Dormancy in Dioscorea: Sprouting promotion by inhibitors of protein synthesis in bulbils and rhizomes

The sprouting of immature and half-dormant mature bulbils andrhizomes in some species of Dioscorea was promoted by treatmentwith inhibitors of protein synthesis, although the sproutingof completely awake mature bulbils was inhibited. Gibberellicacid (GA₃) inhibition of the sprouting was relieved by cycloheximideand some base and amino acid analogues. Polyphenol oxidase activity in the bulbils decreased after incubationwith the protein-synthesis inhibitors. Possible involvement of protein synthesis in inducing dormancyof Dioscorea bulbils is discussed. (Received July 11, 1977; )     

Quantitative changes of batatasins and abscisic acid in relation to the development of dormancy in yam bulbils

As yam bulbils (Dioscorea batatas Decne.) grow on the motherplants, dormancy of the bulbils develops gradually. This isevidenced by the fact that the sproutability of bulbils decreasedin proportion to their sizes, 2–4 mm to 8–12 mmin diameter when bulbils are detached and sown in Petri dishes.This decreasing sproutability was seen until the stratificationat 5°C for dormancy release was completed. During the growth process of attached bulbils, the contentsof batatasins I and II increased markedly per surface area andper bulbil although on a fresh weight basis they remained constant.The increases of batatasin III and abscisic acid (ABA) weremore striking and were evident in all comparisons. These results corresponded to different localization of batatasinsand ABA in bulbil issues. All of 3 batatasins were localizedin the outermost layers of the bulbils, whereas ABA was distributedevenly throughout their tissues. These results suggest differentroles for batatasins and ABA in the regulation of yam bulbildormancy. ¹ This paper concerns K. H.'s Ph. D. dissertation presentedto Tohoku University based on work done at I. P. C. R. ² Present address: Biological Institute, Faculty of Science,Tohoku University, Sendai, Japan. (Received November 15, 1972; )     

Minor differences with big consequences: Reproductive patterns in the genus Gagea (Liliaceae)

Reproductive patterns in ten species of Gagea Salisb. were compared by counts and measurements of bulbs, bulbils and flowers in large cohorts including all life stages. Two types of bulbils were found: taxa with “type I bulbils” start to develop a single to several bulbils as soon as the replacement bulb has reached a certain diameter and then continue to form them indefinitely throughout the life of the plant. “Type II bulbils” are only temporarily produced in immature, non-flowering plants of some species, but not in fully grown, flowering individuals, a phenomenon termed “reproductive switch”. Patterns of bulbil formation are species-specific: G. davlianidzeae, G. nigra, G. peduncularis, G. pratensis, and G. spathacea produce only type I bulbils; G. angelae, G. fedschenkoana and G. lutea develop only type II bulbils. Both bulbil types occur simultaneously in G. fragifera and G. villosa. The quantitative investigations demonstrate the existence of species-specific thresholds for the development of bulbils as well as flowers. Compared to the adult volume of the replacement bulb (where 90% of all plants flower), both types of bulbils have usually low thresholds: 0–5% (type I, all but one species) and 3–13% (type II). Inflorescences develop if plants attain between 38 and 60% of the adult bulb volume. Minor changes in patterns of bulbil formation and thresholds for their development may ensure survival of highly sterile taxa (e.g. G. spathacea, G. fragifera). This, in turn, can facilitate speciation in the genus driven by both hybridization and polyploidization.     

Structures and synthesis of the growth inhibitors batatasins IV and V,and their physiological activities

Two new compounds, batatasins IV and V were isolated from dormant bulbils of Chinese yam (Dioscorea batatas) and shown to be 2′,3-dihydroxy-5-methoxybibenzyl and 2′-hydroxy-3,4,5-trimethoxybibenzyl, respectively. An analogue, 3,4′-dihydroxy-5-methoxybibenzyl was synthesized. Inhibitory activities of these three compounds as well as batatasin I (6-hydroxy-2,4,7-trimethoxyphenanthrene) and batatasin III (3,3′-dihydroxy-5-methoxy-bibenzyl) in lettuce seed germination, lettuce hypocotyl elongation and wheat coleoptile section elongation tests are described.     

Proteinase Inhibitors in Plant Seed

Three different types of proteinase inhibitors, I, II and III, were fractionated from Japanese radish seed by repeated column chromatographies on SE- and CM-cellulose. The finally purified preparation of inhibitor III was found to be homogeneous by both chromatographic and electrophoretic analyses. All three of them strongly and stoichiometrically inhibited trypsin whereas they showed weak inhibition on other proteinases, such as chymotrypsin, Nagarse and Pronase. From nitrogen content and ultraviolet absorption spectra, each of the inhibitors I and III was confirmed to be a protein. The molecular weights of inhibitors I and III were calculated to be 8000 and 12,000, respectively. These inhibitors were stable at temperatures above 90°C in an acidic pH.     

Heterogeneity of the basic pancreatic inhibitor (Kunitz) in various bovine organs

Four protein protease inhibitors (I, II, III, IV) having low molecular weights (10 600-6500) and basic isoelectric points were isolated by affinity chromatography from bovine spleen. Inhibitor IV was identified as the basic pancreatic trypsin inhibitor (Kunitz inhibitor); the presence and distribution of components I, II and III vary in the different bovine organs. Spleen inhibitors I, II, III and IV were purified by ion-exchange chromatography; they form 1:1 complexes with trypsin and inhibit enzymatic activity of trypsin, chymotrypsin and kallikrein. Inhibitors I, II and III contain carbohydrate moieties (7-4%) covalently bound to the polypeptide chain. Specific basic pancreatic trypsin inhibitor antiserum has shown the complete identity between inhibitor IV and the basic pancreatic trypsin inhibitor, while partial cross-reactivity between the basic pancreatic trypsin inhibitor and inhibitors I, II and III can be seen from a double immunodiffusion test.     

Inhibition of photosynthesis and respiration by batatasins

Effects of batatasins I, III and V, phenolic growth inhibitors occuring in dormant bulbils of Dioscorea batatas Decne., on photosynthetic reactions of chloroplasts from spinach (Spinacia oleracea L.) and on respiration of mitochondria from potatoes (Solanum tuberosum L.) were investigated. In chloroplasts, the batatasins effectively inhibited CO₂-dependent oxygen evolution and electron flow from water to acceptors such as dichlorophenolindophenol, ferricyanide and methylviologen. Photosystem-I dependent electron transport from ascorbate to oxygen was stimulated. The proton conductivity of thylakoid membranes was increased and phosphorylation was uncoupled from electron transport. Inhibition of electron transport with water as electron donor appeared to precede uncoupling. In mitochondrial, batatasin I did not much inhibit succinate-dependent O₂ uptake in the absence of ADP, but caused strong inhibition in the presence of ADP. Batatasins III and V inhibited oxygen uptake irrespective of the presence or absence of ADP. Inhibition of chloroplast and mitochondrial reactions by batatasins was shown to be reversible.Abbrevations B-I batatasin I, 6-hydroxy-2,4,7-trimethoxyphenanthrene - B-III batatasin III, 3,3-dihydroxy-5-methoxybibenzyl - B-V batatasin V, 2-hydroxy-3,4,5-trimethoxybibenzyl - Chl chlorophyll - MV methylviologen - DCPIP 2,6-dichlorophenol-indophenol - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - PVP polyvinylpyrrolidone     

Cyanide-resistant Respiration of Sweet Potato Mitochondria

The oxidation of malate and succinate by sweet potato mitochondria (Ipomoea batatas [L.] Lam.) was blocked only partly by inhibitors of complexes III (2-heptyl-4-hydroxyquinoline-N-oxide) and IV (cyanide and azide). The respiration insensitive to inhibitors of complexes III and IV was inhibited by salicylhydroxamic acid. Essentially complete inhibition was obtained with inhibitors of complex I (rotenone, amytal, and thenoyltrifluoroacetone) and complex II (thenoyltrifluoroacetone). The observations indicated that electrons were transferred to the cyanide-resistant pathway from ubiquinone or from nonheme iron (iron-sulfur) proteins of complexes I and II before reaching the b cytochromes. In contrast, the oxidation of exogenous NADH did not involve the alternate pathway, as indicated by complete inhibition by inhibitors of complexes III and IV and the absence of an effect of inhibitors of complexes I and II. Hence, electrons from exogenous NADH appear to pass directly to complex III in sweet potato mitochondria.     

Carbonic anhydrase inhibitors. Inhibition of isozymes I, II, IV, V and IX with complex fluorides, chlorides and cyanides

The inhibition of five human carbonic anhydrase (hCA, EC 4.2.1.1) isozymes, the cytosolic hCA I and II, the membrane-bound hCA IV, the mitochondrial hCA V and the tumour associated, transmembrane hCA IX, with complex anions incorporating fluoride, chloride and cyanide, as well as B(III), Si(IV), P(V), As(V), Al(III), Fe(II), Fe(III), Pd(II), Pt(II), Pt(IV), Cu(I), Ag(I), Au(I) and Nb(V) species has been investigated. Apparently, the most important factors influencing activity of these complexes are the nature of the central metal ion/element, and its charge. Geometry of these compounds appears to be less important, since both linear, tetrahedral, octahedral as well as pentagonal bipyramidal derivatives led to effective inhibitors. However, the five isozymes showed very different affinities for these anion inhibitors. The best hCA I inhibitors were cyanide, dicyanocuprate and dicyanoaurate (K(I)s in the range of 0.5-7.7 microM), whereas the least effective were fluoride and hexafluoroarsenate. The best hCA II inhibitors were cyanide, hexafluoroferrate and tetrachloroplatinate (K(I)s in the range of 0.02-0.51 mM), whereas the most ineffective ones were fluoride, hexafluoroaluminate and chloride. The best hCA IV inhibitors were dicyanocuprate (K(I) of 9.8 microM) and hexacyanoferrate(II) (K(I) of 10.0 microM), whereas the worst ones were tetrafluoroborate and hexafluoroaluminate (K(I)s in the range of 124-126 mM). The most effective hCA V inhibitors were cyanide, heptafluoroniobate and dicyanocuprate (K(I)s in the range of 0.015-0.79 mM), whereas the most ineffective ones were fluoride, chloride and tetrafluoroborate (K(I)s in the range of 143-241 mM). The best hCA IX inhibitors were on the other hand cyanide, heptafluoroniobate and dicyanoargentate (K(I)s in the range of 4 microM-0.33 mM), whereas the worst ones were hexacyanoferrate(III) and hexacyanoferrate(II).     

Iron-containing metallocenes as active site directed inhibitors of the proteinase that cleaves the NH2-terminal propeptides from type I procollagen

Derivatives of ferrocene (dicyclopentadienyliron) (Fc) were examined as active site directed inhibitors of type I procollagen N-proteinase, the enzyme that cleaves the NH2-terminal propeptides from type I procollagen. The compounds were shown here to be reversible, competitive inhibitors of the enzyme. The effectiveness of the Fc inhibitors varied with modification of the cyclopentadienyl (cp) rings. The monocarboxylic acid (I) and the 1,1'-dicarboxylic acid (II) derivatives of Fc inhibited 50% of the enzymic activity (I50) at concentrations of 1.0 and 0.5 mM, respectively. The Ki values were 0.3 mM for both I and II. Derivatization of the carbonyl alpha to the cp ring of compound I (FcCOCH2CH2COOH, III) increased the inhibitory activity (I50 = 0.100 mM; Ki = 0.065 mM). Removal of the carbonyl alpha to the cp ring of III did not improve inhibitory activity: FcCH2CH2COOH, I50 = 2 mM; FcCH = CHCOOH, I50 = 1.5 mM. The active inhibitory species apparently contained iron in the 3+ valence state since two ferrocenium derivatives were very effective inhibitors: ferrocenium tetrachloroferrate, IV (I50 = 0.030 mM; Ki = 0.004 mM), and carboxyferrocenium hexafluorophosphate, V (I50 less than 0.1 mM; Ki less than 0.05 mM). In addition, reduction of III with ascorbic acid abolished its inhibitory activity. Compounds I and III stabilized the enzyme to heat denaturation in the absence of exogenous calcium; compound IV did not stabilize the enzyme. Further observations indicated that Fc derivatives were specific inhibitors of procollagen N-proteinase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Carbonic anhydrase inhibitors: cloning, characterization, and inhibition studies of the cytosolic isozyme III with sulfonamides

The cytosolic human carbonic anhydrase (hCA, EC 4.2.1.1) isozyme III (hCA III) has been cloned and purified by the GST-fusion protein method. Recombinant pure hCA III had the following kinetic parameters for the CO(2) hydration reaction at 20 degrees C and pH 7.5: k(cat) of 1.3 x 10(4) s(-1) and k(cat)/K(M) of 2.5 x 10(5) M(-1) s(-1), being a slower catalyst for the physiological reaction as compared to the genetically related cytosolic isoforms hCA I and II. An inhibition study with a library of sulfonamides and one sulfamate, some which are clinically used compounds, is reported. hCA III is less prone to be inhibited by these compounds as compared to hCA I and II for which many low nanomolar inhibitors were detected earlier. The best hCA III inhibitors were prontosil, sulpiride, indisulam, benzolamide, aminobenzolamide, and 4-amino-6-chloro-benzene-1,3-disulfonamide which showed K(I)s in the range of 2.3-18.1 microM. Clinically used compounds such as acetazolamide, methazolamide, ethoxzolamide, dorzolamide, brinzolamide, topiramate, zonisamide, celecoxib, and valdecoxib were less effective hCA III inhibitors, with affinities in the range of 154-2200 microM. This is the first study in which low micromolar hCA III inhibitors are reported.     

Loss of short-day requirement for dormancy breaking of bulbils from gibberellic acid-treated plants in Sedum bulbiferum

Sedum bulbiferum forms bulbils at superterranean nodes under long-day conditions, and the detached bulbils sprout after exposure to short-days [1]. When gibberellic acid was applied to the mother plant at the start of the long-day induction period, the number of bulbils formed increased slightly and these bulbils sprouted on incubation in the dark but not under short days of continuous light. However, when gibberellic acid was applied directly to detached bulbils during incubation, the short-day requirement for sprouting was conserved. Gibberellic acid application to the mother plants enhanced sprouting ability of detached bulbils when incubated under illumination with blue, green or far-red light. However, presence of gibberellic acid during bulbil exposure to light did not induced marked enhancement in sprouting under blue, green of far-red light. Thus, gibberellic acid application to the mother plant modified light and photoperiodic requirements for the sprouting of detached bulbils of S. bulbiferum.     

Sprouting promotion by inhibitors of nucleic acid and protein synthesis in the bulbils of some herbaceous plants

The sprouting of immature bulbils of Laportea bulbifera andpartially dormant (in-sufficiently chilled) mature bulbils ofL. bulbifera, Elatostema involucratum and E. umbellatum waspromoted by inhibitors of nucleic acid and protein synthesis(8-azaguanine, 5-fluorouracil, 2-thiouracil, ethionine, canavaninesulfate, p-fluorophenylalanine and cycloheximide in Laporteaand 5-fluorouracil, cycloheximide and chloramphenicol in Elatostema).However, the sprouting of nondormant (chilled) mature bulbilsof L. bulbifera was not promoted, but slightly suppressed whenthese inhibitors (especially, 8-azaguanine, cycloheximide andchloramphenicol) were applied either during or after chillingtreatment These results suggest that the two counteracting systems,dormancy-inducing and -breaking which involve nucleic acid andprotein synthesis participate in the dormancy regulation. (Received December 2, 1977; )     

Gibberellin-induced dormancy and batatasin content in yam bulbils

The sprouting of partially stratified yam bulbils was promotedby exogenous benzyladenine, cycocel (CCC) and indole-3-aceticacid and suppressed by gibberellin A₃(GA₃. Light exposure causedslight suppression of sprouting. GA₃ raised the content of batatasinsbut not that of abscisic acid. We suggest that the sprouting-inhibitingactions of GA₃ and light are exerted by raising the batatasinlevel in the bulbils. A possible mechanism of the natural dormancyinduction in yam bulbils was discussed. ¹This paper concerns K. H.'s Ph. D. dissertation presented toTohoku University based on work done at I.P.C.R. ² Present address: Biological Institute, Faculty of Science,Tohoku University, Sendai 980, Japan (Received May 25, 1973; )     

Dormancy in Laportea bulbils: experiments with buds cultured in vitro

Dormancy in bulbils of Laportea bulbifera was investigated usingbuds cultured in vitro. Buds excised from immature bulbils couldsprout in light, but scarcely in the dark. Regardless of lightconditions, buds isolated from mature bulbils failed to sproutwhile those from chilled mature bulbils could. GA and BA stimulatedthe sprouting of buds excised from dormant mature bulbils. Incontrast, ABA and morphactin inhibited the sprouting of budsexcised from nondormant chilled mature bulbils. These resultssuggest that, in dormant Laportea bulbils, the buds themselvesare in a dormant state which may be controlled by a balanceof activity between growth-inhibiting and -promoting substances. (Received October 29, 1976; )     

The ecology of flower and bulbil production in Polygonum viviparum

Polygonum viviparum produces inflorescences which bear both sexual flowers and asexual bulbils in highly variable proportions. This paper investigates the proportion of flowers and bulbils per inflorescence at 12 sites in central Norway. It is shown that there is a 'trade—off between flowers and bulbils so that one cannot be increased without reducing the other'. On average, the balance is tilted in favour of bulbils, but there are significant differences in the proportion of bulbils per inflorescence among sites and among quadrats within 11 of the sites. Of these 11 sites there are 6 in which the proportion of bulbils per inflorescence is correlated with factors of the biotic environment. The factors include: (1) the amount of vascular plant cover, which is negatively correlated with the proportion of bulbils at 2 sites; (2) the cover of P. viviparum , which is positively correlated at 1 site; (3) the number of P. viviparum inflorescences, which is negatively correlated at 3 sites. The results suggest that both intra– and inter–specific factors of the biotic environment play a role in the control of bulbil and flower production in P. viviparum.     

Dormancy in bulbils of several herbaceous plants: Effects of photoperiod,light, temperature,oxygen and gibberellic acid

Effects of some environmental conditions (photoperiod, white and colored lights, temperature, partial oxygen pressure) and growth regulators (gibberellic acid, 2-chloroethyltrimethylammonium chloride) on induction and release of dormancy of the bulbils ofDioscorea batalas, Laportea bulbifera, Elatostema involucratum andSedum bulbiferum were investigated. Bulbils were formed under short-day conditions inLaportea andElatostema, under long-day conditions inSedum, and irrespective of photoperiods inDioscorea. In all species exceptSedum, immature bulbils required light, particularly blue or far red, for sprouting (photo-sprouting stage), and mature bulbils required a cold treatment (thermo-sprouting stage). The duration of photo-sprouting and thermo-sprouting stages and the degree of dependency on light or low temperature of sprouting differed from species to species. Sprouting of chilled mature bulbils of these species was promoted by light, especially by red or green light. Both immature and mature bulbils ofSedum sprouted under short-day conditions. Continuous irradiation with blue, far-red and green light markedly inhibited their sprouting. Oxygen at high concentration inhibited the sprouting of immature bulbils inDioscorea; in the other species it promoted sprouting regardless of the maturation of the bulbils. Applications of gibberellic acid caused the sprouting of bulbils the absence of light, chilling or photoperiodic treatment in all species exceptDioscorea, in which gibberellic acid inhibited sprouting. Polyphenol oxidase activity was very high in the homogenates ofDioscorea bulbils, and increased further when the bulbils had been treated with gibberellic acid. In the other species, little or no such activity was observed.     

Binding of soluble type I collagen to fibroblasts: specificities for native collagen types, triple helical structure, telopeptides, propeptides, and cyanogen bromide-derived peptides

Unlabeled collagenous proteins were quantified as inhibitors of binding of native, soluble, radioiodinated type I collagen to the fibroblast surface. Collagen types IV, V a minor cartilage isotype (1 alpha 2 alpha 3 alpha), and the collagenlike tail of acetylcholinesterase did not inhibit binding. Collagen types II and III behaved as competitive inhibitors of type I binding. Denaturation of native collagenous molecules exposed cryptic inhibitory determinants in the separated constituent alpha chains. Inhibition of binding by unlabeled type I collagen was not changed by enzymatic removal of the telopeptides. Inhibitory determinants were detected in cyanogen bromide-derived peptides from various regions of helical alpha 1 (I) and alpha 1(III) chains. The aminoterminal propeptide of chick pro alpha 1(I) was inhibitory for binding, whereas the carboxyterminal three-chain propeptide fragment of human type I procollagen was not. The data are discussed in terms of the proposal that binding to surface receptors initiates the assembly of periodic collagen fibrils in vivo.     

Physiological activity in persistent bulbils of Agave vilmoriniana (Agavaceae)

Plants of Agave vilmoriniana are unique members of a reproductively diverse genus since they produce prolific numbers of both bulbils and viable seeds. Bulbils were observed to remain attached to the flowering stalk into a second summer, instead of abscising in the year they developed. Hence, a number of hypotheses were tested to establish the physiological basis for their longevity and evaluate their influence upon the plant's reproductive biology. Bulbils produced following anthesis were abundant (>2000 bulbils), large (0.44 g dry mass per individual), and their combined leaf surface area (one side) could exceed 3 m². Thus, the current-year inflorescence may act as a photosynthate source. Second-year, attached bulbils are healthy-looking, even after leaves of the parent plant have senesced, since maternal roots continue to absorb rain water. Rehydrated 2nd-yr bulbils resume Crassulacean acid metabolism, sometimes demonstrating net daytime tissue acidification. Water conservation by bulbils is very efficient, exhibiting very low cuticular conductances (0.016-0.0035 mm/s). Bulbils detach over all climatic seasons after having formed >3 and >18 root primordia in their 1st and 2nd year, respectively. About one-third of bulbils are capable of forming roots after a 1-yr storage at 35°C, when their water contents averaged 37%. Irrigated bulbils produce water-absorbing roots within 2 d, with de novo chlorophyll synthesis and nocturnal acidification commencing within 4 d. Two bulbils were observed to root and become established in shaded microhabitats during this 2-yr study of 29 plants.