Photosynthesis in Aspen Bark Author(s): L. C. Pearson and D. B. Lawrence Reviewed work(s): Source: American Journal of Botany, Vol. 45, No. 5 (May, 1958), pp. 383-387 Published by: Botanical Society of America Stable URL: http://www.jstor.org/stable/2439638 . Accessed: 30/01/2012 13:49

Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at . http://www.jstor.org/page/info/about/policies/terms.jsp

JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range of content in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new forms of scholarship. For more information about JSTOR, please contact [email protected].

Botanical Society of America is collaborating with JSTOR to digitize, preserve and extend access to American Journal of Botany.

http://www.jstor.org PHOTOSYNTHESIS IN ASPEN BARK1

L. C. Pearson and D. B. Lawrence2

QUAKING ASPEN, PopildUs tremuloides Michx., thate production,one which would have meaning is probablythe most widely distributed tree in North even if negativeresults were obtained. America(Harlow and Harrar,1941) and therefore At thebeginning of the observationperiod, April is exposedto a wide varietyof climaticconditions. 1, neitherflowering nor leaf expansionhad begun. Over muchof its range,which is mainlynorth of AboutApril 16, theaspen reached full bloom. Leaf- latitude30 degreesnorth, its forestassociates are ingbegan May 5 on mostof thetrees although a few evergreenscapable of photosynthesisduring the specimensgrowing on higherand sandiersoil were cold monthsof the year, even at temperatures about four days earlierthan the others. By May below the freezingpoint of wateras demonstrated 17 seed dispersal was complete. The leaves had by Parker(1953). Some populationsof aspen have reachedmaximum size by June7 and at this time bark of yellow-greencolor as contrastedwvith the were a brightgreen color. On August3 whenthe morefamiliar powdery-white bark; Cottam(1954) finalobservations were made the leaves had dark- has observedthat the former are foundat thehigh- ened to a deep almost blue-greencolor. At this est elevationsin Utah and neighboringstates and as timethe bark also had changedcolor, being not as suchmust be especiallywell adapted to lifein areas distinctlygreen as beforeon the southside of the of long cold wintersand short summers.Marr stems. (1947) observedin Colorado thatthe aspens with Monthlymaximum and minimumtemperatures greenishbark were much more winterhardy than in the shade at the bark samplingheight above the thenon-green individuals. groundduring the period of observationwere 27? A yellow-greenaspen whichseems quite similar and minus11?C. in April,310 and minus40 in May, to thosedescribed by Marr and by Cottamis com- 28? and 70 in June,and 330 and 12? in July. mon in Minnesota. Sectionsof the bark taken at CHLOROPHYLL DETERMINATIONS.-Materials and breast heightfrom trees a foot in diameterand Methods. Samplesof bark were obtained by stamp- about20 yearsold have invariablyrevealed a bright ing them out with a steel die of 16 mni. diam. greenzone close belowthe surface.The greenpig- Twentydisks, each approximately1 mm. thick, from mentwas foundto be locatedin plastidswhen hand barkof thesouth and of thenorth sides of the stem sectionswere examined microscopically. We are not were collected. The outermostlayer of bark was aware of any publishedstudy of the nature and brownand on May 31 averaged0.5 mm.in thickness functionof thisbark pigment, although the opinion on the northside but only 0.04 mm. on the south had been expressedorally by a pathologistthat it side. Beneath this was a green layer 0.20 mm. was a greenstain producedby a parasiticfungus. thickon the northside and 0.33 mm.thick on the These observationssuggest the possibilitythat south side. Thus all the green pigmentswere in the green pigmentmight be chlorophyllin which the outer 1 mm. of bark fromwhich the samples case it could be capable of carryingon photosyn- were taken. Twentydisks each were also taken thesiseven in maturestems. fromsun-exposed leaves on the south and from shade leaves on the northside of the crown; these To test this hypothesis,three experiments were averaged0.18 mm.and 0.14 mm.thick respectively. conductedduring the springand summerof 1957 All samples of bark and leaf were taken froma Forest at theUniversity of Minnesota'sCedar Creek single pistillatetree which was 19 years old and Natural HistoryArea in northernAnoka County, 16 cm. in diameterat the two-meterlevel above One of Minnesota,30 milesnorth of Minneapolis. groundwhere the bark samples were taken. The these experimentswas designed to ascertainthe bark outside the true cambium averaged 8 mm. to natureof the greenpigments in aspen bark and thickat this level. measure the quantityof these pigmentsper unit area of bark. A second was a simpleand rather To extractchlorophyll, two techniques were used. crudetest to measuredifferences in amountof starch In the experimentof May 31, bark of a knownsur- presentin normallight-exposed and in artificially face area was placed in a Kenmoreblender with a darkenedareas of bark. The third test was de- knownquantity of 95 per cent ethanoland run at signedto give a morerefined measure of photosyn- high speed for one minute.This mixturewas then boiledfor three minutes and storedin flaskscovered 1 Receivedfor publication November 6, 1957. withaluminum foil at I?C. for threedays untilit 2 The authorsare gratefulto ProfessorAlbert W. Frenkel was possibleto examinewith a spectrophotometer. foraid in developingthe chlorophyllextraction techniques At thattime it was filteredand the volumeof the used on August3, and forcriticizing the manuscript, to the greeirs-olutionascertained. Wave lengthsof visible Louis W. and Maud Hill FamilyFoundation for financial lightat whichmaximum and minimumabsorption support,and to ElizabethG. Lawrencefor reviewingthe manuscript.We are gratefulto Miss WilmaG. Monserud occurredand also the amountof absorptionof light at each of fourdifferent wave lengths were measured 383 384 AMERICAN JOURNAL OF BOTANY [Vol. 45

.SUN LEAVES, SOUTH ...... SHADE LEAVES, NORTH ...... NORTH-FACING BARK ...SOUTH-FACING BARK

SUN LEAVES, SOUTH ......

SHADE LEAVES, NORTH.. . < 1.0 * \-. \ SOUTH-FACING BARK.. NORTH-FACING BARK.

0

-j0.5

3500 4000 4500 5000 5500 6000 6500 7000 WAVE LENGTH OF LIGHT IN ANGSTROMS Fig. 1. Absorptionspectra of pigments in aspen dissolved in ether. Data of August 3. Chlorophylla measured at 6600 A. witha BeckmanSpectrophotometer Model B. The to the resultingsolution to bringthe totalvolume quantitiesof chlorophyllsa and b presentwere esti- to 100 cc. mated by comparisonwith absorptioncoefficients Absorptionwas ascertainedwith the spectro- publishedby Zscheileand Comar (1941). Chloro- photometerat wave lengthsof 4300, 4500, 4700, phyllwas also extractedfrom known areas of leaves 6425, 6500, and 6600 A. The concentrationof of aspen by the same technique. In each case for chlorophylla was ascertainedby measuringat 6600 thisexperiment bark or leaves totalling40.20 cm.2 A inasmuchas the absorptionat this wave length in area weremixed with 95 cc. of alcohol. An addi- due to carotinoidsis negligibleand that due to tional 15 cc. alcohol was used to rinse the con- chlorophyllb less than4 per centof thetotal. The tainers. Samples were broughtto the boil at the concentrationof chlorophyllb was ascertainedat same rate as well as being boiled for the same 6425 A after calculatingthe absorptiondue to lengthof time. chlorophylla at this wave lengthand subtracting In the experimentof August3 designedwith the thatfrom the total optical density. help of Dr. Frenkel,a sample of bark of known From a 30 cc. sampleof theether-chlorophyll so- area- again 40.20 cm.2 was placed in the Ken- lution, chlorophyllsa and b were removed by more blenderwith approximately50 cc. acetone saponifyingwith a solutionof 30 per centKOH in plus about 2 g. powderedCaCO3 and run at low methanoland washing with water. The heavier speedfor 3 minutes.The contentsof the blender were chlorophyllcomplex was drawnoff the bottomof a thenplaced in a centrifuge;the blenderwas rinsed separatoryfunnel; the remainingpigments, a mix- withan additional25 to 30 cc. of acetoneand this ture of carotinoids,were studiedwith the spectro- was added to the mixturein the centrifuge.The photometerat severalwave lengthsincluding 4300, mixturewas centrifugedfor about three minutes 4500, and 4700 A and theoptical densities recorded. untilthe chlorophyllsolution was clear. The solu- The resultingvalues gave a comparisonof the rela- tionwas pouredinto a separatoryfunnel containing tiveconcentration of carotinoidsin each of thefour about 50 cc. of ethylether. Water was added by tissuesstudied. applyingsmall quantitiesalong the side of the Experimentalresults.-The absorption spectra for funneland swirlingcarefully. When separation oc- aspen bark extractand for aspen leaf extractare curred, the heavier acetone and water mixture verysimilar as shownin fig.1, havingmaxima near was drawnoff and discarded.The chlorophyll-ether6610 A and 4300 A and a minimumat about 5400 solutionwas washedwith water several times unitil A. In the originalalcohol and acetone solutions, it appearedto be quite freeof acetone,care being thebark extractshowed an additionalmaximum at takento avoid saponifyingthe chlorophyllby too 3860 A not foundin the leaf extracts.This is not directapplication of the water. Ether was added apparentin theether solution from which the curves May, 1958] PEARSON AND LAWRENCE-PHOTOSYNTHESIS IN ASPEN BARK 385

TABLE 1. Milligrams of chlorophyllsa and b per square sultingblue color was comparedwith that of a meter of aspen tissue. Values of May 31 fromalcohol sampleof wheator potato whichhad been solution, those of August 3 from ether starch, treatedwith the same solution,and scoredas a per Leaves Bark centof thelatter to givea measureof starchpresent. Date Sun Shade Sun Shade Because of the possibilitythat this typeof subjec- tivemeasurement might want in precision,random- Chlorophylla May 31 202 147 258 233 ization and replicationprocedures were followed, Aug. 3 300 250 195 193 each treatmentbeing replicatedfour times. The Chlorophyllb May 31 156 116 241 246 randomizationcompletely removed all humanbias Aug. 3 190 130 115 100 fromthe measurements. On April 12, beforethe leaves appeared,masks in fig. 1 were drawn. A water soluble brown pig- made of fourthicknesses of paperpicnic plates were ment was washed out along with the acetone in pre- attachedsecurely by fourcup-hooks to the bark of paring the ether solutions of the bark extract; this southand northsides of two aspens,one of them pigmentwas probably responsible for the increased the same tree fromwhich the chlorophyllsamples absorption at 3860 A noted in the alcohol extrac- weretaken, the othersmaller. The followingweek tion of May 31. a layer of aluminumfoil was placed under each In table 1 are shown estimates of the quantity of maskto cut offcompletely the passage of light. chlorophyllsa and b for each of the four tissues The maskswere left in place forthree weeks after and at each of two dates, the May 31 data from which at weeklyintervals samples of bark were alcohol extracts,those of August -3 from ether. By takenfrom under them and testedfor starch along calculating the optical density at each of three with samples of light-exposedbark. From each wave lengths for the carotinoid solutions and sub- treatment(combination of exposed vs. darkened tracting these values from the total values at the bark,south vs. north-facingbark, and treesof differ- same wave lengths for the chlorophyll solutions entsizes) foursamples were taken and tested.These (which included carotinoids) additional values sampleswere about one mm. thick; theywere re- were obtained which could be used as checks on the movedwith a sharpknife after which a randomized accuracy of the original calculations. This pro- numberwas placed on the under surfaceof each cedure indicated that the values shown for chloro- samplewith a lead pencil. phyll a are accurate within about 5 mg; the values for chlorophyll b, however, are somewhat less ac- curate. When the carotinoid spectra of the four tissues were compared (not shown graphically in fig. 1) LEAFY CROWN they were seen to be similar with maxima at 4200 A, 4400 A, and 4700 A and minima at 4300 A and

4600 A. Comparison of these spectra with the ab- N0 sorption spectrum for ,8-carotene published by Cooley and Koehn (1950) suggests that /3-carotene is not the most abundant of carotinoids present in these tissues. Judging from the total absorption at 0

4700 A, sun and shade leaves contained approxi- DEAD - -GE 6 YEARS mately equal quantities of carotinoids, south-facing ro ~~~~~~TWIG ~ ~~~~~~~ACONTROL: NOT bark about one and one-halftimes as much carotin- DARKENED oid pigment, and north-facingbark about twice as .0 }/. < B DARKENED much carotinoid as leaves. This relationship held C NOT true at both dates. The proportions were in the / DARKENED same order but of somewhat differentmagnitudes D DARKENED at 4300 A and 4500 A indicating the same carotin- E NOT DARKENED oids were not presentin the same proportionsin all four tissues.The absorption spectrafor a-and / v F CONTROL: NOT DARKENED ,8-caro- AGE 8 YEARS tenes, xanthophyll, and lycopene published by Miller (1934) failed to account for the spectral patternobtained fromthe aspen tissues. FIRST PHOTOSYNTHATE MEASUREMENTS.-Materi- als and methods.-To test for starch, samples of Fig. 2. Sketch of the aspen branch girdled in four places to preventtranslocation of foods to the intergirdle bark were boiled in for regions, alcohol a few minutes, labeled to indicate treatmentsduring firstweek of experi- dipped in hot water to soften,then removed and laid mentation. Regions B and D were covered with aluminum on a table top, and to the inner cut surfaces was foil over black paper to exclude light; C and E were added a solution of I *KI. The intensityof the re- exposed to light. 386 AMERICAN JOURNAL OF BOTANY [Vol. 45

Experimental results.-Starch was present in all TABLE 2. Relative starch content of lighted and darkened of the bark samplesand it was impossibleto detect aspen bark one week aftergirdling. Values are given as the amongthem. 'the experi- color intensityof the samples expressed as a per cent of any consistentdifferences the intensityof a pure starch-iodinecomplex mentindicated either that the starch occurring there was not producedin the bark as a resultof photo- Avg of4 synthesisor else that the starchproduced in the Treatment samples exposedbark was translocatedinto areas of dark- ened bark relativelyrapidly; the weaknessof this 1) Control. Proximal region of branch experimentwas thatthese two possibleeffects could (Region F) -100% be separated.Therefore a morecomplex experi- 2) Control. Distal region (Region A) - 93% not 3) Girdle-isolatedregion completely exposed to light mentinvolving removal of phloemtissue by gird- (Region E) -68% ling to preventtranslocation of foods was devised. 4) Coveredportion (D) of the partlyexposed girdle- SECOND PHOTOSYNTHATE MEASUREMENTS.-Ma- isolated region (C-D) -38% terialsand methods.-On May 31 a branch of an 5) Girdle-isolatedregion completelycovered aspentree was girdledin fourplaces along a leafless (Region B) -5% portionas shownin fig. 2. Assumingthat trans- Least significantdifference, 5% probability-- 28% locationof foodstakes place via thephloem, each of the threeintergirdle regions as well as the leafy LSD (least significantdifference) test. All differ- distalregion was thusdependent on its own photo- ences exceptthose between the two controls(treat- syntheticactivities for its food,at least,afterstored ments1 and 2) exceedthe LSD in value. In other food had been used up in respiration.The xylem words,the chances are less than one in 20 that was not damaged; thusan abundantsupply of wa- even the smallestdifferences shown in table 2 are ter and mineralnutrients was assuredeach region. due to chance variationamong the samplestested While therewere no leaves presenton any of the and we can thereforefeel confidentthat the treat- threeintergirdle regions, the distal regionbore a ment differencesare real differences-thatthe normalsupply of photosyntheticleaves. chlorophyllin aspen bark is photosyntheticallyac- ,Oneintergirdle part of the branch (Region B in tive. fig. 2) was completelycovered with black paper DISCUSSION.-Assumingthat the productionof and this in turnwas coveredwith aluminumfoil chlorophyllin a planttissue is moreor less propor- whichwas held in place with plastic electrician's tional to the intensityof the lightfalling on that tape. Anotherregion (C-D) was partly(D) cov- particulartissue, it would be expectedthat plant eredwith black paper and aluminumfoil and partly organs exposed to the sun would contain more (C) exposed,whereas a third(Region E) was left chlorophyllper unit area than similarorgans kept entirelyexposed to light. Afterone week,samples in the shade. This provedto be the case in aspen, of bark wereremoved from each sectionand tested both for leaves and for bark. Also in supportof forstarch; the black paper and aluminumfoil were thisassumption was the observationthat the chloro- thenreplaced but withRegion E coveredand Re- phyllcontent of theleaves increased over the period gion B exposed. Samples were taken again the May 31 to August3 whereasthe chlorophyll content followingweek, but it was notedthat by thattime of bark decreasedduring the same period. In fact, muchof the tissue was dead and it was thereforefelt the differencein chlorophyllcontent between south thatlittle reliance could be placed on the resultsof and north-facingbark which was noted in May, the second sampling. In this experimentas before whenthe difference in lightintensity was also great, randomizationand replicationprocedures were fol- had apparentlybeen erased by Augustalong wvith lowed. the *differencein light intensity,an expectedre- Experimentalresults.-Differences were very pro- sult. However,bark-even fromthe northside of nouincedamong the samplescollected at the end of the tree-proved to containmore chlorophyllper the firstweek following girdling. Among the group unit area of surface3on May 31 than any of the of samplestaken the secondweek, little starch was leaves. To thewriters, this was a ratherunexpected presentin any of the intergirdleregions and the finding,although it is certainlynot in disagreement variationamong treatments(other than the con- withthe assumptionthat more chlorophyllis pro- trols) could all be attributedto chance variation. duced in tissuesexposed to abundantlight than in The day afterthe sample collectionwas made at darkenedtissues. On May 31 bark could well be the end of the second week, the girdled branch expectedto be rich in chlorophyllproduced prior showedmarked wilting and leaf discolorationindi- to leafing, possibly during the winter months, cative of the extentto which necrosis had pro- whereasthe leaveshad notyet had timeto accumu- gressed. The averagesof the foursamples of each late a large storeof thesepigments. treatmenttaken the first week are shownin table2. The photosynthatesproduced in aspen bark are Because the treatmentswere randomized and apparentlytranslocated quite readily from one replicated,it is possible to test the differences 3 Leaf surface area is the value for one side of the leaf amongtreatment averages in table 2 withFisher's only. May, 1958] PEARSON AND LAWRENCE--PHOTOSYNTHESIS IN ASPEN BARK 387 part of the plant to another through the phloem. thesis that the green pigmentin aspen bark is That translocation of foods in aspen bark occurs chlorophylland thataspen bark of maturestems is was indicated in the earlier test in which there were capable of carryingon photosynthesis,at least no differencesbetween covered and exposed bark underthe conditionsin Minnesotain the springof in the amount of starch present. Removal of the 1957. There is a possibilitythat the resultsre- phloem between the differentregions under observa- portedhere may bear a relationshipto conditions tion in the final test, however, resulted in the in the Rocky Mountains as described by Marr appearance among the samples of differencesin (1947), by Cottam (1954), and othersmentioned starch content. Comparison of treatments4 and by him. However,similar experiments would need 5 in table 2 illustrates this further. These two to be conductedin the Inter-mountainarea to verify treatmentswere identical in every respect except theseassumptions. There is also a possibilitythat that in treatment5 (Region B) there was no phloem these resultsobtained in the late springmay bear contact betweenthe darkened bark and either leaves a relationship:to winterconditions and an experi- or bark that were exposed to light, while in treat- mentto measurephotosynthesis in aspen bark at ment4 the darkened bark (Region D) was in direct temperaturesbelow 0?C. is planned. contact with bark that was exposed to light (Region C). The bark under both treatmentswas connected SUMMARY by undamaged xylem to the rest of the tree. In each By use of the the taken spectrophotometer, greenpig- case, the bark from which the samples were mentsin aspen bark wereshown to be case chlorophyll; was covered to exclude all light; also in each it was also ascertainedthat these pignments are more the sampled region was part of ani intergirdle abundantin bark than in leaves of unit these aspen per section. The differencesbetween treatments, area duringthe early part of the growingseason, to the phloem contact which therefore,must be due but more abundantin leaves later in the summer. D with a light exposed Region (treatment4) had By use of the starch-iodinetest, it was ascertained of Region C. As a result of this con- portion bark, thatthe chlorophyllin aspen bark is photosyntheti- the four samples of bark taken from Region D tact, callyactive. It is suggestedthat this ability of aspen all starch whereas no starch at all could contained to carry on photosynthesisin be observed in three of the four samples from its bark might partlyaccount for the Region B and only a small amount in the fourth. ability of this species to thrivein what is otherwisemainly an evergreen Failure to find differencesamong the samples conifer environment. taken from areas of differenttreatments the day before the girdled branch began to wilt points up DEPARTMENT OF AGRONOMY AND PLANT GENETICS, the necessity in experimentsof this type, in which UNIVERSITY OF MINNESOTA drastic treatmentsare imposed upon plants other- SAINT PAUL 1, MINNESOTA wise in their native habitat and under natural con- AND ditions, to complete such experimentsas rapidly as DEPARTMENT OF BOTANY possible. UNIVERSITY OF MINNESOTA These experiments give support to the hypo- MIINNEAPOLIS 14, MINNESOTA

LITERATURE CITED

COOLEY, M. L., AND R. C. KOEHN. 1950. Chromatographic MILLER, E. S. 1934. Quantitativeabsorption spectra of the estimationof carotene in feeds and feed ingredients. commoncarotinoids. Plant Physiol. 9: 693-694. Analyt. Chem. 22: 322-326. PARKER, J. 1953. Photosynthesisof Picea excelsa in winter. COTTAM, W. P. 1954. Prevernal leafing of aspen in Utah Ecology 34: 605-609. mountains. Jour. Arnold Arboretum35: 239-248. HARLOW, W. M., AND E. S. HARRAR. 1941. Textbook of ZSCHEILE, F. P., AND C. L. COMAR. 1941. Influenceof pre- dendrology. McGraw-Hill Co., New York. parative procedure on the purity of chlorophyllcom- MARR, J. W. 1947. Frost injury to aspen in Colorado. (Ab- ponentsas shown by absorptionspectra. Bot. Gaz. 102: stract) Bul. Ecol. Soc. Amer. 28: 60. 463-481.