Studies in Plant Growth Hormones: V. CHROMATOGRAPHY OF HORMONES IN EXCISED AND INTACT ROOTS OF TOMATO SEEDLINGS

1. An examination has been made of the hormones present in extractsof excised roots and intact seedling roots of tomato. Acid andneutral ether-soluble fractions and the ether-insoluble aqueousfraction were chromatographed, and the chromatograms assayedusing oat coleoptile sections. 2. The pattern of hormone activity in excised roots differedlittle from that in seedling roots. 3. On chromatograms of the aqueous fraction developed in isopropanol/ammonia, growth promotion occurred at the position of 3-indolylaceticacid (IAA, Rf 0·5) and sometimes at the position of 3-indolylacetonitrile(IAN, Rf 0·8). When the IAA zone was eluted off the paperand rechromatographed, it formed the IAN zone and another zoneof promotion at Rf 0·1–0·2. 4. When the aqueous fraction was developed in n-butanol/ammonia,promotion occurred at the position of IAA (Rf 0·15),at Rf 0·5, and at the position of IAN (Rf 0-85). Thesezones have been called X, Y, and Z respectively. They were alsoformed when the IAA zone in wopropanol/ammonia was rechromatographedin ammoniacal n-butanol. It is shown that X and Y are interconvertible,and that each can form Z on rechromatography; also, there issome evidence that Z can form X and Y. When Z was separatedinto ether-soluble (acid and neutral) and ether-insoluble fractionswith sodium bicarbonate solution and chromatographed in iiopropanol/ammonia,growth resulted at the position of Z in the neutral and aqueousfractions, but in the acid fraction it occurred at Rf 0·24–O·35.Comparison with other chromatograms indicates that this lastzone does not occur in the aqueous fraction but has been formedas a result of extraction with sodium bicarbonate solution. 5. The zones found in the aqueous fraction also occurred insmall quantities in acid and neutral ethereal fractions in anumber of experiments. 6. The ethereal fractions gave no chromogenic reactions withferric chloride/ perchloric acid, nitrous/nitric acid, or p-dimethylaminobenzaldehyde(MeAB). In the aqueous fraction only MeAB gave a reaction (yellow)which showed any consistent correlation with biological activity.A yellow colour with this reagent is not a characteristic chromogenicreaction of indole compounds. It is suggested that a non-indolehormone system may be operating in tomato roots.     

Inhibitor and Auxin Activity in the Avocado Fruit

The wheat coleoptile elongation bioassay was used for determination of growth-promoting and growth-inhibiting substances in avocado (Persea americana Mill.) fruit tissues, during development and maturation. Growth-promoting activity was found in two zones on paper chromatograms developed with isopropanol: ammonia : water (10:1:1 v/v): Rf 0.30–0.50 and Rf 0.8–0.9. Growth inhibiting activity was found in three different zones: “A” Rf 0.0–0.2, “B” (abscisic acid) Rf 0.6–0.8, and “C” (l-acetoxy-2,4-dihyroxy-n-hepta-deca-16-ene) Rf 0.85–1.0. Higher levels of auxins were found in seed tissues than in the mesocarp. No correlation was found between fruit growth rate and level of extractable auxins in the mesocarp. The amount of abscisic acid (ABA) in the mesocarp was constant during fruit growth. A gradual and consistent increase in 1-acetoxy-2,4-dihydroxy-n-hepta-deca-16-ene was found during fruit growth, reaching a maximum when the fruit attained maturity.     

Growth Regulators in Picea abies

The occurrence of growth regulators active in the Avena coleoptile straight-growth test in sprouting buds and seedlings of Norway spruce (Picea abies Karst.) was investigated. The acid ether fraction contained a growth stimulator, the Rf of which in isopropanol: ammonia: water was 0.2–0.4. This substance behaved as indole-3-acetic acid (IAA) in elec-trophoresis, in chromatography in various solvent systems on paper and on a Sephadex column. It gave the colour typical of IAA when sprayed with Ehrlich reagent and its fluorescence characteristics corresponded to IAA. Acid ether-soluble inhibitors showed most activity at Rf 0.4–0.7, but due to tailing they interfered with the determination of the stimulator at the Rf of IAA in the bioassay. They also masked the activity of other stimulators. Colour reactions were obtained with Ehrlich reagent in the inhibiting chromatogram zone. When eluates from this zone were tested in high dilutions or after gel filtration growth stimulation was obtained. The acid fraction of seedling shoots also contained a stimulator with Rf 0.7–0.8. In the neutral-basic ether-soluble fraction growth stimulation was obtained at Rf 0.5–0.7. The extracts also contained stimulatory substances insoluble in ether but soluble in n-butanol and partly in ethyl acetate. When the butanol fraction was hydrolyzed in 1 M NaOH a substance behaving as IAA when chromatographed was released.     

Evidence for Naturally Occurring Inhibitors of Archegonial Differentiation in Lygodium japonicum

Archegonial differentiation in prothallia of Lygodium japonicum was inhibited when the filtrate of conditioned medium or the extracts of prothallia with organic solvents were added to the medium. By varying the timing of treatment with the methanol extract, archegonial differentiation was shown to start at least 4 days before microscopically detectable change. The inhibitory effect of methanol extract was nullified by transferring the treated plants to a fresh medium omitting the methanol extract, so that the archegonial formation became discernible 6 days after the transfer. The inhibitory activity was stable in both acidic and basic solutions at room temperature, and was partially lost by boiling at pH 3 or 11 for 30 min. The inhibitor, which could be retrieved from the filtrate and the methanol extract, was fractionated into the neutral ethyl acetate fraction, but was not found in the acidic ethyl acetate fraction and in the aqueous residue. At least two active zones were separated on thin layer chromatograms of the ethyl acetate extracts from the filtrate and the methanol extract, and the relative flow-rates of each active zone from these two sources were very similar. The evidence described above indicates that specific inhibitors of archegonial differentiation may be produced in the tissue of prothallia of Lygodium and eventually be secreted to the medium.     

Growth Substances in the Roots of Vicia faba

An investigation has been made into the growth regulators presentin ethanol extracts of the seedling roots of Vicia faba afterseparation on paper partition chromatograms, using segmentsof Avena coleoptiles and mesocotyls and of Pisum sativum.rootsas biological assay material. Acetonitrile purification shows the presence of at least threeauxins running in isobutanol: methanol: water, at Rfs of 0–0·25,0·4–0·6, and 0·65–0·95;the latter may represent two different auxins. A similar, butclearer, picture is shown by the ether-soluble acid fraction.Here an auxin at Rf 0–0·25 also stimulates rootgrowth and could be ‘accelerator ’. A second atRf 0–0·25 is an indole compound which inhibitsroot growth and does not seem to be be IAA. A third at Rf 0·8–1·0is also a root-growth inhibitor and gives no indole reaction.The ‘inhibitor ß’ complex was demonstrated(Rf 0·65–0·85) together with a number ofother inhibitors at lower Rf value. The ether-soluble neutral component also contains auxins orauxin precursors. The water-soluble, ether-insoluble fraction contains four readilyinterconvertible substances with auxin properties. They allappear to inhibit root growth and give no indole reaction.     

Identification of (+)-abscisic acid in strawberry leaves

Summary The acidic ether extract of leaves collected in October from dormant strawberry plants outdoors at Invergowrie inhibited coleoptile growth in germinating wheat embryos. On paper chromatograms developed either with isopropanol: ammonium hydroxide: water (10:1:1) or isopropanol: 1% ammonium hydroxide (4:1), the zones possessing inhibitory activity had R_f's 0.6–0.7 and 0.6–1 respectively. Fractions possessing comparable activity were eluted from granular animal charcoal columns by 10% and 20% acetone in water, and, on further purification, eluted only by 10% ethyl acetate in chloroform from columns of celitesilicic acid (2:1). The inhibitor in this fraction was identified as (+)-abscisic acid (abscisin II, dormin) by spectropolarimetry.     

Root-promoting Substances in Salix alba

Root-promoting substances were extracted from softwood cuttings of Salix alba L. by centrifuging them with water or by shaking the ground freeze-dried stems with water. Rooting substances were partitioned by paper chromatography or chemical fractionation and their rooting activity was tested by mung bean cuttings. Both extracts indicated three major root -promoting fractions at Rf 0-0.1, 0.7-0.8, and 0.3-0.4 in a decreasing order of their activities when paper chromatographed with isopropanol:ammonia:water 8:1:1 v/v. The strongest one indicated an apparent synergistic rooting effect with indol-3yl-acetic acid (IAA) regardless of the extraction method. These results indicate that water can extract from freeze -dried sample the similar rooting substances found in the centrifugal diffusates. The Rf 0–0.1 fraction consisted of at least four fractions and the strongest one did not move from the starting line on the chromatogram when isopropanol:ammonia:water 8:1:1 was used. This starting line fraction was extremely strong in rooting activity and its highest concentration resulted in 8.7 times as many roots as controls. More thain additive rooting effect between IAA and the fraction was found only at the highest concentration. The fraction was very soluble in water but insoluble in chloroform or ethyl ether and only stimulated rooting of mung bean cuttings when it was applied within 3 days after cuttings were made. It had no effect in lengthening roots. The starting line fraction was further found to have four root-promoting subfractions at Rf 0.05, 0.35, 0.65, and 0.85 when it was chromatographed in 60 % isopropanol. Among these four, the subfractions at Rf 0.65 and 0.35 were strongly root promotive and displayed more than additive root promotion with IAA at the highest concentrations studied.     

Studies in Plant Growth Hormones: IV. CHROMATOGRAPHY OF HORMONES AND HORMONE PRECURSORS IN CABBAGE

1. Acid and neutral ethereal fractions and the non-ether-solubleaqueous fraction of an extract of cabbage leaves were chromatographed,and the chromatc-grams assayed using oat coleoptile sections. 2. In the aqueous fraction an acidic precursor of 3-indolylacetonitrile(IAN) was found. When chromatographed in isopropanol/ammonia,the precursor travelled at the position of 3-indolylacetic acid(IAA), but in n-butanol/ammonia it was much closer to the starting-line.IAN is liberated from the precursor under conditions of alkalinehydrolysis including ammoniacal chromatography, and is alsoliberated by heat. Precursor and IAN zones promoted coleoptilegrowth, but the former when sprayed with ferric chloride/perchloricacid or nitrous/nitric acid gave a yellow colour showing thatthere was no free IAN on this part of the chromatogram. Hypothesesto account for this activity are discussed. 3. A neutral inhibitor was present in the aqueous fraction.It is volatile, ether-soluble, and is thought to have been liberatedfrom a water-soluble substance. 4. The neutral fraction, chromatographed in isopropanol/ammonia,contained IAN and a growth promoter at Rf o–o·1:thelatter stimulates cress root growth above that in water. Thispromoter could be formed from a precursor in the aqueous fractionby heat treatment followed by shaking with sodium bicarbonatesolution. It is suggested that this neutral hormone is the accelerator-of Bennet-Clark and Kefford. The data of these workers are analysedto show that this interpretation is consistent with their results. 5. The acid fraction contained IAN but no IAA. The former isthought not to have been liberated from the precursor in thisfraction but to have entered into it from the neutral fractionduring separation with sodium bicarbonate solution. AlthoughIAA may have been absent from the plant material, it is possiblethat any present was destroyed during the process of extraction. 6. Evidence is presented that there are other growth promoterspresent at low concentration in the extract in addition to thosealready mentioned.     

Interaction of Kinetin and Abscisic Acid in the Avena Straight-Growth Test

Different amounts of abscisic acid, 2.7 × 10^?9? 5 × 10^?8 moles, were chromatographed in isopropanol: ammonia: water (100:14:6), firstly alone and secondly together with 5 × 10^?8 moles kinetin. The same amount of kinetin was also chromatographed alone. The chromatograms were tested biologically with the Avena straight-growth test. Whereas a large part of the chromatograms of kinetin gives growth stimulation, the Rf region 0.4–0.6 of abscisic acid chromatograms is strongly growth-inhibiting. The inhibition within this Rf region does not become less if abscisic acid and kinetin are chromatographed together.     

Growth and Dormancy in Lunularia cruciata (L.) Dum: VII. THE ISOLATION AND BIOASSAY OF LUNULARIC ACID

The extraction, purification, and isolation of the growth inhibitorpreviously postulated are described. Methanol extraction andseparation into acid, neutral, and basic fractions was followedby paper chromatography of the acid and neutral fractions withdistilled water, re-extraction with methanol, and thin-layerchromatography, the peak of inhibition being located at Rf 0.7–0.8(isopropanol: ammonia: water, 100:5:5), or Rf 0.3–0.4(chloroform: ethyl acetate: acetic acid, 60:40:5) Lunularia gemmae, grown directly on the chromatographic stripwith added nutrient solution, served as the most appropriateand direct bioassay. Area measurements after 5–10 days'growth yielded significant differences. Other bioassays included:Marchantia polymorpha gemmae, lettuce hypocotyl growth, cress-seedgermination, oat coleoptile, and radish cotyledon disc tests.An active inhibitor, i.e. dihydrohydrangeic acid, now named‘lunularic acid’, was isolated in crystalline form.Lunularic acid was found to increase with long-day treatmentof Lunularia thalli, though present even in short-day. Its concentrationcould be altered rapidly when daylength conditions were changed.The growth inhibition was linearly related to concentrationover the range from 0.1 to 10 ppm, very high concentrationsbeing lethal. Abscisic acid, though inhibitory to Lunulariain low concentrations, was not detected in extracts, and couldeasily be separated from lunularic acid.     

Auxin in the Cambium and its Differentiating Derivatives

Cambium and differentiating xylem and phloem tissues from thetrunks of trees of Acer pseudoplatanus L., Fraxinus excelsiorL., and Populus tremula L. were extracted with ether and testedfor auxin, which was found on chromatograms of the acidic fractionat an Rf corresponding to that of indol-3yl-acetic acid in fivesolvent systems. In addition, small amounts of auxin with ahigher Rf in ammoniacal isopropanol were found in phloem samples.The amounts of auxin were greatest in xylem samples, less inthe cambium, and least in phloem. The differences, which cannotbe explained in terms of differential losses during extractionand purification, suggest that auxin is actually formed in differentiatingxylem tissue. The significance of these results is discussed.     

Synthesis of auxins from tryptophan and tryptophan-precursors by fungi isolated from mycorrhizae of pine (Pinus silvestris L.).

Fungi isolated from mycorrhizae of pine required tryptophan for auxin synthesis. More auxins were found in culture grown with pyrogallol than in those without this compound. The fungi studied produced also auxins from other than tryptophan compounds. Indole employed with serine was more suitable for the production of auxins than indole or anthranilic acid used separately. The active compounds showing auxin activity were located on the chromatograms at Rf 0.2--0.4 and 0.3--0.5 with the solvent system isopropanol, ammonia, water (10:1:1 v/v).     

Synthesis of auxins by fungi isolated from the roots of pine seedings (Pinus silvestris L.) and from soil.

Synthesis of auxins by fungi grown with and without tryptophan has been studied. 26 out of 30 fungal strains produced detectable amounts of auxins in tryptophan contain media. 18 strains produced but very small amounts of auxins in media without this amino acid. By means of paper chromatography, chromogenic reagents and biotest three active substances could be distinguished. They were found on the chromatograms run with isopropanol, ammonia, water (10:1:1 v/v) at Rf 0.05--0.2, 0.3--0.5 and 0.8--1.0. Most strains produced active substances with Rf 0.3--0.5.     

Production of auxins by bacteria isolated from the roots of pine seedlings (Pinus silvestris L.).

Qualitative and quantitative studies were carried out on the production of auxins by Coryneform bacteria, the only bacterial types isolated from roots of pine seedlings. Almost all isolates were capable of producing auxins in tryptophan containing media. In media without this amino acid only trace or no auxins were produced. Most of the bacteria studied synthesized auxins located on the chromatograms run with isopropanol, ammonia, water (10:1:1 v/v) at Rf 0.3--0.5. Moreover substances with Rf values 0.05--0.2 and 0.8--1.0 were produced by some strains. No plant growth inhibitors detected with the Avena coleoptiles biotest were produced by the bacteria studied.     

Seed dormancy in Acer: Endogenous germination inhibitors and dormancy in Acer pseudoplatanus L.

Summary Dormant seeds of Acer pseudoplatanus L. contain two zones of inhibition on paper chromatograms in 10:1:1 as detected by the lettuce and cress seed germination, and the wheat coleoptile bioassays. One zone at R_f 0.6–0.8 was partitioned into ethyl acetate at acid pH and was shown to contain ABA by its behaviour on GLC and isomerization under ultra-violet light. The other zone at R_f 0.9 was detected only in the germination bioassays and was partitioned into ethyl acetate over a range of pH indicating the presence of one or more neutral compounds.The inhibitors present in the embryo of dormant sycamore seeds inhibited the germination of non-dormant sycamore seeds at relatively low concentrations. A comparison with the effects of application of exogenous ABA indicated that endogenous ABA could not solely account for the inhibitory activity of seed extracts, which appeared to be due partly to the presence of ABA and partly to that of neutral compounds present in the embryo. Leaching treatments that removed dormancy led to a decrease in the level of inhibitors present mainly in the basic fraction. The exogenous application of kinetin to dormant sycamore seeds increased germination whereas gibberellic acid had no effect. Similar responses were obtained with lettuce seeds inhibited by the basic fraction of dormant sycamore seeds.It is suggested that an inhibitor-cytokinin interaction may be involved in the dormancy of sycamore seeds.     

Gibberellins in the embryonic axes of tall and dwarf beans and their changes with initial growth

Ten gibberellin-like activities were detected in the dry embryonicaxes of tall (cv. Kentucky Wonder) and dwarf (cv. Masterpiece)beans (Phaseolus vulgaris L.) using the lettuce hypocotyl assayof thin-layer chromatograms; 2 in the non-acidic ethyl acetatefraction (NEI, NEII), 3 in the acidic ethyl acetate fraction(AEI, AEII, AEIII), 2 in the non-acidic n-butanol fraction (NBI,NBII) and 3 in the acidic n-butanol fraction (ABI, ABII, ABIII).There was no qualitative difference in these gibberellins betweenthe tall and dwarf axes, but all, particularly AEIII, NBII andABIII as the main gibberellins in the axes, were contained muchmore abundantly in the tall axes. In both axes the gibberellinactivities of most fractions decreased during germination.Theamounts of some gibberellins in tall axes without cotyledonswere greater than those in axes with cotyledons at 48–72hr of germination. Neither AMO-1618 nor CCC caused significantreduction in the levels of the gibberellins. Axis growth inthe early germinating period depended on the gibberellins storedin the axis, itself. (Received November 26, 1974; )     

Characterization of antimicrobial compounds from a common fern, Pteris biaurita

Methanol extract was prepared from the fronds of Pteris biaurita and partial purification was done by solvent partitioning with diethyl ether and ethyl acetate, followed by hydrolysis and further partitioning with ethyl acetate. The three fractions, thus obtained were bioassayed separately against five test fungi--Curvularia lunata, Fomes lamaoensis, Poria hypobrumea, Fuasrium oxysporum and a bacterium--Bacillus pumilus, by spore germination, radial growth and agar cup techniques. Results revealed that ethyl acetate fraction (III) contained the active principle. TLC plate bioassay of the active fraction revealed inhibition zone at an Rf of 0.5-0.65. Silica gel from this region was scraped, eluted in methanol and subjected to UV-spectrophotometric analysis. An absorption maxima of 278 nm was recorded. HPLC analysis of TLC-eluate revealed a single peak with retention time of 8.1 min. GC-MS analysis revealed six major peaks in the retention time range of 7.2-10.9 min. Comparison with GC-MS libraries revealed that the extracts may contain a mixture of eicosenes and heptadecanes.     

Characterisation of polyphenols by HPLC-PAD-ESI/MS and antioxidant activity in Equisetum telmateia

The antioxidant activity of an aqueous extract (infusion) and respective ethyl acetate fraction of Equisetum telmateia Ehrh. (Equisetaceae), a plant used in traditional medicine for its anti-inflammatory and diuretic properties, has been evaluated by DPPH, TEAC and TBARS assays. A high and significant antioxidant activity was detected in the ethyl acetate fraction. Analysis of the aqueous extract and the ethyl acetate fraction by HPLC-PAD-ESI/MS allowed the identification of the major phenolic compounds as flavan-3-ol, kaempferol and phenolic acid derivatives. Among the flavan-3-ols, A-type proanthocyanidins and afzelechin derivatives were detected as well as the more common B-type procyanidins, B2 and C1, whose identification was further confirmed by HPLC using detection involving chemical reaction with p-dimethylamino-cinnamaldehyde. The results suggest that the anti-inflammatory activity of E. telmateia could be due, at least in part, to the presence of compounds with antioxidant activity.     

Retaining the viability of Camellia japonica pollen in various organic solvents

Pollen viability in 31 organic solvents was studied. Pollengrains of Camellia japonica which had been soaked in 28 organicsolvents for 3 days retained their viability and grains in 19solvents such as ethyl acetate, n-amyl alcohol and petroleumether grew longer pollen tubes than fresh pollen. (Received July 20, 1972; )     

Enhancement of anti-candidal activity of endophytic fungus Phomopsis sp. ED2, isolated from Orthosiphon stamineus Benth, by incorporation of host plant extract in culture medium

This study examined the effect of host extract in the culture medium on anti-candidal activity of Phomopsis sp. ED2, previously isolated from the medicinal herb Orthosiphon stamineus Benth. Interestingly, upon addition of aqueous host extract to the culture medium, the ethyl acetate extract prepared from fermentative broth exhibited moderate anti-candidal activity in a disc diffusion assay. The minimal inhibitory concentration of this extract was 62.5 μg/ml and it only exhibited fungistatic activity against C. albicans. In the time-kill study, a 50% growth reduction of C. albicans was observed at 31.4 h for extract from the culture incorporating host extract. In the bioautography assay, only one single spot (Rf 0.59) developed from the extract exhibited anti-candidal activity. A spot with the a similar Rf was not detected for the crude extract from YES broth without host extract. This indicated that the terpenoid anti-candidal compound was only produced when the host extract was introduced into the medium. The study concluded that the incorporation of aqueous extract of the host plant into the culture medium significantly enhanced the anti-candidal activity of Phomopsis sp. ED2.