Received May 21, 1979; )     

Rapid auxin-induced root growth inhibition requires the TIR and AFB auxin receptors

We investigated the relation between auxin-induced gene expression and the rapid auxin-induced growth inhibition in Arabidopsis thaliana roots. The natural auxin indole-3-acetic acid (IAA) induced a strong activation of gene expression as visualized by the DR5rev::GFP reporter gene technique. This effect was specific for active auxins and was abolished in knockout mutants of the F-box auxin receptors. We measured the IAA-induced growth inhibition at high time resolution and show that the F-box auxin receptor mutants failed to display this effect. We conclude that the F-box auxin receptors are needed for the response. In hypocotyls, auxin induces an increase in elongation growth, and this effect has been earlier shown to be independent of the F-box receptors. Based on these findings, we discuss differences in the growth control modes in roots and shoots. We demonstrate that the rapid auxin-induced root growth inhibition, unlike the induction of growth in hypocotyls, requires the presence of the F-box auxin receptors.     

Calcium Involvement In the IAA-induced Leaflet Opening of Cassia fasciculata

Opening of Cassia fasciculata leaflets was induced in darknessafter application of indole-3-acetic acid (IAA). This movementwas obtained with concentrations from 10^–6 M to 10^–4M, after a corresponding time-lag ranging from 120 to 30 min.IAA (5x10^–5 M) allowed leaflet opening at all the pH valuestested (from 3·5 to 7·5), the largest aperturebeing obtained at pH 60 in MES 2·5 mM. Our data suggesta functional involvement of calcium in the regulation of theturgor variations occurring in the pulvinar motor cells duringIAA-induced leaflet opening which occurs in darkness: indeed,this movement was inhibited by the Ca²⁺ chelator EGTA (thisinhibition was reversed by CaCl²) or by antagonists (LaCl³,TMB-8); on the contrary, the IAA-opening was enhanced by ionophoreA 23187. Calcium mobilization through specific channels was tested usingantagonists such as verapamil and nifedipine: at physiologicaldoses, these compounds did not significantly affect leafletresponse. The possibility that calcium could originate frominternal stores was checked using lithium chloride which isknown to block the phosphatidylinositol cycle in animal cells.This compound hindered auxin-induced opening for concentrationshigher than 5x10^–4 M. The calcium-binding protein calmodulinwas shown to be implicated in the IAA-induced response sinceopening was inhibited in a concentration-dependent manner aftertreatment with compound 48/80 and with W-7. Key words: Cassia fasciculata, auxin, calcium, second messenger, turgor regulation     

Rapid induction of specific mRNAs by auxin in pea epicotyl tissue

DNA sequences complementary to three indoleacetic acid (IAA)-inducible mRNAs in pea epicotyl tissue were isolated by differential plaque filter hybridization of cDNA libraries constructed in the vector lambda gt10. Clone pIAA6 hybridized to an mRNA encoding the previously identified translational product polypeptide 6 (Mr 22,000), and clone pIAA4/5 hybridized to one or two mRNAs, encoding polypeptides 4 and 5 (Mr 23,000 and 25,000, respectively). The cDNA clones were subsequently used to characterize the hormonally mediated mRNA accumulation. The induction of the mRNAs was rapid, within 15 minutes of exposure to the IAA, and specific to auxins. Anaerobiosis, heat and cold stress did not induce the mRNAs. Other plant hormones, such as gibberellic acid, kinetin, abscisic acid and ethylene were also unable to cause or interfere with the IAA-induced mRNA accumulation. The hormonally regulated mRNAs were induced at least 50 to 100-fold above control levels after two hours of treatment with IAA and the accumulation was (1) independent of protein synthesis, (2) completely abolished by alpha-amanitin, (3) not due to polyadenylylation of pre-existing RNAs, and (4) independent of IAA and fusicoccin-induced H+ secretion. The IAA-induced mRNAs returned to control levels within three hours after removal of IAA, and the hormonally regulated genes were primarily expressed in the third and second internode of the seven-day-old etiolated pea seedling. The data indicate that IAA increases the amount of specific mRNAs rather than alters the translatability of pre-existing mRNAs. Auxin-induced H+ secretion appears not to have a potential role in mediating the induction and perhaps is a consequence of the enhanced biosynthetic activity induced by the hormone. The IAA-mediated mRNA induction is the fastest known for any plant growth regulator and may represent a primary hormonal response to auxin.     

STUDIES ON THE GROWTH OF FRUIT BODY OF FUNGI: III. OCCURRENCE, FORMATION AND DESTRUCTION OF INDOLE ACETIC ACID IN THE FRUIT BODY OF AGARICUS BISPORUS (LANGE) SING

Considerable amounts of auxin, mostly IAA, in acid and boundforms, occur in gills, pilei and stipes of the mushroom, Agaricusbisporus. Stipe elongation is not stimulated by applicationof IAA. The juice squeezed from the fruit body has an activity to convertL-tryptophan into IAA. This activity is not lost by heating.The substance(s) responsible for it passes through cellophane,and is insoluble in some organic solvents, such as petroleumether and benzene. The amount of IAA produced depends exactlyon the amount of the extraction residue used for the reaction.Since the activity decreases as thiosulfate is added increasingly,the active principle seems to be some strong oxidizing substance(s). ¹ This paper was read on October 25th, 1958, at the 23rd annualmeeting of the Botanical Society of Japan, held at Fukuoka,Japan. ² This investigation has been aided by a grant from the ROCKEFELLERFoundaion. ³ Present Address: J. W. GIBBS Research Laboratory, Departmentof Botany, Yale University. New Haven, Conn., U.S.A. (Received June 6, 1961; )     

Role of calcium–calmodulin in auxin-induced somatic embryogenesis in leaf base cultures of wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> var. HD 2329)

Wheat leaf bases cultured for 1 day on 2,4-d (10 μM) display the induction of somatic embryogenesis. The induction of somatic embryogenesis by 2,4-d appears to be calcium-mediated as treatment of leaf bases with the calcium chelator, EGTA, prior to 2,4-d treatment, inhibited the induction of somatic embryogenesis. This sensitivity of auxin to reduced calcium levels can be reversed by calcium ions alone and not any other divalent cation like magnesium or zinc. Additionally, the expression of the three calcium-regulated genes, Triticum aestivum calmodulin binding protein kinase, calcium-dependent protein kinase, and putative calcium binding protein was analyzed in wheat leaf bases which suggest a specific role for Ca²⁺ in somatic embryogenesis. Application of the calcium ionophore, A23187, either alone or along with 2,4-d, induced somatic embryogenesis. This specificity for calcium was verified both by treatment with the calcium antagonist TMB8, and the elimination of calcium from the medium, resulting in reduction of somatic embryogenesis by 80%. Treatment with calcium channel blockers like verapamil and nifedipine, calcium antagonist, lanthanum, and calmodulin inhibitors chlorpromazine and fluphenazine, prior to the 2,4-d treatment, inhibited induction of somatic embryogenesis. The present study thus provides evidence for the involvement of calcium–calmodulin in the stimulus–response coupling of auxin-induced somatic embryogenesis in wheat leaf base system.     

Auxin Enhancement of mRNAs in Epidermis and Internal Tissues of the Pea Stem and Its Significance for Control of Elongation

The epidermis has been considered the site of auxin action on elongation of stems and coleoptiles. To try to identify mRNAs that might mediate auxin stimulation of cell enlargement, we compared, using in vitro translation assays, mRNA enhancement by indoleacetic acid (IAA) in the epidermis, with that in the internal tissues, of pea (Pisum sativum L., cv Alaska) third internode segments. We used seedlings that had been grown under red light, which enables the epidermis to be peeled efficiently from the internode. Most of the `early' IAA enhancements previously reported using etiolated peas, plus several hitherto undescribed enhancements, occur in both the epidermis and the internal tissue of the light-grown plants after 4 hours of IAA treatment. These enhancements, therefore, do not fulfill the expectation of elongation-specific mRNAs localized to the epidermis. One epidermis-specific IAA enhancement does occur, but begins only subsequent to 1 hour (but before 4 hours) of auxin treatment. Similarly, the previously mentioned IAA enhancements common to epidermis and internal tissue do not begin, in the light-grown plants, within 1 hour of IAA treatment. Since IAA stimulates elongation in light-grown internodes within 15 minutes, it appears that none of these mRNAs can be responsible for auxin induction of elongation. We confirmed, with our methods, the previous reports that some of these mRNAs are enhanced by IAA within 0.5 hour in etiolated internodes. This indicates that we could have detected an early enhancement in light-grown tissue had it occurred.     

Interaction of D-600 with the transmembrane domain of the sarcoplasmic reticulum Ca(2+)-ATPase

Experiments were performedto determine whether the organic Ca²⁺ channel blocker D-600(gallopamil), which penetrates into muscle cells, affects sarcoplasmicreticulum (SR) Ca²⁺ uptake by directly inhibiting the lightSR Ca²⁺-ATPase. We have previously shown that at 10 µM,D-600 inhibits LSR ATP-dependent Ca²⁺ uptake by 50% buthas no effect on ATPase activity (21). These data suggestthat the SR Ca²⁺-ATPase might be a potential target forD-600. The ATPase activity of the enzyme is associated with itshydrophilic cytoplasmic domain, whereas Ca²⁺ binding andtranslocation are associated with the transmembrane domain(18). In the present experiments, we determined which of the two domains of the ATPase is affected by D-600. Thermalinactivation experiments using the SR Ca²⁺-ATPasedemonstrated that D-600 decreased the thermal stability ofCa²⁺ transport but had no effect on the stability of ATPaseactivity. In addition, D-600 at a concentration of 160 µM did nothave any leaking effect of Ca²⁺ on theCa²⁺-loaded SR. Thermal denaturation profiles of SRmembranes revealed that D-600 interacts directly with the transmembranedomain of the Ca²⁺-ATPase. No evidence for interaction withthe nucleotide domain was obtained. We conclude that theCa²⁺ blocker D-600 inhibits the SR Ca²⁺ pumpspecifically by interacting with the transmembraneCa²⁺-binding domain of the Ca²⁺-ATPase.

     

Effects of Growth Regulators on the Induction of Anthocyanin Synthesis in Carrot Suspension Cultures

The effects of plant growth regulators were investigated onanthocyanin synthesis induced by removing auxin from carrotsuspension cultures. Of the auxins tested, 2,4-D showed thestrongest inhibiting effect on anthocyanin synthesis and hadthe strongest promoting effect on undifferentiated growth. When2,4-D was added to anthocyanin synthesizing cells, in whichcell division had ceased, anthocyanin synthesis was repressedimmediately, accumulated anthocyanin disappeared and cell divisionresumed. All cytokinins examined promoted anthocyanin synthesisin the absence of auxin. Both gibberellic acid (GA₃) and abscisicacid inhibited anthocyanin synthesis in media lacking 2,4-D,though GA³ showed no effect on cell division. These effectsof growth regulators on anthocyanin synthesis are similar tothose reported for their effects on embryogenesis [Fujimuraand Komamine (1975) Plant Sci. Lett. 5: 359, (1979) Z. Pflanzenphysiol.95: 13, (1980) Z. PJlanzenphysiol. 99: 1]. The relationshipbetween the induction of anthocyanin synthesis, metabolic differentiation,and embryogenesis are discussed. ¹ Present address: Department of Biology, College of Arts andSciences, The University of Tokyo, Komaba, Meguro-ku, Tokyo153, Japan. ² Present address: Biological Institute, Faculty of Science,Tohoku University, Sendai, Miyagi 980, Japan. (Received November 28, 1985; Accepted July 23, 1986)