Bailey, Lowell F.

Tackett, Dewey L.

English

ScholarWorks@UARK

Journal of the Arkansas Academy of ScienceVolume 11 Article 61958Growth Regulating Substances in Extracts of Mapleand Pear Flower BudsLowell F. BaileyUniversity of Arkansas, FayettevilleDewey L. TackettUniversity of Arkansas, FayettevilleFollow this and additional works at: http://scholarworks.uark.edu/jaasPart of the Botany Commons, and the Horticulture CommonsThis article is available for use under the Creative Commons license: Attribution-NoDerivatives 4.0 International (CC BY-ND 4.0). Users are able toread, download, copy, print, distribute, search, link to the full texts of these articles, or use them for any other lawful purpose, without asking priorpermission from the publisher or the author.This Article is brought to you for free and open access by ScholarWorks@UARK. It has been accepted for inclusion in Journal of the Arkansas Academyof Science by an authorized editor of ScholarWorks@UARK. For more information, please contact scholar@uark.edu, ccmiddle@uark.edu.Recommended CitationBailey, Lowell F. and Tackett, Dewey L. (1958) "Growth Regulating Substances in Extracts of Maple and Pear Flower Buds," Journal ofthe Arkansas Academy of Science: Vol. 11 , Article 6.Available at: http://scholarworks.uark.edu/jaas/vol11/iss1/615GROWTH REGULATING SUBSTANCES INEXTRACTSOF MAPLE AND PEAR FLOWER BUDSDewey L. Taokett and Lowell F. BaileyUniversity of ArkansasIn recent years several investigators have sug-gested that growth inhibitors may be responsiblefor dormancy in plants. Evanari(4) has called at-tention to the wide distribution of growth inhibi-tors in various plant parts, and Stewart and Cap—lin(13 ) have reported inhibiting substances inmaple buds, potato tubers, and onion bulbs. Hem-berg(7»8) proposed that growth inhibitors in potatotubers and Fraxinus buds are directly involved withdormancy, since these substances disappeared whendormancy was broken either naturally or artifici-ally. Hendershott and Bailey(9) extracted a sub-stance from peach flower buds which inhibited theelongation of pea epicotyl sections. However, theamount of inhibitor did not decrease as the end ofthe dormant period approached; in fact, the levelof inhibition did not decrease when the buds hadopened and flowers were evident. A later reportidentified this substance as a cyanide compound(lO).In the present study, dormant maple and pear flowerbuds have been shown to contain a substance whichinhibits the elongation of etiolated pea epicotylsections and does not give a test for cyanide. Pa-per chroma tography has been used to separate natur-ally—occurring growth regulators in bud extracts.Dormant buds of Acer saccharum Marsh, and Pyruscommunis L. var. JC. J_. Taylor were extracted at1°C. for twenty hours with three changes of anhy-drous ethyl ether. The combined ether extractswere evaporated to dryness and the residue storedat 5°C. in a dessicator over calcium chloride. Thepea straight growth test was used to detect growtheffects of substances obtained from buds. Pea seed-lings were grown in white sand in a darkroom main-tained at 25°C. and 80% relative humidity. A sec-tion was cut just below the plumule of an epicotylwhen the third internode was two to five cm. long.Cutting was done in weak red light, using a toolconsisting of two razor blades spaced 4.4 ± 0.2mm. apart. Sections were randomly distributed in15Journal of the Arkansas Academy of Science, Vol. 11 [1958], Art. 6Published by Arkansas Academy of Science, 1958ARKANSAS ACADEMY OF SCIENCE->v>>I16experimental solutions.The standard solution used in growth tests con-tained 1.0 p.p.m. 3-indoleacetic acid (IAA), 2.0%sucrose, and 0.05 M. phosphate buffer at pH 5»7«The experimental solutions contained, in addition,a measured quantity of bud extract or a zone cutfrom a paper chromatogram of a bud extract. Tenpea sections "were used with each test solution andafter twenty hours in a darkroom growth was meas-ured by us ing a photographic enlarger which magni-fied ten times .Growth Tests . Growth tests were made in Petridishes conta ining a filter paper disc and five ml.of test solution. Bud extracts were suspended infive ml. of distilled water and filtered before ad-dition to the standard solution at rates of 0.5,1.0, and 2.0 ml. The results of typical tests us-ing maple buds collected February 6, 1957 an(* pearbuds collected February 2, 1957 are summarized inTable I. These tests demonstrate the amount ofgrowth inhibition of pea sections obtained withmaple and pear bud extracts.Chromatography and Growth Tests. Bud extractswere fractionated by means of ascending paper chro-matography using lo x 2-§--inch strips of unwashedWhatman #1 filter paper. Good separation was ob-tained with 80% isopropanol as the developing sol-vent. A mixture of water, ethanol, butanol, andisopentanol (4:2-§-:l:l) also gave satisfactory re-sults. Ether extracts were redissolved in etherwith one ml. of water added. The ether was evap-orated and the water extract was streaked acrossone end of a paper strip and allowed to dry. Asa standard practice the development was carriedout in the darkroom and strips were equilibratedfor at least four hours prior to lowering into thesolvent .Growth tests were conducted in small covereddishes containing zones cut from a chromatogram,two ml. of the standard solution, and ten pea epi-cotyl sections. Results of a typical test usingan extract of dormant maple flower buds chromato-graphed in 80% isopropanol are presented in Fig-ure 1.The inhibition zones cover a wide range of Rfs,but those statistically s ignif icant are found onlybetween Rfs 0.50 and 0.80. An improved separation16Journal of the Arkansas Academy of Science, Vol. 11 [1958], Art. 6http://scholarworks.uark.edu/jaas/vol11/iss1/6ITABLE IEFFECT OF MAPLE AND PEAR FLOWER BUD EXTRACTS ON GROWTHOF PEA SECTIONS, INMILLIlvlETERS AND PERCENTMl. of extract addedPlant0.0 0.5 1.0 2.0Maple 6.92 ± 0.13* 5-80 ± 0.03 5.72 ± 0.04 5.29 ± 0.04(400 buds/ml. ) 100. 0$ 56.6$ 52.5$ 35. 3$Pear 8.31 ± 0.01 7.33 ± 0.09 7.18 ± 0.09 6.93 ± 0.09(l60 buds/ml.) 100.0% 75-0% 71-1$ 64.8%17Journal of the Arkansas Academy of Science, Vol. 11 [1958], Art. 6Published by Arkansas Academy of Science, 1958>••i)>>I18IARKANSAS ACADEMY OF SCIENCE4#0p3^5—j-- - -control____«_lsopropenol| 2.5 - I 12.0—1|? 1 1 , 1 . I , I , I i I ¦ 1 . 1 ¦ t . I .1.0 .9 .8 .7 .6 .5 .4 .3 .2 .1 0.0Figure 1. Results of a growth test using a chroma tograraof an extract of dormant maple flower budedeveloped in Qo% iaopropanol.18Journal of the Arkansas Academy of Science, Vol. 11 [1958], Art. 6http://scholarworks.uark.edu/jaas/vol11/iss1/6GROWTH REGULATING SUBSTANCES••>>>I19¦was obtained by eluting these zones overnight withwater at 5°^., evaporating the eluate to drynessover steam, and rechromatographing in the alcoholmixture. Growth tests with zones cut from thesechromatograms demonstrated that inhibiting sub-stances had completely masked the presence of agrowth—promot ing substance, as shown in Figure 2.Compounds fluorescing in ultraviolet light werelocated at the higher Rf s and these zones were usedfor separate growth tests.Results of growth tests using six chromatogramsprepared by two-step chroma tography are presentedin Table II.Two features are noteworthy: A growth-promotion zone most prominent at Rf 0.97* and agrowth-inhibition zone between RfS 0. oO and 0.80.No significant activity was recorded below Rf 0.50.CHARACTERIZATION OF THEGROWTH-ACTIVE SUBSTANCESGrowth Promotion Zone. The zone above Rf 0.97fluoresced pink under ultra-violet light, gave anacid reaction when streaked with a universal in-dicator, and an ash color when sprayed with Sal—kowski reagent(ll). The zone just beneath fluor—esced light purple, gave no acid reaction, and gavea faint pink color with Salkowski reagent. Syn-thetic IAA chromatographed with the alcohol mixturegave a pinkish fluorescence inultra-violet and wasacidic at Rf 0.97* Apparently the growth-promotionobtained at Rf 0.97 was a response to IAA in thebud extract.The same growth-promoting zone was evident inchromatograms of non-dormant buds, with the growthresponse being more pronounced. Apparently, thelevel of active IAA increases as buds pass from thedormant to the non— dormant state, as reported pre-viously! 6).Growth Inhibition Zone. Salkowski tests andexamination in ultra-violet light failed to giveany suggestion of an auxin type of compound in thezone 0 f inhibition. Tests for cyanide were madeafter hydrolysis with sodium hydroxide and the ad-dition of ferric chloride, by adding hydrochloricacid and ferrous sulfate. The presence of cyanidein this test is indicated by the formation of prus—19Journal of the Arkansas Academy of Science, Vol. 11 [1958], Art. 6Published by Arkansas Academy of Science, 1958>••i)>>>•I20J-RKANSAS ACADEMY OF SCIENCE4.03»5 - - • • control—m_mmmmmisopropanol*3.0—L.. _-U I 1g j I 1c 2.5-i I2.0_151 1 i I i I , I i 1 i 1 i I i I i 1 , 1 i1.0 .9 .8 .7 .6 .5 .* .3 .2 .1 0.0Figure 2. Growth teat results with an extract of dormantmaple flover buds using a two- step chroma togrephicprocess. The inhibition zone, between Pfs 0.500.80, of the 80/£ isopropanol chrome togrem wasrechroma tographed in the alcohol mixture.20Journal of the Arkansas Academy of Science, Vol. 11 [1958], Art. 6http://scholarworks.uark.edu/jaas/vol11/iss1/6TABLE IIGROWTH TEST RESULTS OF SIX CHROMATOGRAMS OF 100-150DORMANT AND NON-DORMANT MAPLE FLOWER BUDSDEVELOPED IN THE ALCOHOL MIXTURE AFTERA PRELIMINARY CHROMATOGRAPHINGIN80$ ISOPROPANOLkRfTest -.50 .60 .65 .70 .75 .80 .85 .90 .95 .97Dormant buds#1 I* I I I P* P P»2 I I I I P3 I I I I I I4 I I I I P P PNon-dormant buds(#4 I III P6 I P P*I — Inhibition; P — Promotion. Results significant at the one per cent1OTTO1 • •21Journal of the Arkansas Academy of Science, Vol. 11 [1958], Art. 6Published by Arkansas Academy of Science, 1958ARKANSAS ACADEMY OF SCIENCEI>)22sian blue. No evidence of cyanide compounds wasobtained with eluates of the inhibition zones.Elliott and Leopold(2) proposed that dormancyin oats may be due to inhibitors that antagonizesulfhydryl-containing enzymes. An —SH compoundcould competitively inhibit a sulfhydryl enzymenecessary for growth. When a drop of iodine-azidesolution! 5) ws applied to the chromatogranas in thezones of inhibition, the deep brown color of thesolution quickly disappeared. This is a specificreaction for thicketones (R=o) and thiols (R-SH).Zones on chromatograms that indicated the presenceof thicketones and/or thiols are given inTable III,as are those that gave evidence of being acid.With dormant buds, zones showing growth inhi-bition gave a positive test for divalent sulfurcompounds. Also, there was a close correlation be-tween the presence of sulfur and acidic substances,suggesting that thiols rather than thicketones wereinvolved. This was substantiated by the failureof the zone of inhibition to catalyze the iodine-azide reaction after mild oxidation with hydrogenperoxide. Apparently, a, thiol compound occurs inthe zone of inhibition.The close correlation between inhibition and thepresence of thiol compounds in dormant buds was notevident with non-dormant buds (Table III). "Thelevel of inhibition in non-dormant buds dropped,but there was no corresponding loss in thiols.Similar solubility properties and Rf values indi-cate that the thiol compound in both dormant andnon-dormant buds may be glutathione. Glutathioneis soluble * *», water and insoluble inether, occurredbetween RfS d. J>8 and O.65 when chroma tographed inthe alcohol mixture, and catalyzed the iodine—azidereaction.DISCUSSIONConsiderable evidence exists which indicatesthat dormancy in buds results from the presence ofsubstances specific for growth inhibition* Theearlier suggestion that auxins at relatively highconcentrations cause dormancy has been discreditedby several inve s t igators ( 12 j. Chan-Thom(l) showedthat auxin concentrations necessary for inhibitionnever occurred in dormant pear buds. Hemberg {*],8)22Journal of the Arkansas Academy of Science, Vol. 11 [1958], Art. 6http://scholarworks.uark.edu/jaas/vol11/iss1/6TABLE IIICHROMATOGRAPHIC SPECTRUM OF EXTRACTS OF DORMANT AND NON-DORMANTMAPLE FLOWER BUDS FOR ACIDITY AND IODINE-AZIDE TESTS.RESULTS ARE FOR SIX CHROMATOGRAMS DEVELOPEDIN THE ALCOHOL MIXTURE WITH A PREVIOUSCHROMATOGRAPHING IN 80# I30PR0PAN0LDormant No n-DormantTest No. Acidity Io"dine-az ide Acidity Iodine-azide1234 34 56 56Rf0.97 xxxx xx-.95 ..... x . x.90 . , x x.85 x xx xx.80 x xx xxi.75 xxxx xx xx x x.70 xxxx xx xx x x.65 xxxx xx xx xx.60 XXXX XX XX X X.55 xx x x x.50 " x x x23Journal of the Arkansas Academy of Science, Vol. 11 [1958], Art. 6Published by Arkansas Academy of Science, 1958>24ARKANSAS ACADEMY OF SCIENCEfound a connect ion between inhibitors and dormancyin potato and Fraxinus buds and established thatthe auxin content does not change either duringdorroanov or immediatelv thereafter. According tot tm cy y t r. r iLeopold(ll) dormancy results from the occurrenceof acidic inhibitors in buds. In the presenoe ofthese inhibitors endogenous auxin is ineffective.The onset of bud development is accompanied withthe loss of growth inhibitors and the productionof glutathione.In this study, dormant flower buds of maple areshuwn to contain substances that promote and in-hibit elongation of pea epicotyl sections. Thegrowth—promo ting substance apparently is 3— indole—acetic acid which increases in growth activity asdormancy is lost. The growth— inhibiting substanceis not a cyanide-containing compound. On paperchromatograms growth inhibition is a s socia ted withan acidic zone of one or more compounds which con-tain thiol groups. As dormancy passes, inhibitionin this zone disappears but the sulfhydryl reac-tion remains and extends over a wider zone on thechromatograms .The persistence of the sulfhydryl reaction inchromatograms of non— dormant buds suggests that thethiol detected in dormant buds may not be relateddirectly to the inhibitor. This thiol may be glu-tathione since these compounds give similar Rf val-ues when chromatographed in the same manner andpossess similar solubilitypropertie s . Glutathioneincrease has been reported as dormancy is lost(3)and this could account for the broad sulfhydrylzone on the chromatograms.The possibility exists that the thiol presentin dormant buds is not glutathione but a specificsubstance responsible for dormancy — acting as agrowth inhibitor by competitive interference withphysiological activityof intracellular SH—groups.Since the two compounds chromatograph similarly,the proposed thiol inhibitor couldbe structurallysimilar to glutathione and serve as a precursorfor this compound.SUMMARYEther extracts of dormant flower buds of mapleand pear contain one or more substances which in—24Journal of the Arkansas Academy of Science, Vol. 11 [1958], Art. 6http://scholarworks.uark.edu/jaas/vol11/iss1/6GROWTH REGULATING SUBSTANCES25Ihibit the elongation of etiolated pea epicotyl sec-tions. Paper chromatograms of bud extracts con-tained a growth-prom.o ting substance, probably IAA,and a growth-inhibiting zone. The latter was aci-dic, contained no cyanide, and gave a reactioncharacteristic of thiols. As dormancy passed, in-hibition disappeared but the sulfhydryl reactionremained. Glutathione may be involved in the sul-fhydryl reaction, although the thiol occurring dur-ing dormancy may not be identical with that occur-ring after dormancy ends.LITERATURE CITED(l) Chan-Thorn, L. A., 1951. A study of the res-piration of hardy pear buds in relation torest period. Doctoral thesis, Univ. ofCalif., Berkeley. (Cited in reference 12.)(2) Elliott,B. B. and A. C. Leopold, 1953. Aninhibitor of germination and of amylase ac-tivity in oat seeds. Physiol . Plantarum 6:66-78.(3) Emilsson, B., 1949. Studies on the rest per-iod and dormant period in the potato tuber.Ac ta . Agri. Sueo . _3.:189. (Cited in refer-ence 11.)(4) Evanari, M., 1949. Germination inhibitors.Bot. Rev. 15:153-194.(5) Feigl, P., 1954. Spot Tests. Organic Appli-cations. Vol. II.Elsevier Publishing Co.,Amsterdam.(6) Hemberg, T., 1947. Studies of auxins andgrowth-inhibiting substances in the potatotuber and their significance with regard toits rest period. Aeta . Eort. Berg . 14:133*(7) , 1949. Significance of growth-inhibit-ing substances and auxins for the rest-periodof the potato tuber. Physiol . Plantarum 2:24-36.(8) , 1949. Grov/th-inhibiting substances interminal buds of Fraxinus . Phys iol. Plan-tarum 2:37-44.(9) Hendershott, C. H. and L. F. Bailey, 1955.Growth inhibiting substances in extracts ofdormant flower buds of peach. Proc. Amer.Soc. Hort. Sci. 65: 85-92.25Journal of the Arkansas Academy of Science, Vol. 11 [1958], Art. 6Published by Arkansas Academy of Science, 195826ARKANSAS ACADEMY OF SCIENCE(lO) Jones, M. B., J. W.Fleming and L.F. Bailey,1957* Cyanide as a growth-inhibiting sub-stance in extracts of peach leaves, bark,and flower buds, Proc. Amer* Soc. Hort.Sci. 69:152-157.(ll) Leopold, A. C.| 1955* Auxins and Plant Growth.Univ. of Calif, Press, Berkeley.(- — _ _-—*-12) Samish, R. M.,1954. Dormancy inwoody plant sAnn. Rev. Plant Physiol. 5;183-204.(13) Steward, F. C. and S. M. 1952. In-vestigations of growth and metabolism ofLplant cells, III.Evidence for growth in-hibitors incertain mature tissues. AnnalsBot. l6:477-489 026Journal of the Arkansas Academy of Science, Vol. 11 [1958], Art. 6http://scholarworks.uark.edu/jaas/vol11/iss1/6

Growth Regulating Substances in Extracts of Maple and Pear Flower Buds

UARK (University of Arkansas )

Journal of the Arkansas Academy of Science Volume 11 Article 6 1958 Growth Regulating Substances in Extracts of Maple and Pear Flower Buds Lowell F. Bailey University of Arkansas, Fayetteville Dewey L. Tackett University of Arkansas, Fayetteville Follow this and additional works at: https://scholarworks.uark.edu/jaas  Part of the Botany Commons, and the Horticulture Commons Recommended Citation Bailey, Lowell F. and Tackett, Dewey L. (1958) "Growth Regulating Substances in Extracts of Maple and Pear Flower Buds," Journal of the Arkansas Academy of Science: Vol. 11 , Article 6. Available at: https://scholarworks.uark.edu/jaas/vol11/iss1/6 This article is available for use under the Creative Commons license: Attribution-NoDerivatives 4.0 International (CC BY-ND 4.0). Users are able to read, download, copy, print, distribute, search, link to the full texts of these articles, or use them for any other lawful purpose, without asking prior permission from the publisher or the author. This Article is brought to you for free and open access by ScholarWorks@UARK. It has been accepted for inclusion in Journal of the Arkansas Academy of Science by an authorized editor of ScholarWorks@UARK. For more information, please contact scholar@uark.edu. 15GROWTH REGULATING SUBSTANCES INEXTRACTSOF MAPLE AND PEAR FLOWER BUDSDewey L. Taokett and Lowell F. BaileyUniversity of ArkansasIn recent years several investigators have sug-gested that growth inhibitors may be responsiblefor dormancy in plants. Evanari(4) has called at-tention to the wide distribution of growth inhibi-tors in various plant parts, and Stewart and Cap—lin(13 ) have reported inhibiting substances inmaple buds, potato tubers, and onion bulbs. Hem-berg(7»8) proposed that growth inhibitors in potatotubers and Fraxinus buds are directly involved withdormancy, since these substances disappeared whendormancy was broken either naturally or artifici-ally. Hendershott and Bailey(9) extracted a sub-stance from peach flower buds which inhibited theelongation of pea epicotyl sections. However, theamount of inhibitor did not decrease as the end ofthe dormant period approached; in fact, the levelof inhibition did not decrease when the buds hadopened and flowers were evident. A later reportidentified this substance as a cyanide compound(lO).In the present study, dormant maple and pear flowerbuds have been shown to contain a substance whichinhibits the elongation of etiolated pea epicotylsections and does not give a test for cyanide. Pa-per chroma tography has been used to separate natur-ally—occurring growth regulators in bud extracts.Dormant buds of Acer saccharum Marsh, and Pyruscommunis L. var. JC. J_. Taylor were extracted at1°C. for twenty hours with three changes of anhy-drous ethyl ether. The combined ether extractswere evaporated to dryness and the residue storedat 5°C. in a dessicator over calcium chloride. Thepea straight growth test was used to detect growtheffects of substances obtained from buds. Pea seed-lings were grown in white sand in a darkroom main-tained at 25°C. and 80% relative humidity. A sec-tion was cut just below the plumule of an epicotylwhen the third internode was two to five cm. long.Cutting was done in weak red light, using a toolconsisting of two razor blades spaced 4.4 ± 0.2mm. apart. Sections were randomly distributed in15Journal of the Arkansas Academy of Science, Vol. 11 [1958], Art. 6Published by Arkansas Academy of Science, 1958ARKANSAS ACADEMY OF SCIENCE->v>>I16experimental solutions.The standard solution used in growth tests con-tained 1.0 p.p.m. 3-indoleacetic acid (IAA), 2.0%sucrose, and 0.05 M. phosphate buffer at pH 5»7«The experimental solutions contained, in addition,a measured quantity of bud extract or a zone cutfrom a paper chromatogram of a bud extract. Tenpea sections "were used with each test solution andafter twenty hours in a darkroom growth was meas-ured by us ing a photographic enlarger which magni-fied ten times .Growth Tests . Growth tests were made in Petridishes conta ining a filter paper disc and five ml.of test solution. Bud extracts were suspended infive ml. of distilled water and filtered before ad-dition to the standard solution at rates of 0.5,1.0, and 2.0 ml. The results of typical tests us-ing maple buds collected February 6, 1957 an(* pearbuds collected February 2, 1957 are summarized inTable I. These tests demonstrate the amount ofgrowth inhibition of pea sections obtained withmaple and pear bud extracts.Chromatography and Growth Tests. Bud extractswere fractionated by means of ascending paper chro-matography using lo x 2-§--inch strips of unwashedWhatman #1 filter paper. Good separation was ob-tained with 80% isopropanol as the developing sol-vent. A mixture of water, ethanol, butanol, andisopentanol (4:2-§-:l:l) also gave satisfactory re-sults. Ether extracts were redissolved in etherwith one ml. of water added. The ether was evap-orated and the water extract was streaked acrossone end of a paper strip and allowed to dry. Asa standard practice the development was carriedout in the darkroom and strips were equilibratedfor at least four hours prior to lowering into thesolvent .Growth tests were conducted in small covereddishes containing zones cut from a chromatogram,two ml. of the standard solution, and ten pea epi-cotyl sections. Results of a typical test usingan extract of dormant maple flower buds chromato-graphed in 80% isopropanol are presented in Fig-ure 1.The inhibition zones cover a wide range of Rfs,but those statistically s ignif icant are found onlybetween Rfs 0.50 and 0.80. An improved separation16Journal of the Arkansas Academy of Science, Vol. 11 [1958], Art. 6https://scholarworks.uark.edu/jaas/vol11/iss1/6ITABLE IEFFECT OF MAPLE AND PEAR FLOWER BUD EXTRACTS ON GROWTHOF PEA SECTIONS, INMILLIlvlETERS AND PERCENTMl. of extract addedPlant0.0 0.5 1.0 2.0Maple 6.92 ± 0.13* 5-80 ± 0.03 5.72 ± 0.04 5.29 ± 0.04(400 buds/ml. ) 100. 0$ 56.6$ 52.5$ 35. 3$Pear 8.31 ± 0.01 7.33 ± 0.09 7.18 ± 0.09 6.93 ± 0.09(l60 buds/ml.) 100.0% 75-0% 71-1$ 64.8%17Journal of the Arkansas Academy of Science, Vol. 11 [1958], Art. 6Published by Arkansas Academy of Science, 1958>••i)>>I18IARKANSAS ACADEMY OF SCIENCE4#0p3^5—j-- - -control____«_lsopropenol| 2.5 - I 12.0—1|? 1 1 , 1 . I , I , I i I ¦ 1 . 1 ¦ t . I .1.0 .9 .8 .7 .6 .5 .4 .3 .2 .1 0.0Figure 1. Results of a growth test using a chroma tograraof an extract of dormant maple flower budedeveloped in Qo% iaopropanol.18Journal of the Arkansas Academy of Science, Vol. 11 [1958], Art. 6https://scholarworks.uark.edu/jaas/vol11/iss1/6GROWTH REGULATING SUBSTANCES••>>>I19¦was obtained by eluting these zones overnight withwater at 5°^., evaporating the eluate to drynessover steam, and rechromatographing in the alcoholmixture. Growth tests with zones cut from thesechromatograms demonstrated that inhibiting sub-stances had completely masked the presence of agrowth—promot ing substance, as shown in Figure 2.Compounds fluorescing in ultraviolet light werelocated at the higher Rf s and these zones were usedfor separate growth tests.Results of growth tests using six chromatogramsprepared by two-step chroma tography are presentedin Table II.Two features are noteworthy: A growth-promotion zone most prominent at Rf 0.97* and agrowth-inhibition zone between RfS 0. oO and 0.80.No significant activity was recorded below Rf 0.50.CHARACTERIZATION OF THEGROWTH-ACTIVE SUBSTANCESGrowth Promotion Zone. The zone above Rf 0.97fluoresced pink under ultra-violet light, gave anacid reaction when streaked with a universal in-dicator, and an ash color when sprayed with Sal—kowski reagent(ll). The zone just beneath fluor—esced light purple, gave no acid reaction, and gavea faint pink color with Salkowski reagent. Syn-thetic IAA chromatographed with the alcohol mixturegave a pinkish fluorescence inultra-violet and wasacidic at Rf 0.97* Apparently the growth-promotionobtained at Rf 0.97 was a response to IAA in thebud extract.The same growth-promoting zone was evident inchromatograms of non-dormant buds, with the growthresponse being more pronounced. Apparently, thelevel of active IAA increases as buds pass from thedormant to the non— dormant state, as reported pre-viously! 6).Growth Inhibition Zone. Salkowski tests andexamination in ultra-violet light failed to giveany suggestion of an auxin type of compound in thezone 0 f inhibition. Tests for cyanide were madeafter hydrolysis with sodium hydroxide and the ad-dition of ferric chloride, by adding hydrochloricacid and ferrous sulfate. The presence of cyanidein this test is indicated by the formation of prus—19Journal of the Arkansas Academy of Science, Vol. 11 [1958], Art. 6Published by Arkansas Academy of Science, 1958>••i)>>>•I20J-RKANSAS ACADEMY OF SCIENCE4.03»5 - - • • control—m_mmmmmisopropanol*3.0—L.. _-U I 1g j I 1c 2.5-i I2.0_151 1 i I i I , I i 1 i 1 i I i I i 1 , 1 i1.0 .9 .8 .7 .6 .5 .* .3 .2 .1 0.0Figure 2. Growth teat results with an extract of dormantmaple flover buds using a two- step chroma togrephicprocess. The inhibition zone, between Pfs 0.500.80, of the 80/£ isopropanol chrome togrem wasrechroma tographed in the alcohol mixture.20Journal of the Arkansas Academy of Science, Vol. 11 [1958], Art. 6https://scholarworks.uark.edu/jaas/vol11/iss1/6TABLE IIGROWTH TEST RESULTS OF SIX CHROMATOGRAMS OF 100-150DORMANT AND NON-DORMANT MAPLE FLOWER BUDSDEVELOPED IN THE ALCOHOL MIXTURE AFTERA PRELIMINARY CHROMATOGRAPHINGIN80$ ISOPROPANOLkRfTest -.50 .60 .65 .70 .75 .80 .85 .90 .95 .97Dormant buds#1 I* I I I P* P P»2 I I I I P3 I I I I I I4 I I I I P P PNon-dormant buds(#4 I III P6 I P P*I — Inhibition; P — Promotion. Results significant at the one per cent1OTTO1 • •21Journal of the Arkansas Academy of Science, Vol. 11 [1958], Art. 6Published by Arkansas Academy of Science, 1958ARKANSAS ACADEMY OF SCIENCEI>)22sian blue. No evidence of cyanide compounds wasobtained with eluates of the inhibition zones.Elliott and Leopold(2) proposed that dormancyin oats may be due to inhibitors that antagonizesulfhydryl-containing enzymes. An—SH compoundcould competitively inhibit a sulfhydryl enzymenecessary for growth. When a drop of iodine-azidesolution! 5) ws applied to the chromatogranas in thezones of inhibition, the deep brown color of thesolution quickly disappeared. This is a specificreaction for thicketones (R=o) and thiols (R-SH).Zones on chromatograms that indicated the presenceof thicketones and/or thiols are given inTable III,as are those that gave evidence of being acid.With dormant buds, zones showing growth inhi-bition gave a positive test for divalent sulfurcompounds. Also, there was a close correlation be-tween the presence of sulfur and acidic substances,suggesting that thiols rather than thicketones wereinvolved. This was substantiated by the failureof the zone of inhibition to catalyze the iodine-azide reaction after mild oxidation with hydrogenperoxide. Apparently, a, thiol compound occurs inthe zone of inhibition.The close correlation between inhibition and thepresence of thiol compounds in dormant buds was notevident with non-dormant buds (Table III). "Thelevel of inhibition in non-dormant buds dropped,but there was no corresponding loss in thiols.Similar solubility properties and Rf values indi-cate that the thiol compound in both dormant andnon-dormant buds may be glutathione. Glutathioneis soluble**», water and insoluble inether, occurredbetween RfS d. J>8 and O.65 when chroma tographed inthe alcohol mixture, and catalyzed the iodine—azidereaction.DISCUSSIONConsiderable evidence exists which indicatesthat dormancy in buds results from the presence ofsubstances specific for growth inhibition* Theearlier suggestion that auxins at relatively highconcentrations cause dormancy has been discreditedby several inve s t igators ( 12 j. Chan-Thom(l) showedthat auxin concentrations necessary for inhibitionnever occurred in dormant pear buds. Hemberg {*],8)22Journal of the Arkansas Academy of Science, Vol. 11 [1958], Art. 6https://scholarworks.uark.edu/jaas/vol11/iss1/6TABLE IIICHROMATOGRAPHIC SPECTRUM OF EXTRACTS OF DORMANT AND NON-DORMANTMAPLE FLOWER BUDS FOR ACIDITY AND IODINE-AZIDE TESTS.RESULTS ARE FOR SIX CHROMATOGRAMS DEVELOPEDIN THE ALCOHOL MIXTURE WITH A PREVIOUSCHROMATOGRAPHING IN 80# I30PR0PAN0LDormant No n-DormantTest No. Acidity Io"dine-az ide Acidity Iodine-azide1234 34 56 56Rf0.97 xxxx xx-.95 ..... x . x.90 . , x x.85 x xx xx.80 x xx xxi.75xxxx xx xx x x.70 xxxx xx xx x x.65 xxxx xx xx xx.60 XXXX XX XX X X.55 xx x x x.50 " x x x23Journal of the Arkansas Academy of Science, Vol. 11 [1958], Art. 6Published by Arkansas Academy of Science, 1958>24ARKANSAS ACADEMY OF SCIENCEfound a connect ion between inhibitors and dormancyin potato and Fraxinus buds and established thatthe auxin content does not change either duringdorroanov or immediatelv thereafter. According tot tm cy y t r. r iLeopold(ll) dormancy results from the occurrenceof acidic inhibitors in buds. In the presenoe ofthese inhibitors endogenous auxin is ineffective.The onset of bud development is accompanied withthe loss of growth inhibitors and the productionof glutathione.In this study, dormant flower buds of maple areshuwn to contain substances that promote and in-hibit elongation of pea epicotyl sections. Thegrowth—promo ting substance apparently is 3— indole—acetic acid which increases in growth activity asdormancy is lost. The growth— inhibiting substanceis not a cyanide-containing compound. On paperchromatograms growth inhibition is a s socia ted withan acidic zone of one or more compounds which con-tain thiol groups. As dormancy passes, inhibitionin this zone disappears but the sulfhydryl reac-tion remains and extends over a wider zone on thechromatograms .The persistence of the sulfhydryl reaction inchromatograms of non— dormant buds suggests that thethiol detected in dormant buds may not be relateddirectly to the inhibitor. This thiol may be glu-tathione since these compounds give similar Rf val-ues when chromatographed in the same manner andpossess similar solubilitypropertie s . Glutathioneincrease has been reported as dormancy is lost(3)and this could account for the broad sulfhydrylzone on the chromatograms.The possibility exists that the thiol presentin dormant buds is not glutathione but a specificsubstance responsible for dormancy—acting as agrowth inhibitor by competitive interference withphysiological activityof intracellular SH—groups.Since the two compounds chromatograph similarly,the proposed thiol inhibitor couldbe structurallysimilar to glutathione and serve as a precursorfor this compound.SUMMARYEther extracts of dormant flower buds of mapleand pear contain one or more substances which in—24Journal of the Arkansas Academy of Science, Vol. 11 [1958], Art. 6https://scholarworks.uark.edu/jaas/vol11/iss1/6GROWTH REGULATING SUBSTANCES25Ihibit the elongation of etiolated pea epicotyl sec-tions. Paper chromatograms of bud extracts con-tained a growth-prom.o ting substance, probably IAA,and a growth-inhibiting zone. The latter was aci-dic, contained no cyanide, and gave a reactioncharacteristic of thiols. As dormancy passed, in-hibition disappeared but the sulfhydryl reactionremained. Glutathione may be involved in the sul-fhydryl reaction, although the thiol occurring dur-ing dormancy may not be identical with that occur-ring after dormancy ends.LITERATURE CITED(l) Chan-Thorn, L. A., 1951. A study of the res-piration of hardy pear buds in relation torest period. Doctoral thesis, Univ. ofCalif., Berkeley. (Cited in reference 12.)(2) Elliott,B. B. and A. C. Leopold, 1953. Aninhibitor of germination and of amylase ac-tivity in oat seeds. Physiol . Plantarum 6:66-78.(3) Emilsson, B., 1949. Studies on the rest per-iod and dormant period in the potato tuber.Ac ta . Agri. Sueo . _3.:189. (Cited in refer-ence 11.)(4) Evanari, M., 1949. Germination inhibitors.Bot. Rev. 15:153-194.(5) Feigl, P., 1954. Spot Tests. Organic Appli-cations. Vol. II.Elsevier Publishing Co.,Amsterdam.(6) Hemberg, T., 1947. Studies of auxins andgrowth-inhibiting substances in the potatotuber and their significance with regard toits rest period. Aeta . Eort. Berg . 14:133*(7) , 1949. Significance of growth-inhibit-ing substances and auxins for the rest-periodof the potato tuber. Physiol . Plantarum 2:24-36.(8) , 1949. Grov/th-inhibiting substances interminal buds of Fraxinus . Phys iol. Plan-tarum 2:37-44.(9) Hendershott, C. H. and L. F. Bailey, 1955.Growth inhibiting substances in extracts ofdormant flower buds of peach. Proc. Amer.Soc. Hort. Sci. 65: 85-92.25Journal of the Arkansas Academy of Science, Vol. 11 [1958], Art. 6Published by Arkansas Academy of Science, 195826ARKANSAS ACADEMY OF SCIENCE(lO) Jones, M. B., J. W.Fleming and L.F. Bailey,1957* Cyanide as a growth-inhibiting sub-stance in extracts of peach leaves, bark,and flower buds, Proc. Amer* Soc. Hort.Sci. 69:152-157.(ll) Leopold, A. C.| 1955* Auxins and Plant Growth.Univ. of Calif, Press, Berkeley.(- — _ _-—* -12) Samish, R. M.,1954. Dormancy inwoody plant sAnn. Rev. Plant Physiol. 5;183-204.(13) Steward, F. C. and S. M. 1952. In-vestigations of growth and metabolism ofLplant cells, III.Evidence for growth in-hibitors incertain mature tissues. AnnalsBot. l6:477-489 026Journal of the Arkansas Academy of Science, Vol. 11 [1958], Art. 6https://scholarworks.uark.edu/jaas/vol11/iss1/6

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Growth Regulating Substances in Extracts of Maple and Pear Flower Buds

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