Efficiency of Countercurrent Distribution, Sephadex G-10, and Silicic Acid Partition Chromatography in the Purification and Separation of Gibberellin-like Substances from Plant Tissue

The effectiveness of countercurrent distribution, Sephadex G-10column chromatography and silicic acid partition column chromatographyin the purification of gibberellin-like substances from extractsof etiolated Phaseoltu multiflorus seedlings, and elongatingvegetative shoots of Arizona cypress (Cupressus arizonica Greene)and coastal Douglas-fir (Pseudotsuga menziesii var. menziesii)was followed by the use of the barley half-seed -amylase bioassayand dry-weight measurements. Countercurrent distribution resultedin a 10- to 30-fold reduction in the dry weight of the acidic,ethyl acetate-soluble fraction. Sephadex G-10 column chromatographyfurther lowered the dry weight by about two-thirds. Silicicacid partition column chromatography separated gibberellin-likesubstances from each other and again reduced the dry weight.Enhancement of bioassay activity was noted at each step of thepurification procedure. It is concluded that the above proceduresconstitute useful and efficient tools for the initial purificationof gibberellin-like substances from plant tissue.     

CHANGES IN THE AMOUNT OF GIBBERELLIN-LIKE SUBSTANCES IN THE SEEDLING OF PHARBITIS NIL WITH SPECIAL REFERENCE TO EXPANSION OF COTYLEDON

Two gibberellin-like substances were separated chromatographicallyfrom the ethanol-extract of germinating cotyledons of Pharbitisnil. In the dark-grown seedling, these substances in the cotyledonincreased in amount gradually with the slow increase in freshweight and area of the tissue, and attained, 4 days after sowing,a maximum level of 0.03 µg gibberellin A₃ equivalent pera couple of cotyledon; in the light-grown seedling, they increasedrapidly with the rapid increase in the fresh weight and area,and attained a maximum content of 0.07 µg gibberellinA₃ equivalent per cotyledon on the 6th day. When the etiolatedseedling was exposed to light, the amount of the gibberellin-likesubstances in the cotyledon increased in parallel with openingof the hypccotyl hook and expansion of the cotyledon. The openingof the hook and increase of the substances occurred in red lightas well as in far-red. Relatively small amounts of the gibberellin-like substanceswere also contained in the germinating hypocotyl and root. The growth of cotyledonary disks was promoted by gibberellinA₃ and the gibberellin-like substances from the expanding cotyledon. The dwarf rice seedling and dwarfs 1, 3 and 5 of maize respondedto the gibberellin-like substances, but dwarf Pharbitis didnot. (Received August 20, 1963; )     

Evaluation of a Bioassay for Cytokinins Using Soybean Hypocotyl Sections

The dose-response curves of several cytokinins were investigatedin a soybean hypocotyl bioassay. Zeatin riboside, zeatin-O-ß-D-glucoside,dihydrozeatin, and dihydrozeatin riboside produced linear responsesparallel to that for zeatin. The hypocotyl section assay wassuperior to the conventional soybean callus assay because theresponse (log₁₀ transformed data) was linear, exhibited lowvariability, and was more reproducible and more sensitive. Theassay was quicker to perform and required less cytokinin.     

Gibberellin-like Substances in Immature Seeds of Guava (Psidium guajava L.)

From immature seeds of guava fruits, Psidium guajava, L. eightgibberellin-like substances designated X1, X2, X3, X4, X5, X6,X7, and X8 were isolated. Chemical and biological evidence ledto the tentative identification of X1, X2, X3, X4, X5, X6, andX7 as gibberellins A₆, A₅, A₁, A₃, A₇, A₉, and A₄ respectively.Support for such identification was obtained from paper chromatography,thin-layer chromatography, gradient elution column chromatography,and the lettuce hypocotyl test. The compound X8 has not beenidentified chemically.     

Cytokinins in Gladiolus (Gladiolus grandiflorus) Corms

Ten cytokinin-like substances, termed X₁, X₂, X₃, X_4a, X_4b,X_5a, X_5b, X₆, X₇, and X₈, active in the soya bean hypocotyltest, were detected in gladiolus corms. The factors X_4a, X_5aand X₄ were tentatively identified as zeatin (Z), isopentenyladenosine (iPA) and isopentenyl adenine (iP), respectively,based on their behaviour in Sephadex LH-20 column chromatography,paper chromatography and high pressure liquid chromatography.Factor X₃, which behaved like zeatin riboside (ZR) in the abovesystems could be ZR and/or dihydrozeatin riboside (DHZR). Thebehaviour in Sephadex and ion-exchange column chromatographysuggested that X₁ and X₂ may be cytokinin glucosides and X₅a cytokinin nucleotide or a cytokinin conjugate similar to lupinicacid. The total cytokinin content and the concentration of Z,ZR/DHZR and iPA were higher in non-dormant than in dormant corms.The concentrations of X₁ and X₂ were higher in dormant corms. Gladiolus grandiflorus, gladiolus, dormancy, cytokinins, Sephadex column chromatography, high pressure liquid chromatography     

A Comparative Study of Multiple: Acid Phosphatases in Cellular Fractions of Bean Hypocotyl

Six kinds of acid phosphatases were solubilized with TritonX-100 from the cell wall (W-I, W-II), mitochondrial (M-I, M-II,M-III) and microsomal (Ms) fractions of bean hypocotyl, andthey were partially purified by using Sephadex gel filtrationand DEAE-cellulose column chromatography. Acid phosphatasesfrom the soluble fraction were also fractionated into 12 isozymesby electrophoresis, and the properties of the isozymes werecompared. The soluble isozymes showed pH optima at 5·0,5·3 and 5·6; the isozymes possessed high affinityto p-nitrophenyl phosphate and ADP, and their mol. wts rangedfrom 30000 to 45000. Among the solubilized phosphatases, W-I,M-I and M-III showed maximum activity at pH 5·0 and theirmol. wts were between 50000 and 110000. W-I proved to have highaffinity to ATP and bis-p-nitrophenyl phosphate as M-I did top-nitrophenyl phosphate and M-III to phenyl phosphate. The characteristicsof these solubilized isozymes were different from those of thesoluble isozymes. On the contrary, W-II, M-II and Ms were quitesimilar to those of the solubles in pH optima, substrate specificity,K_m value, affinity to DEAE-cellulose and gel electrophoreticpatterns. These results suggest that W-II, M-II and Ms werederived from the soluble isozymes. Phaseolus vulgaris L., bean, hypocotyl, acid phosphatases, Michaelis constant     

Tuberization in Potato at High Temperatures: Gibberellin Content and Transport from Buds

Warm temperatures (35°C day/30°C night) which inhibittuberization in potato (Solanum tuberosum L., cv. Sebago) increasedgibberellin activity in crude extracts from buds, but not frommature leaves, as determined by the lettuce hypocotyl bioassay.Changes in the growth of tubers and stolons indicate the occurrenceof basipetal movement of GA₃ applied to the terminal bud ora mature leaf. ¹⁴C labelling from GA₃ or mevalonic acid injectedjust below the terminal bud was recovered in the lower shoot,stolons and tubers, but the amount transported was greater atcool temperatures (20/15°C). It is concluded that high temperaturespromote the synthesis of gibberellin in the buds rather thantransport to the stolons. Solanum tuberosum L., potato, tuberization, gibberellin     

Effect of darkening the cotyledons on the growth and curvature of the sunflower hypocotyl: evidence of hydraulic signalling

When both cotyledons of light-grown sunflower seedlings (Helianthusannuus L.) were darkened by covering them with aluminium foil,the resulting increase in the rate of elongation of the hypocotylwas closely proportional to the associated reduction in seedlingtranspiration and to the increase in xylem water potential (_PX.Covering only one cotyledon induced curvature away from thatside. This response was associated with a higher _PX in the vascularbundles connected directly with the covered cotyledon than inthose connected with the illuminated cotyledon. The water contentof peripheral tissues of the hypocotyl below the covered cotyledonwas also higher than that of similar samples from below theilluminated cotyledon. These results are consistent with thehypothesis that the effect of darkening the cotyledons on thegrowth and curvature of the hypocotyl is mediated by hydraulicsignalling, characterized by transmission to the hypocotyl ofan increase in _PX resulting from the reduction in cotyledontranspiration. Key words: Cotyledons, Helianthus annuus, hydraulic signalling, hypocotyl, transpiration, water potential     

Enhancement of Gibberellic Acid-stimulated Hypocotyl Elongation of Lettuce (Lactuca sativa L. cv. Grand Rapids) by Phlorizin and Light

The interaction of light, gibberellic acid (GA₃), and phlorizinin the growth of lettuce (Lactuca sativa L. cv. ‘GrandRapids’) hypocotyls was investigated. At all concentrationsof GA₃, phlorizin enhanced GA₃-induced growth at luminous intensitiesabove 50 ft-c (continuous light). Without GA₃, phlorizin hadno effect on hypocotyl growth in the light but it inhibitedgrowth in the dark. Both seedlings and hypocotyl sections respondedto phlorizin in the presence of GA₃. There was no iteractionbetween phlorizin and KCl. Water-growth was severly inhibitedby light. GA₃,-induced growth was slightly inhibited by light,and then only at luminous intensities above 50 ft-c. Thus, relativeto H₂O-growth, GA₃-induced growth increased with increasingluminous intensity up to 450 ft-c, where it reached saturation.It seems that a synergism may exist between light and GA₃ aswell as between phlorizin and GA₃. Lactuca sativa L, lettuce, hypocotyl elongation, gibberellic acid, phlorizin, light     

Partial Purification and Some Properties of Flavonol 3-O-Glycosyltransferases from Seedlings of Vigna mungo, with Special Reference to the Formation of Kaempferol 3-O-Galactoside and 3-O-Glucoside

A novel enzyme, UDP-D-galactose:flavonol 3-O-galactosyltransferase(F3GaT), catalyzing the transfer of D-galactose from UDP-D-galactoseto the 3 position of 5,7,4'-trihydroxyflavonol (kaempferol),was detected in and purified about 404-fold from seedlings ofVigna mungo by precipitation with ammonium sulfate, chromatographyon Sephadex G-100 and chromatofocusing. The enzyme was separatedby this procedure from a coexisting UDP-D-glucose:flavonol 3-O-glucosyltransferase(F3GT), which was simultaneously purified about 189-fold. F3GaTwas isolated as a soluble enzyme with pH optima of 8.0 in imidazole-HClbuffer and 7.5 in histidine-HCl buffer. F3GT had the same pHoptima. The M_r of both F3GaT and F3GT, which had isoelectricpoints of 5.1 and 6.1, respectively, was estimated by elutionfrom a column of Sephadex G-100 to be about 43,000. The activitiesof F3GaT and F3GT were stimulated by 14 mM 2-mercaptoethanoland strongly inhibited by 1 mM Cu²⁺, 1 mM Zn²⁺, and variousreagents that react with sulfhydryl groups. Among various possiblesubstrates for F3GaT that were tested, kaempferol, isorhamnetinand quercetin were the best. The K_m values for kaempferol andUDP-D-galactose were determined to be 0.40 µM and 125µM, respectively. Similarly, F3GT had low K_m values of0.69 µM for kaempferol and 1.67 mM for UDP-D-glucose.F3GaT and F3GT mediated the transfer of galactose and glucose,respectively, to the 3-hydroxyl groups exclusively of kaempferol,isorhamnetin and quercetin. Rhamnetin also functioned as a galactosylacceptor though less efficiently. (Received October 12, 1992; )     

Control by Light of Hypocotyl Elongation and Levels of Endogenous Gibberellins in Seedlings of Lactuca sativa L.

Four 13-hydroxygibberellins, gibberellin A₁ (GA₁), 3-epi-GA₁,GA₁₉ and GA₂₀ were identified by full-scan GC/MS in extractsof lettuce seedlings (Lactuca sativa L. cv. Grand Rapids). Theresults suggest that the early-13-hydroxylation biosyntheticpathway to GA₁ functions in the lettuce seedlings. It was alsofound that GA₁ is active per se in the control of hypocotylelongation in lettuce seedlings. To investigate the relationshipbetween control by light of hypocotyl elongation and levelsof endogenous GAs in lettuce, endogenous levels of GAs werequantified by radioimmunoassay in seedlings that had been grownfor 5 days in the dark (5D) and in those that had been grownfor 4 days in the dark and then under white light for 1 day(4D1L). The endogenous level of GA₁ in the upper and lower partsof hypocotyls in 5D seedlings was about four times higher thanthat in 4D1L seedlings. The response of explants (hypocotylsegments with cotyledons) from dark-grown seedlings to GA₁ isknown to be similar in the dark and under white light when theexplants are treated with inhibitors of the biosynthesis ofGA. Therefore, the above information suggests that the highlevel of GA₁ in hypocotyls of dark-grown seedlings is responsiblefor the rapid elongation of hypocotyl, while irradiation bywhite light decreases the endogenous level of GA₁ in the hypocotylswith a resultant decrease in the rate of hypocotyl elongation. (Received March 13, 1992; Accepted May 21, 1992)     

Genetic Variation of Hypocotyl Length in the Brussels Sprout

A study was made of variation in the hypocotyl length of twoinbred lines of Brassica oleracea gemmifera (Brussels sprout).The pattern of means and variances obtained from parental, F₁,F₂ and backcross generations suggested polygenic control ofhypocotyl length. Measurements of parental seed weights, embryocell numbers, and the growth of seedlings from seeds of knownweight support the view that variation between the lines inhypocotyl length, when the seedlings were exposed to light,was determined by differential extension rather than by initialdifferences in seed weight. The effect of reduction in lightintensity on hypocotyl extension was studied and the differencesbetween the lines was maintained under all light intensities.Differential reaction of the lines was only observed when theperiod allowed for growth was short. The inbred line with thelonger hypocotyl was shown to suppress the other under competitiveconditions.     

Activity of Gibberellins A1 to A0 in the Avena First-leaf Bioassay, and Location after Chromatography

Activity curves are determined for gibberellins A₁ to A₀ bythe Avena first-leaf bioassay method. Gibberellins A¹, A₄ andA₅ can be detected at 10^-11 or 10^-10 g/ml and give optimum activityof approximately 230 per cent elongation (water controls = 100per cent). Gibberellins A²A³, and A⁹ can be detected at 10^-3g/mland give optimum activity of approximately 200 per cent. GibberellinsA₆ and A₇ can be detected at 10^-5g/ml; GA₇ gives optimum activityof around 190 per cent. All the gibberellins except GA₈ canbe detected by this bioassay method after chromatography inn-butanol: 1.5 N ammonia (3: 1) and benzene: acetic acid: water(4: 2: 1) when applied to the paper at concentrations from O.Ito µg. The sensitivity of the method is compared withthat of other gibberellin bioassay methods.     

Carbonic Anhydrase from the Blue-Green Alga (Cyanobacterium) Anabaena variabilis

Carbonic anhydrase (CA) activity was detected in homogenatesfrom Anabaena variabilis ATCC 29413, M-2 and M-3, but not inthe suspension of the intact cells. Activity was higher in cellsgrown in ordinary air (low-CO₂ cells) than in those grown inair enriched with 2–4% CO₂ (high-CO₂ cells). Fractionationby centrifugation indicated that the CA from A. variabilis ATCC29413 is soluble, whereas both soluble and insoluble forms existin A. variabilis M-2 and M-3. The addition of dithiothreitoland Mg^{2 $} greatly decreased the CA activity of A. variabilisATCC 29413. The specific activity of the CA from A. variabilis ATCC 29413was increased ca. 200 times by purification with ammonium sulfate,DEAE-Sephadex A-50 and Sephadex G-100. Major and minor CA peaksin Sephadex G-100 chromatography showed respective molecularweights of 48,000 and 25,000. The molecular weight of the CAdetermined by polyacrylamide disc gel electrophoresis was 42,000?5,000.The activity of CA was inhibited by ethoxyzolamide (I₅₀=2.8?10^-9M), acetazolamide (I₅₀=2.5?10^-7 M) and sulfanilamide (I₅₀=2.9?10^-6M). (Received January 5, 1984; Accepted April 26, 1984)     

Purification and properties of cytochrome f from Brassica komatsuna leaves

Cytochrome f was extracted from leaves of Brassica komatsuna(Brassica Rapa L. var. pervidis Bailey) in an aqueous solutionusing methyl ethyl ketone and was purified by the followingsteps: (i) acetone precipitation, (ii) ammonium sulfate fractionation(0.33–0.7 saturation), (iii) DEAE-cellulose column chromatography,and (iv) Sephadex G-100 column chromatography. Characteristic spectroscopic properties and the midpoint potentialof the cytochrome were essentially identical with those of thecytochrome f from parsley reported by Bendall et al. Molecular weight of the cytochrome determined by gel filtrationwas close to 32,000 and it contained one haem per molecule ofprotein. The ferro-cytochrome was oxidized by potato polyphenol oxidasein the presence of chlorogenic acid. Under light-aerobic conditions, the ferro-cytochrome was rapidlyoxidized by the chlorophyll-protein CP743 from Chenopodium albumin the presence of menadione. Under light-anaerobic conditions,the oxidized cytochrome was reduced at a considerable rate. ¹ Cytochrome c₆ according to the enzyme nomenclature recommendedby I.U.P.A.C.-I.U.B. (5). (Received November 7, 1974; )     

Inhibition of oyster drill chemotaxis

A bioassay was developed by Rittschof el al. (1983) to examinedistance chemoreception in the predatory marine gastropod, Urosalpinxcinerea. This bioassay was used to test the effect of a senesof low mol. wt. organics on the ability of newly hatched oysterdrills to detect a prey odor released from barnacles, Balanusbalanoides. Two series of low mol. wt. organics were testedusing methanol as the reference compound. In one series, R-OH,the carbon chain length was varied from 1 to 4. In the secondseries, CH³-R, the chain length was held constant while thefunctional group, R, was varied. When these compounds were presentin the rnM range, they inhibited the creeping response of oysterdrills towards barnacle prey odor. In the CH₃-R series, inhibitionincreased in the following order: sodium acetate > ethylacetate > acetonitnle > methanol; and, in the alcoholseries C₁ to C₄, inhibition increased with increasing chainlength. No creeping response was observed when these compoundswere tested in the absence of prey odor.     

ISOLATION OF AN INHIBITOR OF GROWTH AND ROOT FORMATION FROM PORTULACA GRANDIFLORA LEAVES

1. From leaves of Portulaca grandiflora, a substance which inhibitedthe IAA-induced elongation of Avena coleoptile sections andthe adventitious root formation of Raphanus hypocotyl cuttingswas separated by means of thin layer chromatography. It wasisolated and crystallized. 2. On paper chromatograms, this substance gave the same R_f valuesas the inhibitor from leaves of Xanthium strumarium and thatfrom leaves of Helianthus tuberosus ("heliangine"), namely,R_f0.9 in ammoniacal isopropanol, R_f 0.85 in methanol-water andR_f 0.0 in n-hexane-water. On thin layer chromatograms, however,these inhibitors were clearly separated from each other. 3. Infra-red absorption spectrum also indicated that this substanceis identical with neither xanthinin nor heliangine. ¹ Contribution No. 8 from the Botanical Gardens, Faculty ofScience, University of Tokyo, Koishikawa, Tokyo     

Purification of cytochrome f from Japanese-radish leaves

Cytochrome f was purified (A_420.5/A₂₇₃, 2.0) from acetone extractsof Japaneseradish leaves without use of detergent. By gel filtrationwidi Sephadex G-100 the molecular weight was estimated to be33,000 daltons. (Received September 17, 1974; )     

Al Binding in the Epidermis Cell Wall Inhibits Cell Elongation of Okra Hypocotyl

Al inhibits root elongation at micromolar concentrations, butthe mechanisms leading to this process are unknown. In thesestudies, Al-induced inhibition of cell elongation was examinedusing hypocotyl of okra (Abelmoschus esculentus Moench cv. ClemsonSpineless) as an experimental model. One-h exposure to Al (0.5mM A1Cl₃) in the presence of 10 µM auxin in 0.5 mM CaCl₂,pH 4.0 significantly inhibited auxin-induced cell elongationof okra hypocotyl segments. Elongation was further suppressedwith increasing Al concentrations up to 1 mM. Treatment of thehypocotyl with 1 mM citrate for 10 minutes after 2-h exposureto Al resulted in significant recovery of elongation. The amountof Al in the cell wall relative to the total in the tissue was96.0, 96.2, and 85.4%, respectively, following 1-, 2-, and 3-hexposure to the Al solution. The total and cell wall Al contentwas decreased by half after the citrate desorption treatment.Further-more, 95% of Al was found in the epidermis, and 95%of the Al in the epidermis was associated with the cell wall.Experiments using split hypocotyl segments showed that Al exposureincreased the outward bending of hypocotyl segments, suggestingthat the epidermis elongation was specifically inhibited byAl. Al inhibited the autolysis of epidermis by about 20%, buthad little effect on the autolysis of core tissue. Taken together,these results suggest that Al binding in the epidermal cellwall inhibits critical components in cell wall loosening mechanism,resulting in inhibition of cell elongation.     

Factors Affecting Shedding of Flowers in Soybean (Glycine max (L.) Merrill)

Flower shedding in soybean, Glycine max (L.) Merrill, was studiedusing cultivar ‘Clark’, isoline E₁t, which has relativelylong racemes for convenient identification and observation ofindividual flowers. On each raceme studied, pod set was greatestat the proximal (basal) positions, whereas shedding was greatestat the most distal positions. When proximal flowers were removedas they reached anthesis, pod set increased at the more distalpositions. Pod set was increased in some instances by applicationof water directly to the ovaries as a drop in the calyx cup.Peroxidase activity changed in parallel with ovary development,increasing rapidly in growing pods but not in shedding flowers.Increases in flower peroxidase was mainly in ovary walls. Flowerstaken at or near anthesis from positions with high percent podset could be grown in vitro with especially good ovary enlargement,whereas ovaries in flowers taken from positions of low pod setdid not enlarge in culture. Unidentified substances were extracted from young pods which,when incorporated into lanolin and tested in an in situ bioassay,could mimic the effect of proximal flowers in inducing sheddingof distal flowers. Indole-3-acetic acid resembled the extractedmaterials in inducing shedding, but differed by eliciting side-effectsthat extracts did not. The growth substances abscisic acid,gibberellic acid, and benzyladenine did not promote sheddingin the in situ test. The evidence was taken to indicate that soybean flower sheddingis induced in distal flowers by substances from the more proximal,fertilized ovaries, and that this is possibly due to interferencewith some of the intense metabolic changes that follow pollinationand fertilization.