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The Effect of Certain Metabolic Inhibitors on Vascular Differentiation in Isolated Pea Author(s): John G. Torrey Source: American Journal of , Vol. 40, No. 7 (Jul., 1953), pp. 525-533 Published by: Botanical Society of America Stable URL: http://www.jstor.org/stable/2438502 . Accessed: 23/08/2011 15:21

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http://www.jstor.org THE EFFECT OF CERTAINMETABOLIC INHIBITORS ON VASCULAR TISSUE DIFFERENTIATION IN ISOLATED PEA ROOTS' JohnG. Torrey

THE SEQUENCE of eventsoccurring during cellu- thatthe mechanical inhibition of rootelongation by lar differentiationmay be describedand, to some solidifiedgypsum involved inhibition of elon- degree,understood in termsof manifeststructural gation,cell division,and cell differentiation.He changeswhich the individualcell undergoesduring demonstratedthat the high osmoticpressures of its ontogeny. Yet littleis knownconcerning the concentratedsucrose solutionscaused essentially biochemicalprocesses in tissues which are similarinhibitions of thesecell processesin these fundamentallyrelated to theseontogenetic changes. roots. Isolated pea tips,cultured in the dark in a Severalworkers have concernedthemselves with sterilesynthetic nutrient medium, grow in a re- the effectsof chemicalinhibitors on cell elongation produciblemanner, showing usual patternsof tis- and differentiation.Cholodny (1931), in his an- sue differentiation.Inhibition of elongationin cul- alysis of the action of auxin on root elongation, turedroots may be producedby a varietyof meta- suggestedthat applied auxin so speedsup thematu- bolic inhibitorswhich act throughtheir effect on ration of cells that the phase of growthduring biochemicalprocesses essential for normal root whichelongation usually occurs is omitted. Noir- growth. This paper describesthe effectsof three falise (1940) reportedthat treatmentof roots of inhibitors,indole-3-acetic acid, iodoaceticacid, and Vicia faba with relativelyhigh concentrationsof 2,4-dinitrophenolon primaryvascular tissue dif- indoleaceticacid (1:40,000) causes cessation of ferentiationin isolatedpea rootswhich have been mitoticactivity in the apical meristemfollowed by subjected in each case to reagentconcentrations prematurevacuolation of the apical initials. The causing approximately90 per cent inhibitionof rapid appearanceof lignifiedvessels closely adja- rootelongation. The purposeof such a studyis to cent to the apical meristemwas interpretedas an attemptto relatebiochemical processes to theknown accelerationof cellulardifferentiation. Barghoorn anatomicalstages in primaryvascular tissue dif- (1942) was unable to demonstrateany effectof ferentiation. applied 1-prolinein stimulatingprotoxylem dif- Attemptsto relate root elongationand cellular ferentiationin the rootsof beans or cotton. Hay- differentiationhave been made in severalstudies of ward and Blair (1942) illustratedan interesting inhibitedroot elongation. As earlyas 1893 Pfeffer case of naturallyoccurring inhibition of root elon- reportedapparent acceleration of acropetaldiffer- gationin thedormant roots of Valencia orange. In entiationof vasculartissue elements in the rootsof the dormantcondition, the apical meristembe- a numberof when root elongationwas in- comes relativelyinactive and reduced. Continued hibitedmechanically by plasterof Paris encasement differentiationof vascular tissues results in the of the root. Using Vicia faba rootsgrown in solid appearanceof matureelements closely adjacent to gypsum,Pfeffer found acropetaldifferentiation of the apical region. The environmentalconditions pittedvessel elements to within1 mm.from the tip normallyinducing this dormant root condition were at the end of 15 days, as comparedto 25-35 mm. reproducedexperimentally by treatmentof theroots in uninhibitedroots. Pfeffer'sfinding has beensub- of Valencia orange seedlingswith high chloride stantiatedby numeroussubsequent workers (Na- solutionsat a high pH. Heimsch(1951) reported thansohn,1898) . More recentlyKojima (1931, thatin rapidlyelongating barley roots, maturation 1933), also usingthe technique of Pfeffer,reported of vascular elementsoccurs at a greaterdistance thatsuch mechanicalpressure inhibits cell division fromthe meristemthan in slowlyelongating roots. in the root tip as early as 16 -hr. afterbeginning In his studiesof mineralnutrition of wheatroots, treatment.In inhibitedroots, elements were Burstrom(1947) showedthat high phosphateen- apparentat an average distanceof 1.4 mm. from hancescell multiplication.Under conditions of low the root apex at the end of one week,while in con- phosphate(1/10,000 M) for 5 days, the number trol roots, xylemwas apparentfirst at 13.7 mm. of cell divisionsin theapical meristemis sufficient- Kojima (1931) pointedout that in termsof posi- ly reducedto resultultimately in the disappearance tion, the inhibitedroots appeared to show accel- of the meristemas the cells are "used up" by elon- erated differentiationof xylemcells, but in terms and differentiation.In this the mer- of age gation case, of cells, cellular differentiationin the in- istem disappears due to an unfavorablebalance hibitedroots was actuallyhindered. He concluded amongcell division,elongation, and differentiation. 1 Receivedfor publication January 30, 1953. Inhibitionof root elongationhas been produced This investigationwas supportedin part by a research by X-irradiation(Smith and Kersten,1942) and grantfrom the NationalInstitutes of Health,Public Health P32 et al., Service. recentlyby radiation from (Mackie The writeris indebtedto Dr. Tova Arzeefor her compe- 1952). In each case an upset in normal differ- tent-technical assistance. entiationprocesses has also been observed. Ac- 525 526 AMERICAN JOURNAL OF BOTANY [Vol.40 cordingto Smithand Kersten,dry of Vicia considerabledistance proximal to the firstmature faba pretreatedwith soft X-rays showed markedly elements. Subsequent centripetaldiffer- inhibitedroot elongation upon .Pitted entiationof the primaryxylem progresses without vessel elementswere observedclosely adjacent to interruptionfrom these outer small protoxylem ele- the apical meristemin all treatedroots at the end mentsuntil the completetriarch xylem pattern is of 5 days. The authorsconcluded that in the ab- formed. The level of the firstmature protophloem sence of elongation,only pitted vessel elements are and protoxylemelements can be quite accurately formed. determinedby carefulstudy of serialtransverse sec- In all the cases cited above, inhibitionof root tions. Using the criteria adopted by Heimsch elongation,whether by mechanical or chemical (1951), mature protophloemelements are those means,resulted in the ultimateappearance of ma- identifiedby scarcityof stainablecontents and lack ture vascular elementsmuch closer to the apical of nucleus; matureprotoxylem elements are desig- meristemthan in untreatedroots. Apparently,in nated as thosecontaining no nucleus. Throughout all cases in whichcell divisionsin the these discussions,the term "differentiation"in- ceased, resultingultimately in cessation of root cludes all stages in the ontogenyof the cell from elongation,the differentiationprocesses continued its initial formationat the apical meristemto its uninterruptedin an acropetaldirection. In none ultimatematuration into a characteristicanatomical of thesecases, however,is thereconclusive evidence cell type. Each stage of differentiationmay in it- foreither acceleration or inhibitionof vasculartis- selfcomprise a sequenceof separaterelated events. sue differentiationunder conditionsof root inhi- RESULTS.-The effectivenessof indoleaceticacid bition. as an inhibitorof root elongationwas recognized MATERIALS AND METHODS.-Throughout all ex- by Kogl et al. (1934) soon afterits chemicalisola- periments,sterile root tips excisedfrom 48-hr. ger- tion. Lane (1936), usingAvena seedlings,showed minatedseeds of the gardenpea, Pisum sativum, thatthe inhibition of rootelongation is proportional varietyAlaska (Asgrow strain No. 44007) were to auxin concentration.Subsequent workers have culturedin pea rootmedium (Bonner and Devirian, studied extensivelythe auxin inhibitionof root 1939) in Petridishes in the darkat 26?C. as previ- elongation,but no clear understandingof themech- ously reported(Torrey, 1950). Five or ten mm. anismof its actionhas been achieved. The inhibi- tips were excised asepticallyand transferredim- tory action of iodoaceticacid on root elongation mediatelyto liquid mediumto which appropriate was shownby Albaumand Commoner(1941) who additionsof the inhibitorhad been made. One ml. reportedconsistent inhibition of rootelongation in additionsof the inhibitorsolutions, sterilized by Avena seedlingsgrown in 10-4 M solutionsof Seitz-filtration,were made to 100 ml. amountsof iodoaceticacid. Kandler (1950) recentlyreported the nutrientmedium and thepH was adjustedwith the inhibitionby 2,4-dinitrophenolof root elonga- sterile0.05N NaOH to the appropriatepH noted tionin isolatedcorn roots grown in syntheticnutri- below for each inhibitor. At the end of the treat- ent mediumcontaining 10-4 M concentrationof mentperiod, root tips were killed in formalin-aceticthe inhibitor. acid-alcoholwith aspiration, dehydrated in tertiary In orderto studyprimary tissue differentiation butylalcohol series, and embeddedin "Tissue-mat." under conditionsof inhibitedroot elongation,it Serial sectionswere cut at 8,Aand stainedwith Hei- was necessaryto establishsuitable inhibitorcon- denhain'shematoxylin and safranin.Measurements centrationswhich would produce comparablein- of sectionedroots were made by an actualcount of hibitionof pea root elongation.Serial dilutionsof sectionsmultiplied by sectionthickness. For m-ea- each of the inhibitorswere testedin liquid nutri- surementsof longitudinalsections a suitablycali- ent mediumfor effectiveinhibition of root elonga- bratedocular micrometer was utilized. tion. In fig. 1 are plottedrepresentative data for As has been reportedearlier (Torrey, 1951), iso- inhibitionof pea root elongationby indoleacetic lated pea rootsgrowing in a syntheticnutrient me- acid (IAA), iodoaceticacid (IODOAC), and 2,4- dium exhibitthe typicalsequence of primaryvas- dinitrophenol(DNP). The minimalconcentration cular tissue differentiationfound in the roots of a of each inhibitorproducing approximately 90 per numberof dicotyledonousplants (Esau, 1943; Wil- centinhibition of elongationwas establishedas the liams, 1947). Immediatelyproximal to the apical concentrationto be used in theseexperiments. initials,the enlargementand vacuolationof pro- Root elongationand primaryvascular tissue dif- cambialcells progressively"blocks out" or delimits ferentiationin controlmedium.-Five mm. root the futuretriarch arrangement of xylemtissue, ac- tips grownin controlmedium elongate to an aver- cording to the pattern described by Wetmore age lengthof 50 mm. in one week. Representative (1947). The differentiationof primary phloem and tips were fixed at daily intervals,embedded and xylem proceeds acropetally,with mature proto- sectioned.Exact levels of the firstmature phloem phloem elementsbecoming apparent before the and maturexylem elementswere determinedbv completexylem pattern has been blockedout. The microscopicexamination of serial cross-sections. firstmature protoxylem elements are seen at the Averagefigures for each day werecalculated, using outermostpoles of the xylemstrands, usually at a a minimumof fiveroots for each figure. In fig.2 July,1953] TORREY--DIFFERENTIATION IN PEA ROOTS 527

100 maturexylem, relative to the apical meristem,re- main fairlyconstant. First maturephloem occurs between330-550,u from the apical initials,whereas ROOT the firstxylem, showing wider variation, is appar- ELONGATION o ent from4250-5800. Changesin the rate of root elongationwhich occur duringthe period are ac- PERCENT \Nv companiedby correspondingchanges in the rate of tissue maturation,so that the relativeposition of CONTROL \x IODOAC matureprimary vascular elements in theelongating rootis maintained. 50 Root elongationand tissuematuration during in- IAA hibitionof root elongation:Inhibition by IAA.- Amongthe mosteffective inhibitors of rootelonga- tion are the auxins. At pH 5.0, 1 p.p.m. (5.7 X x 10-6 M) IAA inhibitsthe elongationof pea roots about90 per cent. Duringthe treatment, secondary effects,including an increasein root diameterand lateralroot initiation,may be noted. The striking 10 90 Inhibition 7 effectof IAA on vasculartissue maturation becomes 10 evidentvery early in treatmentwhen microscopic examinationof the tissuesis made. In fig. 3 are presenteddata on rootelongation and primaryvas- io 8 1o07 1o06 IO-5 10-4 10-3 cular tissuematuration in IAA-treatedroots during MOLAR CONCENTRATIONOF INHIBITOR the first3 days of treatment.Data for control Fig. 1. The inhibitionof elongationof isolated pea roots roots (untreated)are also includedso that exact grown in nutrientmedium with differentgrowth inhibi- comparisonsmay be made. The effectiveinhibi- tors. Five-mm.root tips excised from germinatingseeds and grown in the dark at 25?C. for 3 days. Each point representsaverage of 20 roots. All solutions adjusted to 5000 /CONTROL/ pH 5.0 with 0.05 N NaOH. Ix\

x CONTROL are presentedtypical measurements of rootelonga- tion and the locationof maturevascular tissue in 4000 / rootsgrown in the controlmedium. Distancesto U) the first mature vascular tissue are presented zO | x XYLEM throughoutin micronsalong the lengthof the root o / o PHLOEM fromthe apical meristem(exclusive of the root 3000 / TISSUE MATURATION cap). During the course of root elongation,the 2 I -- -ROOT ELONGATION w positionsof the firstmature phloem and the first QC

_ \

4 2000 TISSUE MATURATION -- ROOT ELONGATION D#STANCE8T 40OOT < X ~~~/1 / RO 2 V /w FROM 7 LENGTH O I AA APICAL 6 . IN MM. U. MERISTEM 3 0 0 1000 IN MMR 7 z~~~

XYLEM co / X_ x IAA

/- e esCONTROL IAA

2 , 10 0 24 48 72 -V~~~~~~~~~~~~~1 TIME IN H OU R S _ -_ ~~-0 PHLOEM Fig. 3. Primaryvascular tissue maturationand increase 0 I 2 3 4 5 6 7 in root length of isolated 5-mm.pea roots grown in nutri- TIME IN DAYS ent medium with and without5.7 X 10-6 M indoleacetic Fig. 2. Root elongation and primary vascular tissue acid during 3 days. Averages were determined from 2 maturationof 5-mm.pea root tips grownin controlnutrient separate experiments. Root elongation curves represent medium for 7 days. Tissue maturationrepresents the aver- the increase in root length in micronsduring the treatment age level at which the firstmature tissue can be distin- period. Curves for control roots are equivalent to those guished. in fig.2. .528 AMERICAN JOURNAL OF BOTANY [Vol. 40 tion of root elongationis clearly evident. Little celerationeffect of auxinon therate of phloemmat- differencein the levels of firstmature phloem in urationwas evident,nor was thereany apparent treatedand untreatedroots can be noted. Very effectof auxin on the initial delimitationof the striking,however, is the decreasein the distanceof xylempattern by procambialcell enlargementand the firstmature xylem from the apical meristemin vacuolation. Longitudinalsections of typicalcon- the IAA-treatedroots. troland auxin-treatedroots are seen in fig.4 and 5 The auxin effectis most strikingduring the in- respectively.In the latterfigure, the proximityof itial 24-hr.period of IAA treatment.In table 1 is matureprimary xylem to the apical meristemis presentedthe detailedanalysis of root elongation strikinglyevident. A comparisonof relativematu- and tissuematuration in one representativeexperi- ration in controland IAA-treatedroots may be mentwith auxin-treated and untreatedroots. It is made by comparingfig. 6 and 7, transversesections evidentfrom the figurespresented that the accel- cut in each case at a levelof 1840kproximal to the eratedxylem maturation in the 24-hr.period can- apical meristem. not be accountedfor alone by the decreasedroot A similarstudy of the effectof IAA on 10-mm. elongation,as was shownto be the case for me- tips excised frominitial tips grownone week in chanical inhibitionover longerexperimental peri- control medium,then excised and treated with ods by Kojima (1931). During the initial 24-hr. auxin forsimilar periods gave essentiallythe same period,xylem maturation in the treatedroots pro- resultsas above. Thus, an auxin concentration gresseda distanceof over 5 mm. (5240u) com- which preventselongation of the root accelerates pared to 1940u in controlroots. Root elongation, the rate of maturationof primaryxylem elements. however,was inhibitedonly to the extentof about It is interestingto note thatthis same auxin con- 6OO in comparisonto controlroots. The discrep- centrationis about optimalfor maximumelonga- ancy of about 3300, cannotbe attributedto differ- tionof pea stem sections(Galston and Hand, 1949). ences in root elongation,but such differenceis ap- In fig. 10, the spatial relationshipsbetween the parentlydue to thespecific effect of IAA in increas- primaryvascular tissues withinthe 24-hr. IAA- ing therate of xylemmaturation. treated root are diagrammaticallyrepresented, In the last columnof table I are presentedfor showingthe position of firstmature xylem elements each 24-hr.period the averagedistances over which acropetallydifferentiated to withinabout 1 mm.of xylemmaturation progressed. In thecontrol roots, the apical meristem. The protoxylemelements it is clear thatthe rate of xylemmaturation closely whichare producedunder these conditions are typi- reflectsthe rate of root elongation. Thus, during cally short,reticulately-pitted elements (fig. 5)- the third 24-hr. period, primaryxylem elements characteristicof xylemelements differentiated un- achieveda maturestate along a lengthof root al- der conditionsof inhibitedgrowth (Smith and most6?/2 mm. long. In the auxin-treatedroot, de- Kersten,1942). Althoughthe centrifugaldelimi- spitealmost complete inhibition of rootelongation, tation of the xylem,which proceeds in the pro- nearlythis same rate was achievedduring the first cambiumimmediately proximal to the apical mer- 24-hr. period of treatment. No comparable ac- istem,is apparentlyunaffected by the auxin treat-

TABLE 1. Root elongation and distance of mature primary vascular tissue from the apical meristemin isolated pea roots grownin nutrientmedium with and withoutIAA. Initial root length was 5.0 mm.

Hr. Distance fromapical meristem,A Average increase Incrementof maturedtissue in ,u/day 1st mature phloem 1st maturexylem in root length,A Phloem Xylem Roots grownin controlmedium at pH 5.0 Av. Av. 0 664) 6156) 640} 650 6224 6190 ---- 24 59608265 180 5150 900 950 1940 48 490 4690 408 {5408)455s 5050 4600 4750 4700 72 384 340 5130 4910 6200 6310 6340 286 j3046965 Rootstreated with 5.7 X 10-6 M IAA at pH 4.8 0 650 6190 24 330 380 1183 1250 300 570 5240 4325 1320j 48 320 320 750 820 400 460 830 320 896j 72 258 270 645 750 400 450 470 286. 856. July.1953 ] TORREY-DIFFERENTIATION IN PEA ROOTS 529

*,,v''314 [5 ~'.

Fig. 4-5.-Fig. 4. Longitudinal section of isolated pea rout grown in controlnutrient medium for 48 hr. Total length of root in figureequals about 13501At.Note lack -ofmature primaryxylem. X70.-Fig. 5. Longitudinalsection of isolated pea root grownin nutri-entmedium containing5.7 X 10-6 M IAA for 72 hr. Total length in figureequals about 1350/t.4 Note mature primary xylem strands approaching the apical meristem. In this root, the firstprimary xylem occurred lateral to this section at 645Asfrom the apical meristem. The -enlargementof the cortical cells at the proximal end of the root is a characteristicresponse to auxin treatment. X70. ment (fig. 10), the rate of centripetalmaturation that produced by IAA treatment,although less of thesexylem elements in the xylemstrands is ac- marked. Progressive centripetalmaturation of celeratedby thetreatment with IAA. xylemelements is not as rapid as in IAA-treated Inhibitionby iodoaceticacid.-Iodoacetic acid is roots (fig. 8). In all the rootstreated with iodo- reportedto inhibitrespiration by inactivating-SH acetate,no mitoticdivisions in the apical meristem groups. The inhibitoris consideredto be a non- nor along the lengthof the rootwere observed. specific inhibitorof sulfhydrylenzymes and is Secondarywall formationin theseroots was ab- especiallyeffective on certain enzymesof aerobic normal in that xylem elements,especially newly respiration.At pH 5.0, a concentrationof 10-4 M formedprotoxylem, had very thin, dark-staining iodoacetate almost completelyinhibits pea root cell walls, showinglittle or no lignification.The elongation (fig. 1). Machlis (1944) reporteda maturestate of such cells was evidentonly by the greaterthan 60 per cent inhibitionof aerobic res- loss of the cell contents.Maturation of sieve-tube pirationin barleyroots treated with 5 X 10-5 M elementsof the phloemappeared to be normal,but iodoacetateat pH 5.0. Salt accumulationwas cor- in matureregions of theroot phloem fibers, like the respondinglyreduced in the presence of the in- elementsof the xylem,showed only slightsecon- hibitor.Laties (1949) also reportedthe strongin- darywall thickening.The inhibitionof usual sec- hibitoryeffect of iodoaceticacid on roottissue res- ondarywall thickeningmust be attributableto some piration.Morphologically, no macroscopicchanges director indirecteffect of the inhibitoron cellu- are apparentin the treatedroots, which remain lose depositionby thesecells with the resultthat white,turgid and normalin appearance. this normalphase of xylemmaturation is omitted. In table 2 are summarizedthe resultsof mea- Inhibition by 2,4-dinitrophenol.-Thisreagent surementsmade on roots treated48-72 hr. with has been shownto be active in animal and plant 1O-4 M iodoacetateat pH 4.9, in the nutrientme- tissues(Loomis and Lipmann,1948) in uncoupling dium. It is evidentthat thereis an apparentac- aerobic respirationfrom the phosphorylativesys- celerationof xylem tissue maturation,similar to tem,producing a concomitantstimulation of oxygen 530 AMERICAN JOURNAL OF BOTANY [Vol. 40

......

Fig. 6-9.-Fig. 6. Transversesection of isolatedpea rootgrown for 48 hr. in controlnutrient medium, cut at 1840,u proximalto the apical meristem,showing tissues of the centralcylinder. Note delimitationby cellularenlargement and vacuolationof the triarchpattern of the futureprimary xylem tissue (protoxylempoles of futurexylem strands indi- cated by X). Matureprimary phloem elements are evidentin the peripheryof the cylinderat alternateradii withthe xylempoints. Level of sectioncomparable to thatin fig.6-8. X144.-Fig. 7. Transversesection of isolatedpea root grownfor 48 hr. in nutrientmedium containing 5.7 X 10-6 M IAA, showingcentral cylinder of root. Sectioncut at 1840,uproximal to the apical meristem.Note maturationof primaryxylem elements (X) in triarchxylem tissue. X144. -Fig. 8. Transversesection of isolatedpea root grownfor 48 hr. in nutrientmedium containing 10-4 M iodoacetic acid, showingcentral cylinder of the-root. Sectioncut at 1840,uproximal to the apical meristem.Note beginningof primaryxylem maturation at each of the,three protoxylem points (X). X144. Fig. 9. Transversesection of isolated pea rootgrown for 48 hr. in nutrientmedium containing 10 -4 M 2,4-dinitrophenol,showing central cylinder of theroot. Sectioncut at 1840A.proximal to the apical meristem.Note thetriarch patt-ern of thefuture primary xylem (X) evident in the enlargedcentral procambial cells. Matureprimary phloem elements may be seen at alternateradii with the futurexylem poles, but no maturationof primaryxylem is evident.X 144. consumption.Bonner (1949) has shownthat DNP In table 2 are presentedtypical measurements stronglyinhibits the elongationof Avena coleop- fromroots treated with 10-4 M DNP at pH 5.0 in tiles. Stenlid (1949) demonstratedthat DNP in- nutrientmedium. From these data it is apparent hibitedabsorption of glucoseby wheatroots while thatprimary vascular tissue maturation was inark- at thesame timeincreasing 0, consumption.Strong edly affected. The inhibitorcompletely stopped inhibitionof pea root elongationby relativelylow xylemdifferentiation (fig. 9). The distanceof ma- concentrationsof DNP is shownin fig.1. turexylem from the apical meristemin the treated July,1953] TORREY-DIFFERENTIATION IN PEA ROOTS 531

TABLE 2. Root elongation and distance of mature primary vascular tissue fromthe apical meristemin isiolatedpea roots treated in nutrientmedium with growth inhibi- tors. Centripetolmoturation 2000 p + IFfIe im ; tofxylem elements Distance from apical Average root meristem,,u lengthin 1stmature 1stmature g | fi -~~ Hr. mm. phloem xylem Roots grow-nin control medium at pH 5.0 5 0 0 2 . - C or tex 0 5.0 650 >5000- 1500 ~~~~~~~~ 24 5.8 640 >4820 Endodermis 48 9.3 380 >5000 72 15.9 340 >4500

Roots treated with 10-4 M lodoacetate at pH 4.9 -First mature Av. Av. 1000 xylemelemrents 24 5.2 48 5.3 3401 18401 410 340 1260 1710 g 4 A _ 1 260J 2030J i Immature 72 5.4 1901 12201 xylemelements 150 180 710 910 500 190l 800 Roots treated with 10-4 M 2,4-dinitrophenolat pH 5.0 -First maturesieve, 1020 >5000 tube elements 1080 980 >5000 24 5.0 850J >5000 -_Centrifugol blocking outof xylempottern 48 5.3 1250k 1130 >5000 1010 f >5000 Apical meristem 72 5.5 1280 1190 >5000 1100 f >5000 Root cap 300 a Indicates firstmature xylem appears at a distance great- er than the lengthof the root sectioned. Fig. 10. Diagrammaticrepresentation of isolated pea root roots was unchanged during the treatmentperiod. grown for 24 hr. in nutrientmedium containing 5.7 X DNP inhibited those phases of xylem maturation 10-6 M IAA, showingspatial relationshipbetween primary which include secondary wall formation,lignifica- tissues in inhibitedroot. First mature sieve-tubeelements tion and the subsequent loss of cell contents. It is of phloem appear about 350,a proximal to apical meristem. not possible to state from these studies whether First mature xylem elements appear about 1250, proximal to DNP also prevents the earlier stages of xylem dif- apical meristem. The early centrifugalblocking out or delimitationof the triarchprimary xylem is contrastedto ferentiationwhich give rise to the early delimitation the subsequent centripetal maturation of these primary of the patternof the primaryxylem tissue. In none xylem tissues. Based on the type of diagram devised by of the DNP-treated roots was cellular division ob- Esau (1941). served. The differentiationof phloem tissue is also influ- enced by this growthinhibitor. During DNP treat- DISCUSSION.-The differentiationof any cell must ment, the position of the firstmature Fphloemele- involve a large number of biochemical reactions, ments was progressivelymore distant fromthe api- which act together in such a direction as to lead cal meristem-even more distant than at the start ultimatelyto the formationof a given mature cellu- of the experiment. This anomalous condition can lar component. These reactions underlyingcellular be explained in terms of the limited cell elonigation differentiationare modifiedand controlled for each which occurred under these conditions. It is clear cell by the physical and chemical environmentes- that no maturation of phloem tissue occurred after tablished locally by all adjacent differentiatingcells. the onset of inhibitortreatment. That the firstma- For differentcell types,the complexityof the differ- ture elements became progressivelysomewhat more entiation patternmight be expected to differ-from distant from the apical meristemindicates that the relatively simple to extremely complex patterns. limited cell elongation which occurred in the treat- Modification of any single aspect of the chemical ed roots must have occurred in the cells located environment may well modify and change the between the apical initials and the first mature course of the differentiationpattern. phloem. The important conclusion to note, how- By treatmentof elongating roots with chemical ever, is that DNP inhibits root elongation and pre- inhibitors of growth, it has been shown that the vents differentiationof both xylem and p)bloem sequence of differentiationof the vascular tissue elements. may be modified,i.e., accelerated in all its aspects, "32 AMERICAN JOURNAL OF BOTANY [Vol. 40

MATURE XYLEM ELEMENT The phases of xylem elementmaturation con- sideredhere are intimatelytied to thecarbohydrate metabolismof the tissue. wall forma- loss of end walls, Secondary nucleus ond cell contents tion involves a considerabledeposition of cellu- losic materialsat the expenseof the available car-

MATURATION IMMATURE XYLEM ELEMENT bohydratesin themetabolizing cell. Thatiodoacetic PROCESSESI acid does notprevent maturation suggests that these processesare not completelydependent upon the li gnif i cation aerobic respirationof the tissue. On the other hand, DNP, whichinterferes with the phosphorv- secondor wll formation I AA acceleration lative system,prevents the maturationsteps. Al- IODOACETATE inhibition though practicallynothing is known concerning the enzymesystems involved in cellulosesynthesis, XYLEM ELEMENT MOTHER CCti DNP inhibition it is reasonableto expectthat phosphorylated inter- I mediatesare involvedand thatinhibition of cellu- v a c uolation lose synthesisby depletingthe supply of phosphory- lated substratesmight well interferewith the matu- DELIMITATION cell enlargement - IAA effect ? rationprocess. Conclusiveevidence concerning the PROCESSES specificnature of actionof each of theseinhibitors in cell differentiationmust await the development PROCAMBIAL CELL of morecritical methods of attackingthis problem. SUMMARY APICAL INITIAL Five mm. Alaska pea roots excised from48-hr. Fig. -1. Ontogeneticsteps in the differentiationof a ves- germinatedseed and grownin a syntheticnutrient sel element in the primaryxylem of a pea root, from its mediumin Petri dishes in the dark were treated origin at the apical meristemto the formationof the mature withthe knownchemical inhibitors of root elonga- xylem element. Probable site of inhibitoraction is indi- tion, indoleaceticacid, iodoacetic acid and 2,4- cated. dinitrophenol,at concentrationscausing approxi- mately90 per centinhibition of rootelongation. A acceleratedin onlycertain phases, or preventeden- detailedhistological study was made of these in- tirely. From the knownactivity of the growthin- hibited roots, using serial transverseand longi- hibitorsused in this study,one mightmake some tudinalsections, in an attemptto relatebiochemical interestingspeculations concerning the biochemical processesof the root tissuesto the differentiation processesinvolved in primaryxylem differentiation of primaryvascular tissues. At a concentrationof -or at least certainaspects of thatdifferentiation. 5.7 X 10-6 M (1 mg./l.),indoleacetic acid almost Stepsinvolved in the differentiationof a particu- completelyinhibits root elongationbut markedly lar cell typemay be outlinedsomewhat arbitrarily acceleratesthe maturationof primaryxylem ele- in histogeneticterms and each step thensubjected ments,while having little effect on the differentia- to analysis and study. Only by such a step-wise tion of primaryphloem. lodoaceticacid at 10-4 analysiscan one hope to approachan understand- M at pH 4.9, a concentrationreported to inhibit ing of the over-allprocess. In fig.11 is presented stronglyaerobic respirationin root tissues,causes in summaryfashion a statementof the ontogenyof slight accelerationof primaryxylem maturation a vesselelement in the pea root. Each arrowindi- while inhibitingroot elongation. Secondarywall cates a series of chemical reactionswhich must formationof the primaryxylem elements was ab- occur in the directionof differentiation.In this normalunder iodoacetate treatment, with protoxy- schemeare indicatedthe stepsat whichthe chemi- lemelements showing reduced secondary wall thick- cal inhibitorsstudied most probably exert their ening and lignification.2,4-dinitrophenol, an in- influence. hibitorwhich has been shownto uncoupleaerobic No evidenceis given in the presentwork con- respirationfrom the phosphorylativesystem, at a cerningthe role of IAA in cell enlargement.There concentrationof 10-4 M at pH 5.0, completely is evidenceto indicatethat auxin is involvedactive- stopsprimary vascular tissue differentiation as well ly in the maturationof primaryxylem elements- as rootelongation. It is pointedout that the action i.e., the formationof the secondarywall, its ligni- of all threeinhibitors centers around cellular pro- ficationand the subsequentloss of cell contents.All cesses associated with carbohydratemetabolism. of thesephases of differentiationare markedlyac- Thus, by use of chemicalinhibitors, it has been celeratedby IAA. Whetherthis effectis director possibleto modifythe usual sequence of primary indirectcannot be decided fromthe evidencepre- vasculartissue differentiation in these roots. sentedhere. Recently,Jacobs (1952) has shown DEPARTMENT OF BOTANY, auxin to be the factorlimiting xylem regeneration UNIVERSITY OF CALIFORNIA, in woundedstem of Coleuts. BERKELEY 4, CALIFORNIA July,1953] TORREY-DIFFERENTIATION IN PEA ROOTS 533

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