1965_USA_ABSORPTION AND TRANSLOCATION OF BORON BY SUNFLOWER PLANTS.pdf

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Absorption and Translocation of Boron by Sunflower PlantsAuthor(s): John G. Husa and Wayne J. McIlrathSource: Botanical Gazette, Vol. 126, No. 3 (Sep., 1965), pp. 186-194Published by: The University of Chicago PressStable URL: http://www.jstor.org/stable/2473233

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BOTAN. GAZ. 126(3):186-194. 1965.

ABSORPTION AND TRANSLOCATION OF BORON BY SUNFLOWER PLANTS'JOHN G. HUSA2 AND WAYNE J. MCILRATH2

Department of Botany, University of Chicago, Chicago, Illinois 60637ABSTRACT

The absorption and translocation of boron by sunflower plants (Helianthus annuus L.) grown undercontrolled-environment conditions were investigated. Exudates in a 43-hr period from the cut stem endsof the root systems of detopped plants contained the same amount of boron whether the roots were im-mersed in nutrient solutions containing 500 ,lg or 1,000 l.g of boron per liter. Plants given a total of 60 /g ofboron in the substrate, but in varied amounts and times of application, showed no differences among thetreatments in the accumulation of boron by their mature leaves. Two of the four series with multiple appli-cation, however, showed delays in the onset of severe symptoms of boron deficiency as compared withthe series with an initial single 60-/Agapplication. Significant lateral transport of boron occurred at thestem tip and in leaf blades when boron was supplied to only half of a split root system at 500 or 1,000 lig perroot-half. Slight, though not significant, lateral transport occurred across the lower levels of the stems inthese series. Lateral transport of boron at the lower internodes was demonstrated when boron was suppliedto only one-half of the root system in higher quantities (5,000 or 10,000 lMgper root half). Little or no lateraltransport in leaves or stems was observed with boron application of 50 or 100 yg per root-half. Althoughmature leaves sprayed with 10-ppm boron solution (as boric acid) showed an increase in dialyzable boroncontent, this soluble boron was not translocated from the leaves. No differences in growth were evidentbetween plants receiving dialyzable boron extracted from leaf tissues and those receiving boric acid inthe nutrient solutions. Non-dialyzable boron in the non-particulate fraction of leaf homogenate, however,was not utilized by plants when it was added to the nutrient solution. Reversing the transpiration streamof a leaf by placing it in a dilute minus-boron nutrient solution and withholding a water supply from otherorgans of the plant did not result in a movement of boron from the leaf to the apical meristem. Some of theboron in the leaf, however, was readily leached into the nutrient solution. Covering the top surface of aleaf with aluminum foil to limit photosynthesis did not result in a change in its boron content. A nitrogen-deficient nutrient solution adversely affected the capacity of plants to accumulate boron. From the resultsof the experiments described, it is concluded that one of the prime factors controlling the distribution ofboron in sunflower plants is the extent and location of the metabolic requirement for this element. It isfurther proposed that immobility of boron in the leaves of this species results from some type of translocationblock in the vascular tissue and not from a fixation of boron in a soluble but non-utilizable form.

IntroductionSome workers (KOHL and OERTLI, 1961) have con-cluded that boron, once absorbedby the roots of aplant, moves passively in the transpirationstreamandaccumulates n those organsthat are water sinks.The older mature leaves, as the largest sinks, tendthereforeto accumulatethe most boron. A largepor-tion of the accumulatedboron in the leaves of a num-ber of species has been shown to be in the solubleform (EATON,1944). Many investigators have alsoobserved that plants can accumulate boron to atoxicity level in their leaves and still show symptomsof boron deficiency within a very short time after

boronis withheldfrom the substrate.These observa-tions led to the conclusion that boron is highly im-mobile in plants (GAUCH and DUGGER, 1954).Such mmobilityof boron wasquestioned orturnipas early as 1940 by FERGUSONnd WRIGHT 1940).Since then it has been questionedfor several otherspecies of Brassica (CHANDLER, 1941; BENSON, DEG-MAN,and CHMELIR, 961) as well as for the stonefruits (EATON, 1944), grape (SCOTT and SCHRADER,

' This investigation was supported in part by grants fromthe National Science Foundation and the Dr. Wallace C. andClara A. Abbott Memorial Fund of the University of Chicago.2Present address: Northern Illinois University, DeKalb,Illinois 60115.

1947), and cotton (MCILRATH,1965). Boron doesappear, nevertheless, to be relatively immobile incertain species, including sunflower which was usedin the present investigation.This study examines in more detail the distribu-tion and accumulation of boron in the sunflowerplant as well as its apparent immobility once de-posited in the leaf tissues.

Material and methodsSunflower plants (Helianthus annuus L., var.Mammoth Russian) wereused in this study. Acheneswere sown in quartz sand in flats supplied with dis-

tilled water. After 5-10 days uniform seedlings,withroots washed free of sand, were transferred o nutri-ent solutions in 1-liter glazed crocks. The seedlingswere anchored to one-hole Masonite crock coverswith pliable mastic (Kalk-Kord, Sears, Roebuck &Co.). The nutrient solution contained the followingconcentrations of macronutrient salts: 4.5 mMCa(NO3)22.5 mM KH2PO4,and 1.5 mM MgSO4.Iron, supplied as Sequestrene NaFe, was added tothe solution at 5 ppm, and the micronutrients wereadded in the following concentrations: 0.5 ppm B(H3BO3), 0.5 ppm Mn (MnCl2), 0.05 ppm Zn(ZnSO4),0.02 ppm Cu (CuSO4),and 0.01 ppm Mo(H2MoO4). For minus-boron nutrient solutions the186


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HUSA & McILRATH-BORON TRANSLOCATION 187boric acid was omitted. Unless stated otherwise,boric acid was used to supply the indicated levels ofboron.Plants were grown in a controlled-environmentroom on a 20-hr photoperiod.They receivedapproxi-mately 2,000 ft-c of light at the level of the upperleaves from General Electric Power Groove fluores-cent tubes supplemented with 60-w incandescentlamps (approximately 12% of the total wattage).The temperaturewas maintainedat 700 + 20 F andthe relative humidity at approximately50%.Tissues harvestedfor analysis of total boron weredried in a forced-draft oven at 700 C for 48 hr andthen were groundwith mortar andpestle. The tissuepowderswere driedovernight at 900 C and cooled ina desiccator, and weighed samples were ashed in amufflefurnace at 5500 C.Freshlyharvestedmaterialwas used fordetermina-tion of boron in the non-particulatecomponents ofthe tissues. Tissue was homogenized with twice itsweight of distilled water for 5 min in a water-cooledWaring blender. The homogenate that passedthrougha double ayerof cheeseclothwas centrifugedfor 30 min at 34,800 X g at 30 C in a Servall RC-2centrifuge to remove the particulate matter (McIL-RATH, 965).Aliquotsof the centrifugated upernatantwere dried at 900C, ashed at 5500 C, and ana-lyzed forboron;other aliquots weredialyzed in cellu-lose tubing (Visking Co.) against 103volumes of dis-tilled water at 30 C for24 hr to remove the dialyzableboron beforedrying.Dialyzableboron was calculatedas the differencebetween total and non-dialyzableboron.Boron analyses followed the curcuminprocedureof DIBLE, TRUOG,and BERGER (1954). Statisticalanalyses were performed according to SNEDECOR(1946).When plants were transplanted to minus-boronnutrient solution, the roots were thoroughlyrinsedindistilled water. The leaves were always numberedfrom the lowest node upward,the cotyledonarynodebeing designatednode 1.

Incipientborondeficiencywas recordedwhen thevery young apical leaves became silvery gray andexhibited epinasty. A plant was classifiedas havingseveresymptomsof borondeficiencywhen the apicalregionof the stem became necrotic.Experimentationand results

EXPERIMENT1. BORON CONTENT OF EXUDATEFROMCUT STEMS.-The cut stem ends of the root sys-temsof 4-week-oldplants, detoppedbetween the firstand secondnodes,wereattached with rubbertubingto 15-cm engthsof 6-mm diametersoft-glass tubing.The roots were then placed in 1-liter glazed crockscontainingminus-boronnutrient solution. To the cul-ture medium of each of 10 crocks were added 500 Ag

of boron and to a like number, 1,000 ig of boron.Exudate was collected over a period of 43 hr. Ali-quots were evaporated to dryness, ashed, and ana-lyzed for boron.

Although root systems administered 500 ,ug ofboron appeared to exude a slightly greater volume ofsolution containing a lower concentration of boronthan those given 1,000 ,ug, the differences were notstatistically significant (table 1). Similarly, the totalquantities of boron in the exudates from the twoseries were not significantly different. Thus, eventhough the boron level in one nutrient medium wastwice that of the other, over a 2-day period this dif-ference was not reflected in a difference in the ratesof exudation of boron. Possibly in a limited-termexperiment 500 ,ug (or less) of boron in the nutrientmedium were sufficient to saturate the boron absorp-tion sites on the roots. If so, a higher concentrationin the medium would not have resulted in more exu-dation of boron.

TABLE 1BORON CONTENT AND VOLUME OF EXUDATE IN 43 HR

FROM DETOPPED PLANTS WHOSE ROOTS WERE IM-MERSED IN NUTRIENT SOLUTION CONTAINING 500 OR1,000 /AG OF BORON

BORON CONTENT OF EXUDATEAPPLICATION EXUDATE/PLANT

APPLICATION (ML) sg/5 ml Ag/plant500. 6.7+0.7 3.4?0.6 4.9?0.81,000. 6.2?0.4 4.1?0.5 5.5?0.9

It is uncertain whether data obtained in this typeof experiment can be applied directly to interpreta-tion of responses of intact plants, for it is well estab-lished that the concentration of boron in the rootenvironment ultimately determines the total amountof boron absorbed by the plant (EATON, 1944). It isquite apparent from this experiment, however, thatboron is absorbed by the roots and translocated intoand through the vascular tissue to the cut end of adetopped plant. Although the concentration of boronin the exudate in this experiment is comparable tothat found in the vascular elements of potato stems(HAMM, HARVEY, and MILLER, 1941), it is uncertainwhether a greater rate of absorption would occur inintact sunflower plants. Determination of the knownboron requirements of intact sunflower plants of theage used indicate that the rate of boron absorptionhere observed, as calculated from the amount exud-ed, approximates the demands of the plant for nor-mal growth.

EXPERIMENT 2. SINGLE VERSUS MULTIPLE APPLI-CATIONS OF BORON.-Twenty-five 1-week-old plantswere transferred to 1-liter crocks containing a minus-


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188 BOTANICAL GAZETTE [SEPTEMBERboron nutrient solution. The total amount of boronadded to each crock during the experiment was 60Aug,ut the amount added at any given time variedamong five established series:Series 1, 60 Agon thefirst day; Series 2, 30 ,ug on the first and seventhdays; Series 3, 20 4g on the first, fifth, and ninthdays; Series 4, 10 ,ug every other day; and Series 5,5 ig every day. Distilled water and minus-boronnutrient solution were alternately added to thecrocks to bring the solution level to a predeterminedlevel each day.The leaves from the fourth and fifth nodes wereharvested when the plants showed severe symptomsof deficiency. The two leaf blades were pooled forboron analysis.The quantity of boron n the leaf blades was great-est in Series 5 (5 ,tg/day) and the lowest in Series 1(60 mg/day). The differences among the variousseries, however, were not statistically significant(table 2).

TABLE 2EFFECT OF ADMINISTERING 60 yG OF BORON IN VARYING

INCREMENTS UPON INCEPTION OF SEVERE DEFICIENCYSYMPTOMS AND BORON CONTENT OF MATURE LEAVES

Boron Number Days to severe Boron/2 leafSeries applica- of appli- boron-deficiency bladestion (4g) nations symptoms (bg)

1 . 60 1 24.6?0.9 18.3?2.42 . 30 2 25.6?1.1 20.4?4.03 . 20 3 27.8?1.0* 19.9+?1.54 . 10 6 24.8?1.0 19.1+0.85 . 5 12 28.0?1.6** 22.4+3.6

* Significantly different from Series 1 at 5% level.** Significantly different from Series 1 at 1% level.

Plants receiving60 Agof boronas a single applica-tionshowedseveresymptomsofdeficiencyafterabout25 days (table 2). These results appear to conformrather closely with those of previousworkers(SKOK,1957;SKOKnd MCILRATH, 958)whoobservedthat,under somewhat similar experimental conditions,plants given a single application of 50 .tgof boronexhibitedsevere symptoms of deficiencyafter 18-22days. What appears o beof significancen thepresentexperiment s that the onset of severe deficiencywasdelayedwhenthe 60 ,ugof boronwere administered nfractional amountsover a periodof 12days. A statis-tically significant greater number of days to severedeficiency was found for the 20-,ugand S-g treat-ments. It is not clearwhy the 10-yg applicationdidnot also result in a similardelay.One might assume that the earlierexpressionofsevere deficiencyin the 60-Agtreatment was relatedto a rapid and early absorptionof boronin excess ofmetabolicrequirementsand its immobilization n theolder leaves. It has been reportedand there is a gen-

eral belief that boron administered at high levelstends to accumulate in the older leaves (EATON,1944). In this experiment, however, such accumula-tion was not obvious, since the boron contents of theleaves at the fourth and fifth nodes of plants in thevarious series were not significantly different (table2). With total applications of 60 ,ug of boron, littledifference n boron accumulation in the older leaveswas found whether the amount was administered asa single dose or in multiple applications. KOHLandOERTLI (1961) indicated that boron accumulationinleaves is a passive process and is determinedprimari-ly by the transpiration tream. They cautioned, how-ever, that this situation probably obtains only whenplants receive adequate or excessive amounts ofboron. Their caution is probably justified, for itwould appear from the data of experiment 2 that,when boron is in limited supply, its distribution istempered by the boron demands of the metabolicsinks as well as by the transpirationstream.

EXPERIMENT 3. SPLIT ROOT SYSTEM.-The rootsystems of 7-day-old seedlings were removed, and avertical slit was made in the stem base between thetwo cotyledons.The shoots were maintainedin one-fifth completenutrientsolution until vigorousadven-titious roots developed.A double-compartmentvessel was prepared bysuspendingtwo polyethylene bags in a 1-liter crockfroma wiresupport. Equal quantities of minus-boronnutrient solution were added to each bag, and halfthe root system of a plant was immersed in eachsolution. Oneday later six serieswere establishedasfollows: Series 1, 1,000 jig of boron added to thesolution in one bag and none to that in the other;Series2, 500 jig of boron and none; Series3, 100 jigof boronand none;Series4, 50 jig of boronand none;Series5, no boron n eithersolution;and Series6, 500jig of boronaddedto each solution. The level of solu-tion was maintained n eachbag by daily applicationsof distilled water or minus-boron nutrient solution.After 14 days one mature leaf directly above eachroot half (usually at nodes 6 and 7) was harvested,dried, and analyzed for boron. The apical 1.5 cm ofthe shoot was removed and then halved with a razorbladein the planeof the originalcut in the stem, andthe two portionswere dried and analyzedfor boron.Plants in Series 5, with both root portions inminus-boron solution, exhibited symptoms of in-cipient deficiency n 3 days (table 3). Plants of seriesin which boron was supplied to half of the root sys-tem showeda delay in the appearanceof such symp-toms; the largerthe quantity of boronsupplied, thegreater was the delay. In Series 3 and 4 the minus-boron side of the plant exhibited deficiency 2 daysbefore the plus-boronside. Plants given 1,000 jig or500 ,jg of boron (Series1 and 2) to oneportionof theroot system did not exhibit deficiency symptoms in


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1965] HUSA & McILRATH-BORON TRANSLOCATION 189either half during the 14-day period of the experi-ment.There was no significant differencein the size ofthe leaves produced on the plus- and minus-boronsides of the plants in Series 1 and 2. In Series 3 and4, however,very obvious differences n leaf expansionwere noted between the two sides. Figure 1 illus-trates a plant from Series4 in which this differencein size can be noted between leaves at node 6 (plus-boron side) and node 7 (minus-boronside). A verystriking responsewas observedfor leaves so insertedthat they were supplied by vascular elements fromboth the plus- and minus-boronsides. Leaves 8 and9 represent such leaves in which there was a largedifference in the degree of expansion of the twohalves. The half of the plus-boron side appeared toenlargenormally, whereas expansion of the half onthe minus-boronside was greatly inhibited, particu-larly of the portion proximal to the stem. This re-sponse is similar to that observed by SCHOLZ1960)for rooted tobacco leaves. He demonstrated that,whenboronwas suppliedto only half the root systemof suchleaves, growthof the basalpartof the leaf wassuppressed n the half not suppliedwith boron by theroots. It is quite apparentfrom observationssuch asthese that, if boronis in limited supply, little lateral

TABLE 3BORON CONTENT AND DURATION OF FREEDOM FROM

BORON-DEFICIENCY SYMPTOMS OF LEAVES ALIGNEDWITH A ROOT-HALF IMMERSED IN EITHER PLUS- ORMINUS-BORON MEDIUM

BORON DAYS TO IN- BORON CONTENT (MG/KG)CIPIENT BORON-APPLICATION DEFICIENCY

SYMIPTOMS' Mature leaf Young leafSeries 1:1,000 .................. 296.7?29.0** 86.3?8.00 ......... 123.4?20.7 76.7?4.2Series 2:500 ......... 241.5?16.8** 84.3?5.00 ........ 86.8?18.9 74.9?4.4Series 3:100 11 133.0? 8.5** 25.7?1.70 9..... 76.0? 6.4 22.2?3.1Series 4:50 7 . .......5.7? 4.4 17.0?0.8**0 5 . .......0.2- 4.9 12.2?0.7Series 5:0oo J 3 81.5?17.3 9.4?1.0Series 6:500 l 187.4?24.0 72.8?1.8500. r........

** Significantlydifferent rom 0-boronside at 1% level.Where no results are given, deficiency symptoms did not developduring 14-dayperiodof experiment.

s..:..~~~~~~~~~~~~~~.: o.....

_

FIG. 1.-Sunflower plant, 14 days after one half of its splitroot system was placed in a plus-boron (50 Mug)nd the other half in a minus-boron nutrient solution. Numbers indicatenode number of leaf origin.


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190 BOTANICAL GAZETTE [SEPTEMBERtransport of this element occurs even within an in-dividual leaf. On the otherhand, when large quanti-ties of boron are given to one portion of the rootsystem, sufficient lateral movement of boron occursto meet adequately the requirementsfor growth ofall portions of the shoot.The root portions on minus-boronnutrient of allseries exhibited typical symptoms of borondeficien-cy. These symptoms, however, were less severe inSeries 1 than in Series4; perhaps there was a slighttranslocationof boron into the minus-boronroots inthe formerseries. This observationagrees with thatof other investigators (SCHOLZ, 1960; ALBERT andWILSON, 1961).Althoughit appeared hat the boronconcentrationin the matureleaves of the minus-boron ide of plantsincreasedwith an increase n boronon the plus-boronside, no statistical differenceswere found in boroncontent between the leaves of Series5 and the leavesfrom the minus-boronside of any other series. Theboronin young leaves on the minus-boronside of allplants administeredboron on the other side, how-ever, was significantlyhigher than that found in theyoung leaves of plants given no boron (Series5). Theboron content of mature leaves on the plus-boronside in Series1, 2, and 3 was significantlyhigher thanthat in leaves on the minus-boronside; this was nottrue for Series4 (table 3). The only significantdiffer-ence between the boron concentrations of youngleaves on the two sides of a given group was inSeries4; here the leaves on the plus-boronside con-tained significantlymore boron.It can be concludedthat there is a slight, thoughnot significant, lateral transport across the lowerlevels of the stem as evidencedby the boroncontentsof the older leaves. The lack of significant lateraltransport of boron at the lower stem level was notmanifestedin visual symptoms of deficiencybecausethese older leaves contained sufficient boron at theinitiation of the experiment to meet their require-ments adequately. Near the stem tip there appearedto be an appreciablemovement of boron across thestem, particularly when a high level of boron wasadministeredto one side of the root system. Whenboronwas given to one portion of the roots at a lowlevel (for example, 50 or 100 ,ug),however, reducedlateral transport at the stem tip was manifested inabnormal eaf expansionand, in the case of the 50-,ugtreatment, in a lower boron content of the youngleaves on the minus-boronside.

EXPERIMENT 4. SPLIT ROOT-TWO-BRANCHEDSYS-TEM.-Plants with a split root system wereproducedby the technique described in experiment 3. Aftervigorous roots had developed, the plants were sup-plied with completenutrientsolutionuntil the stemswere about 1.5 cm in diameter.At this time the shootwas cut off above node 3, and the lateral buds at

nodes 2 and 3 were permitted to develop. When thetwo opposing side branches were several cm long,each half of the root system of a plant was placed ina polyethylene bag, which was filledwith the minus-boronsolution as describedin experiment3. To thenutrientsolutionin oneof the bags in each crockwereadded 1,000, 5,000, or 10,000 jig of boron.The origi-nal level of the nutrient medium was restoreddailyby the addition of distilled water or minus-boronnutrient solution.Six days after these series were established theplants in the 1,000-,qgeriesexhibitedincipientsymp-toms of boron deficiency on the branch above theroot-half in the minus-boronsolution. Plants in theother series showed no deficiency symptoms duringthe 17 days of the experiment. These observationsappear to substantiate the indication of the data inexperiment 3-that when high levels of boron areadministered, ateral transportof the element in thelower portions of the stem occurs.EXPERIMENT 5. BORIC ACID ABSORPTION BYLEAVES.-Ten-day-old eedlings were transferredto1-liter crocks containing complete nutrient solutionand maintained for a period of 11 days. The plantswere then divided into four series as follows: Series1, plus-boron (500 jig B/crock) nutrient solution;Series 2, minus-boron nutrient solution; Series 3,minus-boronnutrient solution, but the young leavesand apical bud were sprayed once daily with a 10-ppm boronsolution (aqueoussolution of H3BO3 plus

Tween 20 as a spreader);and Series4, minus-boronnutrient solution, but two mature leaves weresprayed once daily with a 10-ppm solution (samesolution as in Series 3).Total boron and non-dialyzable boron in thesupernatant fraction from leaf homogenates weredetermined. The tissues sampled consisted of theapicalbudplusyoungleavesand the bladesof the twomature leaves that had received spray treatment orwere taken from the same nodes as those that had.All sprayed tissue was thoroughlywashed with dis-tilled water beforehand to remove non-absorbedboron. Plants were harvested on the tenth day fol-lowing transfer to the various treatments, at whichtime all plants in minus-boronnutrientsolutionwereshowingsevere deficiencysymptoms.Plants that receivedno boronin the nutrientsolu-tion or as a spray (Series2) were the first to showin-cipient deficiency-the fourth day followingtransfer.Two days later some plants in the two-spray treat-ments were beginning to show deficiency. By theninth day followingtransferall plants of the minus-boron (Series2) and of the mature-leaf-spray(Series4) treatments were showing severe deficiency. Suchsymptoms did not occur on Series 3 plants (youngleaves sprayed) until 1 day later. The appearanceofdeficiency symptoms in the plants of Series 3 is at


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1965] HUSA & McILRATH-BORON TRANSLOCATION 191variance with the results of previous experiments(MCILRATH,unpublished)in which sunflowerplantsgrown in a similar environment on a minus-boronnutrient solution with boron-spray applications tothe young leaves matured and flowered withoutshowing evidence of boron deficiency.Analyses of the supernatant fractions from leafhomogenates suggested that boron-spray treatmentresultedin an increasedconcentrationof this elementwithin the organ sprayed (table 4). It also resultedinan increase n the percentageof boronpresentin thedialyzable form. These increases in concentrationwere statistically significant, however, only in thesupernatantsfrom the matureleaves of Series4. Thelimited quantities of boron absorbed by the youngleaves and terminalbud (Series3) were obviously not

TABLE 4EFFECTS OF BORON SPRAY ON DIALYZABLE AND NON-DIALYZABLE BORON CONTENTS OF YOUNG AND MATURE LEAVES

SUPERNATANT BORON (MG/10 ML)

TREATMENT TOTAL NON-DIALYZABLE DIALYZABLE DIALYZABLE BORON (%)

Young Old Young Old Young Old Young Oldleaves leaves leaves leaves leaves leaves leaves leaves

Series 1:Plus-boron ......... 23.9?3.9 62.3?6.3 2.7?0.1 3.8?1.1 21.2?3.9 58.5?5.7 88.7 93.9Series 2:Minus-boron ....... 1.9?1.6 4.2?1.5 1.7?0.1 1.7?0.0 0.2?0.2 2.5?1.5 10.5 59.5Series 3:Minus-boron, youngleaves sprayed. 3.2 ?1.0 3.6?0.6 1.8?0.2 1.8 ?0.1 1.4 ?0. 7 1.8?0. 7 43.8 50.0Series 4:Minus-boron, matureleaves sprayed .. . 1.9 ?0.2 13.4?0. 7** 1.6?0.0 2.2 ?0. 1* 0.3 ?0.2 11.2 ?0.6** 15.8 83.6* Significantly differentfrom minus-boronseries at 5% level.** Significantly different from minus-boron series at 1% level.

sufficientto meet the metabolic requirement or nor-mal growth. Althoughsignificant quantities of boronwere absorbedby older leaves, little or none of thisappearedto be translocatedto the younger leaves.These results substantiate the observation (McIL-RATH,1965)that dialyzable boronin the olderleavesof sunflower is relatively immobile.

EXPERIMENT 6. UTILIZATION FOR PLANT GROWTHOF BORONIN LEAF HOMOGENATES.-Supernatantfrom the leaves of 6-week-old sunflower plants wasextractedin the mannerpreviouslydescribed,and itstotal and dialyzableboroncontents were determined.Eleven-day-old sunflower seedlings, which had beensupplied complete nutrition solution, were trans-ferred to minus-boronnutrient solution. Two dayslater five series were established as follows: Series 1,10 ,ug boron (as H3BO3) was added to the nutrientsolution; Series 2, 8.7 ,ugboron (as H3BO3) was addedto the nutrient solution; Series 3, supernatant con-

training 0 ,ggtotal boron(8.7 jugof which were in thedialyzable form) was added to the nutrient solution;Series 4, supernatant containing 10 jug of non-dialyzable boron was added to the nutrient solution;and Series5, no boron wasadded to the nutrientsolu-tion. Distilled water or minus-boron solution wasadded daily to bring the solution to its original vol-ume. The experiment was terminated 1 month fromthe time the achenes were sown.This experimentdemonstratedthat the dialyzableboronof the supernatant ractionof leaf homogenateswas equivalent to boric acid as a boron source forplant growth (table 5). No significant differenceswere noted between the plants of Series 1 and 3 inthe number of days to the appearance of deficiencysymptoms, in height, and in the number of leaves

produced. The non-dialyzable component of thesupernatant, however, could not be absorbed andutilized by the plant as evidenced by the similarityof plants given non-dialyzable supernatant boron tominus-boron plants (table 5). It is quite obvious,therefore,that the dialyzable boron in homogenatesof matureleaves can be utilized in plant growth whenabsorbed by the roots of seedling plants. Since sun-flowerplants may show symptoms of boron deficien-cy at the shoot apex while they still have appreciablequantitiesof dialyzable boronin their mature leaves,these leaves are obviously either unable to translo-cate this element or are unable to translocate itrapidly enoughto the terminal meristems to meet themetabolic requirement.

EXPERIMENT 7. LEACHING OF BORON FROM LEAVESBY DILUTE NUTRIENT SOLUTION.Three-week-oldplants, grown on complete nutrient solution, weretransferredto distilled water. Three days later the


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192 BOTANICAL GAZETTE [SEPTEMBERroots and all but the mature leaves at nodes 4 and 5and the young leaves near the apical region were re-moved. In one series of plants the basal cut surfacesof the stems were seared on a hotplate, and the stemswere anchored with mastic to Masonite crock covers.The cut stems were not, however, placed in contactwith nutrient solution. The two mature leaves wereplaced in one 8-oz soft-glass bottle containing a half-strength minus-boron solution. In the second seriesof plants the basal end of the cut stem was immersedin a half-strength minus-boron solution. After 43 hrthe leaves at the fourth and fifth nodes and the tipof the shoot with its young leaves from each plantwere harvested for boron analysis.

TABLE 5RESPONSE OF PLANTS TO THREE BORON SOURCES:BORIC ACID, SUPERNATANT

DIALYZABLE BORON, AND SUPERNATANT NON-DIALYZABLE BORON-

Quantity Days to appearance Plant height Number ofSeries Boron source boron of incipient boron- (cm)b leaves(Mg) deficiency symptoms1. . H3BO.3 10.0 14 8. 7?0.8 21.4?1.32...... .3B3 8.7 14 8.7?0.5 17.2?1.23. Supernatant dialyzable 10.Oc 14 7.5 ?0.4 19.2 ?1.3plus non-dialyzableboron4....... Supernatant non-dia- 10.0 3 3.5 ?0.0 Stem tip deadlyzable boron5....... None 0.0 3 3.2 ?1.2 Stem tip dead

a Plants were harvested 1 month from sowing date.b Measured from cotyledonary node.c 8.7 Mg dialyzable boron plus 1.3 Mgnon-dialyzable boron.

TABLE 6EFFECT OF SUBMERGING MATURE LEAVES IN MINUS-BORON

NUTRIENT SOLUTION ON REDISTRIBUTION OF BORON

BORON CONTENT (AG)SERIES TREATMENT

Young leaves Two matureand apical bud leaves

1...... Mature leaves sub- 5.2 ?0.4 23.9 ?4.7merged in nutri-ent solution2...... Mature leaves in 5.1?0.3 40.4 ?4.4*air

* Significantly different from Series 1 at 5% level.

The young leaves of the plants in Series1 exhibit-ed no wilting, and it is assumed that sufficientwaterwas absorbed via the submerged mature leaves tomeet adequatelythe waterrequirementsof the youngleaves. Boron analysis of the older leaves of the twoseries revealed that the leaves that were submergedcontained significantly less boron than did thoseleaves maintained in air (table 6). Since this reduc-tionin boronin the older leaves of Series1 plants was

not accompaniedby an increaseof this element in theyounger leaves, it is assumed that the boron wasleachedinto the nutrient solution.Althoughthe exact quantity of water that passedthrough the matureleaves in Series 1 was not deter-mined, the visiblereduction n volume of the nutrientsolution was evidence of absorption.Thus, with thenormal orientation of the transpirationstream re-versed,it is of interest that none of the soluble boronin the older leaves was translocated to the youngerleaves. Why this soluble boron apparently could beleached into the nutrient solution but could not betranslocated to otherparts of the plant in the trans-piration stream is not understood.

EXPERIMENT8. INFLUENCE OF CARBOHYDRATESTARVATIONN BORONACCUMULATION.-The ppersurface of leaves at the fourth nodes of 3-week-oldplants was covered with aluminum foil, which wasfolded under only along the margin so that most ofthe lower epidermis was exposed to the atmosphere.The leaves at the fifth nodes wereleft exposedto fulllight intensity. The internodebetween the leaves atnodes 4 and 5 was so short that the leaves were es-sentially located at the same position on the stem.From the seedling stage to the time of harvesting,the plants were maintained on complete nutrientsolution. The leaves wereharvested for boron analy-sis after 6 days of cover by foil.Even thoughthe covered eaves had a significantlylower dry weight at the end of the treatment periodthan did those exposed to light, there was no signifi-cant difference between the two groups in boron con-tent (table 7). This would presumably suggest thatan interruption of the processes of photosynthesis,carbohydrate elaboration, and carbohydrate trans-location in and from the leaf has no direct control-ling influence on the accumulation or loss of boronin the leaf. When boron had earlierbeen supplied tothe plant in adequateamounts,the mobility of boron


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1965] HUSA & McILRATH-BORON TRANSLOCATION 193already present within the leaf obviously was notincreased by interruption of these processes.

EXPERIMENT 9. INFLUENCE OF NITROGEN DEFI-CIENCY ON BORON ACCUMULATION.-Three-week-oldplants, which had been grown on complete nutrientsolution, were divided among four treatments as fol-lows: Series 1, complete nutrient solution (+ N +B); Series 2, plus-nitrogen, minus-boron nutrientsolution (+ N - B); Series 3, minus-nitrogen,plus-boronnutrientsolution (- N + B); Series 4, minus-nitrogen, minus-boronnutrient solution (- N - B).The minus-nitrogensolution was prepared by sub-stituting an appropriateamount of CaCl2 for theCa(NO3)2 n the completenutrient solution.Half of the leaf blade, exclusive of the midrib, atthe fourth node was harvestedfor boron analysis atthe time the plants were transferred to the seriesindicated above; the remaininghalf was harvested 8days later.At the time of the second harvest, the leaves atthe fourth node of plants on minus-nitrogentreat-ments were beginningto show the typical chlorosissymptoms of nitrogen deficiency, and the youngleaves of plants on minus-boronwere showing typicalboron-deficiency ymptoms. Although no differencecould be discerned between the two nitrogen-defi-cient series (Series3 and 4) in the degreeof deficien-cy, the boron-deficiency ymptoms in Series 2 were

TABLE 7EFFECTS OF INHIBITING PHOTOSYNTHESIS ON DRYWEIGHT AND BORON ACCUMULATION

IN MATURE LEAVES

PER LEAFTREATMENTDry weight (mg) Boron (pug)

Covered leaf .......... 235.7 ?15.3 31.2+3.5Exposed leaf.......... 354.3 ?24.7** 36.5?2.9** Significantly different from covered-leaf treatment at 1% level.

slightly more severe than those in Series 4. A com-parison in all series of the dry weights of the twohalves of a given leaf at the two harvests showednosignificantdifferences.The fourth leaf of the plants of all series showedsome increase n boroncontent between the first andsecond harvests (table 8). Only in Series 1 (+ N +B), however,was there a significant increase n boroncontent; this increasewas also significantly greaterthan that of any of the otherseries.What appearstobe of particular ignificance s that nitrogen-deprivedplants supplied with adequate boron (- N + B)failed to accumulateboron n theirfourth leaf. Thus,although nitrogen deficiency did not appear to in-

crease the mobility of boron already present withinthe plant, it did significantly reduce the capacity ofthese plants to accumulate additional quantities ofthis element in the older leaves.Conclusions

KOHL and OERTLI (1961) concluded that whenboron is supplied to roots in adequate or excessamounts, the flow of this element into the leaves iscontrolled by the transpiration stream. The datahere presented do not necessarily contradictthis con-clusion, but they do indicate that the transpirationstream alone is not the determining factor in boronTABLE 8

INFLUENCE OF NITROGEN LEVEL INNUTRIENT SOLUTION ON BORON AC-

CUMULATION OF MATURE LEAVESIncrease in boron contentTreatment per half leaf (jg)Series 1:+N+B. ..... W. 59+1.81Series 2:+N-B ...... 2.37+0.88**Series 3:-N+B ...... 3.09+1.01**Series 4:-N-B ...... 2.04+0.54**

** Significantly differentfrom Series 1 at 1%level.deposition in leaves of the sunflower plant. If thetranspirationstream were the only controllingfactorin boron distribution, it would be difficultto explainthe observed ateral movement of boronin situationswhere it appears highly improbablethat a similarlateral transport of water occurred (experiments 3and 4). Since boron is relatively immobile in theolderleaves of sunflower McILRATH,1965), it wouldalso be difficultto explain the lack of any differencein accumulation of boron in the leaves of plantsamong the various series in experiment2.Similarly, a lack of boron accumulation-n nitro-gen-deficient eaves (experiment9) would be difficultto explain. Although one might argue that nitrogendeficiencyresultedin a reducedabsorptionof boronby the rootsrather than a reducedcapacity of leavesto accumulate the element, it is still apparent,fromthe lack of boron-deficiency ymptomsin the - N +B plants, that adequateboronwas absorbedto meettheir metabolic needs. Water unquestionably wasstill being transported into the nitrogen-deficientleaves, even thoughthis water was essentiallydevoidof boron.It would appear, therefore,that one of the primefactors controlling the distributionof boron in sun-flowerplants is the extent and location of the meta-bolic requirement or the element. Only when boronis suppliedto plants in excess of the requirementsof


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194 BOTANICAL GAZETTEthe boron metabolic sinks does it accumulatein theolder leaves. When the supply is limited, boron doesnot tend to concentrate in the older leaves, eventhough these organs may still be prime water sinksof the plant.When boron is administered n a limited amountto only a portion of the root system, it is utilized inthe metabolic sinks normally supplied via vasculartissue from these roots and little lateral transportoccurs to sinks suppliedby vascular elements fromother roots. If, on the other hand, a portion of theroot system absorbs boronin an excess of the meta-bolic requirementsof tissues normally supplied bysuch roots, lateralmovement of boron will take placeto fill metabolicrequirements n other portions of theplant. Although the occurrenceof such lateral move-ment was demonstrated n this study, nothing wasrevealed as to its mechanism or pathway of trans-port.The immobility of soluble boron within the oldermature leaves of sunflowerplants (as reported byMcILRATH, 1965) was again demonstrated in thisstudy. That this immobility is not primarily the re-sult of a fixation of boronin a solublebut non-utiliz-able form was revealed by experiment6 in whichdialyzable boron n leaf homogenatewas shown to bereadily absorbed by roots and utilized as efficientlyas boric acid in plant growth. The observation thatthis soluble boron could be leached from the leaf(experiment7) by dilute nutrientsolutionbut that itapparentlywas not translocated to otherparts of the

plant by a reversalof the transpirationstream indi-cates that probably characteristics of the vasculartissue prevented boron movement in the latter case.It is well established that drasticanatomical changesmay occur in the vascular tissues of many plantswith the onset of boron deficiency (see PALSER andMcILRATH, 1956), although little is known abouthow such changes influence the physiological func-tioning of such tissues. In view of the above observa-tions it is unlikely that EATON'S (1944) suggestion toaccount for the immobility of soluble boron is valid.He proposedthat the solublemolecule of which boronbecomesa part upon enteringthe leaf mesophyllcan-not diffuse out of such cells.This investigation revealedthat the carbohydrateand nitrogenmetabolismof olderleaves of sunflowerplants probably had little or no influence on themovement of boron from these organs. Similarly,carbohydrate metabolism did not seem to affect ac-cumulation of this element. Nitrogen metabolism,however, appearedto play a definite, but undefined,role in controlling boron accumulation in olderleaves.

AcknowledgmentsThe senior authorexpresseshis appreciation o theDepartment of Botany, University of Chicago, forbeing granted a CharlesL. and FrancesK. Hutchin-son Fellowship and a LaVerne Noyes FoundationScholarship.The authors thank Professor CHARLES

E. OLMSTEDfor his critical reviewof the manuscript.LITERATURE CITED

ALBERT, L. S., and C. M. WILSON. 1961. Effect of boron onelongation of tomato root tips. Plant Physiol. 36:244-251.BENSON, N. R., E. S. DEGMAN, and I. C. CHMELIR. 1961.Translocation and re-use of boron in broccoli. PlantPhysiol. 36:296-301.CHANDLER, F. B. 1941. Mineral nutrition of the genus Brassicawith particular reference to boron. Maine Agr. Exp. Sta.Bull. 404.DIBLE, W. T., E. TRUOG, and K. C. BERGER. 1954. Boron de-termination in soils and plants. Simplified curcumin pro-

cedure. Anal. Chem. 26:418-421.EATON, F. M. 1944. Deficiency, toxicity, and accumulation ofboron in plants. J. Agr. Res. 69:237-277.FERGUSON, W., and L. E. WRIGHT. 1940. Microelement studieswith special reference to the element boron. Sci. Agr. 20:470-487.GAUCH, H. G., and W. M. DUGGER, JR. 1954. The physiologicalaction of boron in higher plants: A review and interpreta-tion. Maryland Agr. Exp. Sta. Tech. Bull. A-80.HAMM, P., R. B. HARVEY, and E. S. MILLER. 1941. Quantita-tive spectroscopic analysis of stem tracheal fluids forinorganic constituents. Soc. Exp. Biol. Med., Proc. 46:347-351.

KOHL, H. C., JR., and J. J. OERTLI.1961. Distribution of boronin leaves. Plant Physiol. 36:420-424.MCILRATH, W. J. 1965. Mobility of boron in several dicoty-

ledonous species. BOTAN. GAZ. 126:27-30.PALSER,BARBARAF., and W. J. MCILRATH.1956. Responses oftomato, turnips, and cotton to variations in boron nutrition.II. Anatomical responses. BOTAN. GAZ. 118:53-71.SCHOLz,G. 1960. tber die Translokation des Bor in Tabak-Blattstecklingen mit geteilten Wurzelsystemen. Flora 148:484-488.SCOTT,L. E., and A. L. SCHRADER.1947. Effect of alternatingconditions of boron nutrition upon growth and boroncontent of grapevines in sand culture. Plant Physiol. 22:526-536.SKOK,J. 1957. Relationship of boron nutrition to radiosensi-

tivity of sunflower plants. Plant Physiol. 32:648-657.SKOK, ., and W. J. MCILRATH. 1958. Distribution of boron incells of dicotyledonous plants in relation to growth. PlantPhysiol. 33:428-431.SNEDECOR,G. W. 1946. Statistical methods. Iowa State Col-lege Press, Ames.

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