The hormonal control of amaranthin synthesis in Amaranthus caudatus seedlings

Summary Exogenous gibberellic acid, A₃ (GA₃) inhibits phytochrome mediated betacyanin synthesis in seedlings of Amaranthus caudatus. The growth retardants, -chloroethyl-trimethylammonium chloride (CCC), 'isopropyl-4-(triethylammonium chloride)-5-methylphenyl piperidine carboxylate (AMO 1618) and tributyl-2,4,-dichlorobenzylphosphonium chloride (phosphon D) enhance pigment synthesis. Retardant stimulation of pigment synthesis is overcome by GA₃ application. Besides lowering endogenous GA levels the retardants inhibit protein synthesis by as much as 25%. Retardant inhibition of protein synthesis is not overcome by GA₃. The results suggest that amaranthin synthesis in Amaranthus caudatus can be directly controlled by endogenous GA. GA₃ has no effect on kinin induced dark pigment synthesis. Kinins, however, do not overcome GA₃ inhibition of pigment synthesis in the light.Abbreviations AMO 1618 2, 'isopropyl-4-(triethylammonium chloride)-5-methylphenyl piperidine carboxylate - CCC -chloroethyltrimethylammonium chloride - GA₃ Gibberellic acid, A3 - Phosphon D tributyl-2,4,-dichlorobenzylphosphoninm chloride     

A possible mechanism of high temperature dormancy regulation in seeds of Avena sativa L. (cv. Moyencourt)

Embryos of Avena sativa L. (cv. Moyencourt) show no high temperature dormancy. The dormancy is induced by the presence of endosperm-aleurone part of the seed. Germination of isolated embryos at 30°C can be prevented by ABA and the inhibition is reversed by GA. Inhibitors of GA synthesis also inhibit embryo germination. The embryos of dormant and non-dormant seeds vary greatly in their sensitivity to exogenous ABA. High temperature dormancy of the entire seeds can be relieved by low concentrations of ethanol. On the basis of these facts a hypothetic model is proposed showing how interaction between endogenous GA and ABA-like inhibitory substance, may regulate the high temperature dormancy of the seeds.     

Dormancy termination of western white pine (<Emphasis Type="Italic">Pinus monticola</Emphasis> Dougl. Ex D. Don) seeds is associated with changes in abscisic acid metabolism

Western white pine (Pinus monticola) seeds exhibit deep dormancy at maturity and seed populations require several months of moist chilling to reach their uppermost germination capacities. Abscisic acid (ABA) and its metabolites, phaseic acid (PA), dihydrophaseic acid (DPA), 7-hydroxy ABA (7OH ABA) and ABA-glucose ester (ABA-GE), were quantified in western white pine seeds during dormancy breakage (moist chilling) and germination using an HPLC–tandem mass spectrometry method with multiple reaction monitoring and internal standards incorporating deuterium-labeled analogs. In the seed coat, ABA and metabolite levels were high in dry seeds, but declined precipitously during the pre-moist-chilling water soak to relatively low levels thereafter. In the embryo and megagametophyte, ABA levels decreased significantly during moist chilling, coincident with an increase in the germination capacity of seeds. ABA catabolism occurred via several routes, depending on the stage and the seed tissue. Moist chilling of seeds led to increases in PA and DPA levels in both the embryo and megagametophyte. Within the embryo, 7OH ABA and ABA-GE also accumulated during moist chilling; however, 7OH ABA peaked early in germination. Changes in ABA flux, i.e. shifts in the ratio between biosynthesis and catabolism, occurred at three distinct stages during the transition from dormant seed to seedling. During moist chilling, the relative rate of ABA catabolism exceeded ABA biosynthesis. This trend became even more pronounced during germination, and germination was also accompanied by a decrease in the ABA catabolites DPA and PA, presumably as a result of their further metabolism and/or leaching/transport. The transition from germination to post-germinative growth was accompanied by a shift toward ABA biosynthesis. Dormant imbibed seeds, kept in warm moist conditions for 30 days (after an initial 13 days of soaking), maintained high ABA levels, while the amounts of PA, 7OH ABA, and DPA decreased or remained at steady-state levels. Thus, in the absence of conditions required to break dormancy there were no net changes in ABA biosynthesis and catabolism.Abbreviations ABA abscisic acid - ABA-GE abscisic acid glucose ester - DPA dihydrophaseic acid - 7OH ABA 7-hydroxy abscisic acid - 8OH ABA 8-hydroxy abscisic acid - MRM multiple reaction monitoring - PA phaseic acid     

Phytohormone-regulated β-amylase gene expression in rice

The expression of -amylase genes in rice (Oryza sativa) and its regulation by phytohormones gibberellic acid (GA) and abscisic acid (ABA) were examined. Upon germination -amylase is synthesizedde novo in aleurone cells and (GA) is not required. Exogenous addition of GA does not enhance the -amylase activity, while ABA inhibits the -amylase activity, mRNA accumulation, and the germination of rice seeds. GA can reverse ABA inhibition of -amylase expression, but not ABA inhibition of seed germination. Such regulation represents a new interaction of ABA and GA.     

Endo-β-mannanase isoforms are present in the endosperm and embryo of tomato seeds, but are not essentially linked to the completion of germination

A current hypothesis is that endo--mannanase activity in the endosperm cap of tomato (Lycopersicon esculentum Mill. cv. Moneymaker) seeds is induced by gibberellin (GA) and weakens the endosperm cap thus permitting radicle protrusion. We have tested this hypothesis. In isolated parts, the expression of endo--mannanase in the endosperm after germination is induced by GAs, but the expression of endo--mannanase in the endosperm cap prior to radicle protrusion is not induced by GAs. Also, abscisic acid (ABA) is incapable of inhibiting endo--mannanase activity in the endosperm cap, even though it strongly inhibits germination. However, ABA does inhibit enzyme activity in the endosperm and embryo after germination. There are several isoforms in the endosperm cap and embryo prior to radicle protrusion that are tissue-specific. Tissue prints showed that enzyme activity in the embryo spreads from the radicle tip to the cotyledons with time after the start of imbibition. The isoform and developmental patterns of enzyme activity on tissueprints are unaffected when seeds are incubated in ABA, even though germination is inhibited. We conclude that the presence of endo--mannanase activity in the endosperm cap is not in itself sufficient to permit tomato seeds to complete germination.Abbreviations ABA cis/trans-abscisic acid - GA(s) gibberellin(s) - IEF isoelectric focussing - pI(s) isoelectric point(s) We thank Dr. Bruce Downie for the seemingly endless but inspiring discussions.     

Responses of florence fennel (Foeniculum vulgare azoricum) seeds to light,temperature and gibberellin A4/7

The percentage and the rate of germination of seeds of three varieties of Florence fennel was higher in the dark than in the light, the high temperature cut-off points being between 27.2 and 29.4°C. The optimum temperature for germination was between 20 and 25°C. Seeds of all three varieties responded to incubation in solutions containing gibberellin A_4/7 mixture (GA_4/7; Regulex), giving higher germination in the light at temperatures from 20 to 30°C. Seeds steeped for 4 h at 25°C or for 24 h at 5°C in GA_4/7 solutions gave a higher percentage and increased rate of emergence as compared with untreated dry seeds, when sown in compost at 25°C; steeping in water alone was also beneficial. In general, drying the treated seeds before sowing reduced the rate but sometimes increased the percentage of germination as compared with seeds sown when still moist. Seeds harvested from secondary umbels of var. Zefa fino germinated better both in the light and dark than those taken from primary umbels.Abbreviations GA_4/7 a commercial mixture of the gibberellins A₄ and A₇ (trade name Regulex)     

Gibberellin involvement in dormancy-break and germination of seeds of celery (Apium graveolens L.)

The temperature-dependent, primary dormancy of cv. Florida 683 celery seeds in darkness was partially broken by a 30 min light exposure on the third day of incubation at 20–22°C, resulting in c 50 percent germination after 20 days. This light stimulation was negated by including different inhibitors of gibberellin biosynthesis in the incubation medium. Subsequent addition of a solution of the gibberellins A₄ and A₇ or of the gibberellin-active compound (1-3-chlorophthalimido)-cyclohexane carboxamide (AC94,377) overcame the inhibitory effects on germination of these GA-biosynthesis inhibitors. It is suggested that light stimulates the biosynthesis of gibberellins which are essential for dormancy-break in celery seeds and that this biosynthesis is either directly or indirectly controlled through phytochrome.Abbreviations AC94,377 1-(3-chlorophthalimido)-cyclohexane carboxamide; ancymidol, -cyclopropyl--(4-methoxyphenyl)-5-pyrimidinemethanol - AMO1618 N,N,N-2-tetramethyl-5-(1-methyl ethyl)-4-(1-piperidinylcarbonyl)oxy-benzenaminium chloride - BTS44584 S-2,5-dimethyl-4-pentamethylenecarbamoyloxyphenyl-SS-dimethyl sulphonium - P toluenesulphonate; chlormequat chloride, 2-chloroethyltrimethylammonium chloride; daminozide - N dimethylaminoscuccinamic acid; paclobutrazol, (2RS, 3RS)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1H-1,2,4-triazol-1-yl pentan-3-ol)     

Increased thermal stability of glucose dehydrogenase by cross-linking chemical modification

A hetero-oligomeric glucose dehydrogenase (GDH) from a moderate thermophilic bacterium, SM4 was cross-linked with glutaraldehyde (GA) and it now showed only one optimum temperature for reaction at around 65°C, which approximately follows the Arrhenius equation. The native enzyme had shown optima at both 45°C and 75°C. In addition to the alteration of the optimum temperature for reaction, GA cross-linked GDH retained more than 90% of its initial activity even after 30 min of incubation at 65°C.     

Reversing the inhibitory effect of paclobutrazol on seed germination of Amaranthus paniculatus by GA3, ethephon or ACC

The germination of Amaranthus paniculatus seeds was inhibited by applying paclobutrazol, a specific inhibitor of gibberellin biosynthesis. This inhibition was markedly counteracted by gibberellin A₃ (GA₃), suggesting that endogenous gibberellins are required for germination in this species. The inhibitory effect of paclobutrazol was also overcome by ethephon (2-chloroethylphosphonic acid) or the precursor of ethylene biosynthesis, ACC (1-aminocyclopropane-l-carboxylic acid). Thus the physiological effect of gibberellin can be mimicked by ethylene released from ethephon or synthesised from exogenous ACC. It is suggested, that endogenous gibberellins are involved in germination of Amaranthus paniculatus seeds and that action of GA₃ can be substituted by ethylene.Abbreviations ACC 1-aminocyclopropane-l-carboxylic acid - AMO-1618 (2-isopropyl-5methyl-4-trimethylammoniumchloride)-phenyl-l-piperidinium-carboxylate - ancymidol -cyclopropyl--(4-methoxyphenyl)-5-pyrimidine methanol - chloromequat chloride (2-chloroethyl)trimethylammoniumchloride - ethephon 2-chloroethylphosphonic acid - GA gibberellin A₃ - paclobutrazol (2RS, 3RS)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-lyl)pentan-3-ol - Phosphon D 2,4,dichlorobenzyl-tributhylphosphoniumchloride - tetcyclacis 5,(4-chlorophenyl)-3,4,5,9,10-pentaaza-tetracyclo)5,4,1,0,Z,6,0^8,11 dodeca-3,9-diene     

Soybean seedling emergence at high temperatures

Bragg and Cobb soybean [Glycine max (L.) Merr.] seeds were germinated in sand at temperatures ranging from 25 to 40°C. Emergence decreased with increasing temperature above 37°C, with virtually no emergence at 40°C. Emergence of 12 other cultivars at 38°C ranged from 25 to 95%. Foster and Coker 338 were more sensitive to high temperature than the other cultivars.     

Endogenous gibberellin A1 level and α-amylase activity in germinating rice seeds

The role of endogenous gibberellin A₁ (GA₁) in the induction of -amylase activity was investigated during germination of rice (Oryza sativa L.) seeds. The level of endogenous GA₁ and the -amylase activity in the seeds of normal rice, cv. Nipponbare, increased simultaneously from 3 days after the imbibition of water. The -amylase activities in the dwarf rice, cv. Waito-C and Tan-ginbozu, were less than that in the normal rice. The level of endogenous GA₁ and -amylase activity were decreased in proportion to the concentration of a growth retardant, uniconazole. The retardation in -amylase activity caused by the treatment of uniconazole was recovered by the application of exogenous GA₁. These results indicate that the endogenous GA₁ biosynthesized de novo regulates -amylase production in germinating rice seeds.Abbreviations GA(s) gibberellin(s) - ABA abscisic acid - AE fraction acidic ethyl acetate-soluble fraction - HPLC high performance liquid chromatography - R _t retention time - GC-SIM gas chromatography-selected ion monitoring     

Interactive effects of GAs,CKs and growth retardants on the germination of celery seeds

The germination of whole seeds of celery (Apium graveolens L.) was inhibited by paclobutrazol, ancymidol and lower concentrations of uniconazole. The growth retardants daminozide, AMO 1618 and chlormequat chloride inhibited the germination of cut seeds only, indicating that the seed coat prevents the penetration of these compounds at the examined concentrations. Application of a mixture of the gibberellins A4 and A7 (GA 4/7) reversed the inhibition of all the examined growth retardants. Cytokinins (artificial or natural) had no effect when applied alone and did not interact with GA4/7 in the light. However, in the dark the cytokinins at some concentrations and GA4/7 had a synergistic effect in reversing the inhibition caused by growth retardants to whole seeds or in promoting the germination of whole seeds. It is therefore suggested that the major effect on seed of exogenous cytokinins when applied together with GA's is to increase the uptake of gibberellins by the seeds.Abbreviations AMO 1618 (2 isopropyl-5-methyl-4-trimethylammonium chloride-phenyl-1-Piperidinium-carboxylate - ancymidol -cyclopropyl-[4-methoxyphenyl]-5-pyrimidinemeth anol] - chlormequat chloride 2-chloroethyltrimethylommonium chloride - daminozide succinin acid 2,2-diamethyl hydrazide - paclobutrazol [2 RS, 3 RS]-1-(4-chlorophenyl)-4,4-dimethyl-2-(1 H-1,2,4-triazol-1-yl) pentan-3-ol - uniconazole (E)-1-(P-chlorophenyl)-4,4-dimethyl-2-(1,2,4 triazol-1-yl)-1 penten-3-ol     

Expression of the β-subunit of β-conglycinin in seeds of transgenic plants

Soybean (Glycine max (L.) Merr.) seeds contain the storage protein -conglycinin, encoded by a multigene family. -Conglycinin consists of three subunits; , , and . A genomic clone for a -subunit of -conglycinin has been characterized by restriction-enzyme mapping and hybrid selected in-vitro translation followed by immunoprecipitation. In order to determine the developmental regulation of this -subunit gene, its expression was studied in seeds of transgenic petunia (Petunia hybrida) and tobacco (Nicotiana tabacum L.) plants. The -subunit expressed in seeds of petunia and tobacco was recognized by anti--conglycinin serum at a relative molecular mass of 53 000, equivalent to that of the native protein. Separation of the petunia-seed proteins by isoelectric focusing followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis showed that multiple isoelectric forms of the -subunit were produced. There was approximately a twofold variation in the accumulation of the -subunit protein in the mature seeds of transgenic petunia plants, each containing a single -subunit gene. However, the level of protein accumulation in mature seeds and the amount of -subunit mRNA in developing seeds was not correlated. Accumulation of the -subunit protein in transgenic seeds was less than the -subunit protein that accumulated in transgenic petunia seeds containing a single -subunit gene and less than the amount of the -subunit in mature soybean seeds which contain 8–13 -subunit genes. In transgenic tobacco plants, the accumulation of the -subunit protein in seeds was generally well correlated with the number of genes that were incorporated in the different transformants.Abbreviations kb kilobase - kDa kilodalton - M_r relative molecular mass - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

Genotypic variation in the germination of common bean in response to cold temperature stress

Beans (Phaseolus vulgaris) are regarded as a susceptible crop to suboptimal temperatures. In temperate regions, low temperatures reduce establishment of beans when planted early in the growing season. Seeds of 14 cultivars/lines or beans were germinated in petri dishes at a constant 8, 10, 12, or 18°C or at 12 h alternating temperatures of 10/8, 12/8 or 18/8°C. Differences in germination percentages and rates between cultivars/lines were significant, especially at low temperatures. Cultivars/lines that germinated best and quickly at constant 8°C were Volare, Great Northern (G.N.) Tara, G.N. Belneb # 1, G.N. Spinel, and San Cristobal. Germination percent and rate of Pinto-UI-111 and Canadian Wonder increased significantly when temperatures were increased by 2 to 4°C for 12 h per 24 h, compared with a constant 8°C. Whereas, germination of G.N. Belneb # 1 was reduced. Polyacrylamide gel electrophoresis was used to study the effect of cold treatment on polypeptide patterns of seven cultivars/lines. Seeds were germinated at 18°C constant for 96 h or at 18°C for 48 h followed by 48 h at 2 or 8°C. During cold treatment the synthesis of some polypeptides increased. Volare, G.N. Tara Pinto-UI-111 and Canadian Wonder showed changes in polypeptide patterns, while Alubia-33-1, Michigan 84100 and BAT-1225 showed no changes in polypeptide patterns if compared to the control (96 h at 18°C in the dark). This suggests a likely essential role of these proteins in the development of chilling tolerance.     

Poly(U)-directed peptide-bond formation from the 2′(3′)-glycyl esters of adenosine derivatives

Summary The self-condensation of 2(3)-O-glycyl esters of adenosine, adenosine-5-(O-methylphosphate) and P₁, P₂-diadenosine-5-pyrophosphate in 6.2 mM solutions at pH 8.0 and -5°C in the presence of 12.5 mM poly(U) yields approximately 3 times as much diketopiperazine as reactions without poly(U). As the concentration of 2(3)-O-(glycyl)-P₁, P₂-diadenosine-5-pyrophosphate is decreased from 6.2 mM to 1.5 mM the yield of diketopiperazine in the presence of poly(U) decreases slightly from 6.6% to 5.2%, whereas, in the absence of poly(U) the yield of diketopiperazine decreases substantially from 2.4% to 0.75%. The enhanced yield of diketopiperazine that is attributed to the template action of poly(U) is temperature dependent and is observed only at temperatures below 10°C (5°C to -5°C) for 6.2 mM 2(3)-O-(glycyl)-adenosine-5-(O-methylphosphate) and below 23°C (15°C to -5°C) for 6.2 mM 2(3)-O-(glycyl)-P₁, P₂-diadenosine-5-pyrophosphate. The absence of a template effect at high temperatures is attributed to the melting of the organized helices. The hydrolysis half-lives at pH 8.0 and -5°C of 2(3)-O-(glycyl)-adenosine, 2(3)-O-(glycyl)-adenosine-5-(O-methylphosphate), 2(3)-O-(glycyl)-P₁, P₂-diadenosine-5-pyrophosphate, and 5-O-(glycyl)-adenosine in the presence of poly(U) are substantially larger than their half-lives in the absence of poly(U). The condensation of 2(3)-O-(glycyl)-adenosine yields 5% of 5-O-(glycyl)-adenosine in the presence of poly(U) compared to 0.7% in the absence of poly(U).Abbreviations DKP diketopiperazine - (gly)₂ glycylglycine - (gly)₃ glycylglycylglycine - AppA-gly 2(3)-O-(glycyl)-P₁, P₂-diadenosine-5-pyrophosphate - MepA-gly 2(3)-O-(glycyl)-adenosine-5-(O-methylphosphate) - Ado-2(3)-gly 2(3)-O-(glycyl)-adenosine - Ado-5-gly 5-O-(glycyl)-adenosine - Boc-gly N-tert-butyloxycarbonylglycine - AppA P₁, P₂-diadenosine-5-pyrophosphate - MepA adenosine-5-(O-methylphosphate) - AppA-Boc-gly 2(3)-O-(Boc-glycyl)-P₁, P₂-diadenosine-5-pyrophosphate - Ado-5-Boc-gly 5-O-(Boc-glycyl)-adenosine - Ado-2(3)-Boc-gly 2(3)-O-(Boc-glycyl)-adenosine     

C(alpha) chemical shift tensors in helical peptides by dipolar-modulated chemical shift recoupling NMR

The C chemical shift tensors of proteins contain information on the backbone conformation. We have determined the magnitude and orientation of the C chemical shift tensors of two peptides with -helical torsion angles: the Ala residue in G*AL (=–65.7°, =–40°), and the Val residue in GG*V (=–81.5°, =–50.7°). The magnitude of the tensors was determined from quasi-static powder patterns recoupled under magic-angle spinning, while the orientation of the tensors was extracted from C–H and C–N dipolar modulated powder patterns. The helical Ala C chemical shift tensor has a span of 36 ppm and an asymmetry parameter of 0.89. Its ₁₁ axis is 116° ± 5° from the C–H bond while the ₂₂ axis is 40° ± 5° from the C–N bond. The Val tensor has an anisotropic span of 25 ppm and an asymmetry parameter of 0.33, both much smaller than the values for -sheet Val found recently (Yao and Hong, 2002). The Val ₃₃ axis is tilted by 115° ± 5° from the C–H bond and 98° ± 5° from the C–N bond. These represent the first completely experimentally determined C chemical shift tensors of helical peptides. Using an icosahedral representation, we compared the experimental chemical shift tensors with quantum chemical calculations and found overall good agreement. These solid-state chemical shift tensors confirm the observation from cross-correlated relaxation experiments that the projection of the C chemical shift tensor onto the C–H bond is much smaller in -helices than in -sheets.     

The double ππ5.6 helix of gramicidin a predominates in unsaturated lipid membranes

The structure of the channel-forming polypeptide gramicidin A (GA) incorporated into phosphatidylcholine (PC) liposomes has been studied as a function of the degree of unsaturation of the acyl chains of PC. The initial conformational state of GA in reconstituted bilayers is determined by the solvent in which the peptide and the lipid are initially co-dissolved, whereas the equilibrium conformational state (after heat incubation) is affected by the lipid structure rather than by the nature of the solvent. The conformational equilibrium of GA has been studied in liposomes prepared from PC having a variable number of double bonds in the fatty acid moiety, by circular dichroism and Fourier transform infrared. Liposomes were prepared from trifluoroethanol or ethanol solutions and incubated at 68°C. GA was shown to retain the conformation of the right-handed .3 .3 helix in PC with saturated acyl chains and with one double bond, whereas in dilinoleoyl-PC, having two double bond in each chain, the thermodynamically preferred structures are left-handed antiparallel and parallel double 5.6 helices. Natural soybean PC also favours left-handed 5.6 helical structures of GA (75%). This finding is discussed in terms of the role of PC unsaturation in the dynamic properties of the lipid matrix. Differences between observed FTIR spectra of the =5.6 helix in solution (and to a larger extent in the membrane) and the calculated IR spectra can be interpreted as resulting from deviation of the real structure from the theoretically derived ideal helix. The data obtained provide grounds for better understanding of a GA channel functioning in lipids of variable degrees of unsaturation.Abbreviations GA gramicidin A - CD circular dichroism - FTIR Fourier transformed infrared - 2D-NMR two-dimensional nuclear magnetic resonance - DSPC distearoylphosphatidylcholine (di-C18:0) - DPPC dipalmitoyl-PC (di-C16:0) - DMPC dimiristoyl-PC (di-C14:0) - POPC palmitoyloleyl-PC (C16:0-C18:1) - DOPC dioleyl-PC (di-C18:1) - DLPC dilinoleoyl-PC (di-C18:2) - TFE trifluorethanol - SDS sodium dodecylsulfate - TMA-DPH 1-[4(trimethylammonio)-phenyl]-6-phenyl-1,3,5-hexatriene - HPLC high-performance liquid chromatography Correspondence to: V.T. Ivanov     

Saponin-like substances inhibit α-galactosidase production in the endosperm of fenugreek seeds

When fenugreek (Trigonella foenum-graecum L.) endosperms plus testa (endosperms), which had been isolated from 5-h-imbibed seeds, were incubated for at least 2 h under germination conditions, they leaked substances which, like exogenous abscisic acid (ABA), inhibited the production of fenugreek endosperm -galactosidase. However, unlike ABA, 8 h treatment with these inhibitors had no effect on fenugreek endosperms which had been isolated from 15-h-imbibed seeds and leached for 2 h. This indicated that either their inhibitory action was on processes which were related to the production of -galactosidase and had been completed by this time, or that there might be factors present which inactivate these inhibitors. It was also concluded that the action of the endosperm leachate could not be attributed to the presence of ABA. The activity of the leachate decreased when it originated from endosperms imbibed for periods longer than 25 h and thin-layer chromatography (TLC) of extracts from these endosperms showed decreased contents of the leachable inhibitors as imbibition proceeded. From the seed leachate, which had a TLC pattern and inhibitory action similar to that of the endosperm, were isolated three substances which, when applied to endosperms, inhibited the production of -galactosidase activity. According to their chromatographic behaviour and their reaction with specific reagents, there are strong indications that these substances are saponins. These diffusible saponin-like substances were located in both endosperm and perisperm and their physiological role is discussed.Abbreviations ABA abscisic acid - PEG polyethylenglycol - TLC thin-layer chromatography We wish to thank the Alexander S. Onasis Public Benefit Foundation for a grant to K.Z. and Dr. J.S.G. Reid (University of Stirling, Scotland) for a kind gift of fenugreek seeds.     

Enzymic activities and galactomannan mobilisation in germinating seeds of fenugreek (Trigonella fœnum-graecum L. Leguminosae)

Summary The activities of -galactosidase, -mannosidase and -mannosidase were determined in extracts from the endosperm and from the embryo of fenugreek seeds at different stages of germination. Endosperm homogenates contained little or no activity of the above enzymes in the early stages of germination, before the reserve galactomannan began to be mobilised. The onset of galactomannan breakdown coincided with the appearance of -galactosidase and -mannosidase activities, which increased throughout the period of galactomannan degradation and then remained constant. A similar rise in -galactosidase and -mannosidase activities occurred during galactomannan breakdown in dry-isolated endosperms incubated under germination conditions. The increase could be suppressed by metabolic inhibitors which also inhibit galactomannan breakdown. Embryo homogenates contained high -galactosidase, high -mannosidase and some -mannosidase activity at all stages of germination.No oligomannosyl -1,4 phosphorylase activity could be detected either in the endosperm or in the embryo.It is concluded that the galactomannan of fenugreek is broken down by a series of hydrolases secreted by the aleurone layer of the endosperm. They include -galactosidase, -mannosidase and probably also endo--mannanase.This is part four in a series of papers dealing with galactomannan metabolism. Part three: Planta (Berl.) 106, 44–60 (1972).     

Prebiotic Formation of ADP and ATP from AMP,Calcium Phosphates and Cyanate in Aqueous Solution

Adenosine-5-triphosphate was synthesized by the phosphorylation of adenosine-5-diphosphate in aqueous solution containing cyanate as a condensing reagent and insoluble calcium phosphate produced from phosphate and calcium chloride. In a similar manner, adenosine-5-diphosphate was synthesized from adenosine-5-monophosphate. When the experiment was carried out in the conditions of 4 °C and pH 5.75, the formation of adenosine-5-diphosphate and adenosine-5-triphosphate from adenosine-5-monophosphate was observed in the yields of 19 and 7%, respectively. The other nucleoside-5-triphosphates were also produced from their respective diphosphates.