Plant Physiol. (1970) 46, 64-68

The Time Course of Xylem Differentiation and Its Relation to

Deoxyribonucleic Acid Synthesis in Cultured ColeusStem Segments

Received for publication June 30, 1969

D. E. FosKirThe Biological Laboratories, Harvard University,

ABSTRACT

The relationship between DNA synthesis and woundxylem differentiation was investigated in cultured stem seg-ments of Coleus blumei. The addition of 50 micrograms ofindoleacetic acid per liter to the culture medium resultedin a 400 to 500% increase in the number ofwound vessel mem-bers formed in 7 days. However, the time course of woundvessel member formation was similar in segments culturedin the presence and absence of auxin. In either case, nowound vessel members appeared before the 3rd day of cul-ture, while the majority of wound vessel members appearedon the 4th and 5th days of culture. 3H-Thymidine incorpora-tion into DNA was used to measure changes in the DNAsynthetic activity of the tissues during the culture period.Comparatively little 3H-thymidine incorporation occurredduring the 1st day of culture. Maximum 3H-thymidineincorporation was observed on the 2nd day of culture, 2days before the peak period of xylem differentiation. Therate of incorporation of 3H-thymidine into DNA decreasedwith increasing time in culture after the 2nd day. Auxin at50 micrograms per liter had no effect on the time course of'H-thymidine incorporation, although somewhat more 3H-thymidine was incorporated into DNA throughout theculture period in the presence of auxin. The magnitude ofthis effect was small when compared to the effect of auxinon xylem differentiation. The antimetabolite 5-fluorodeoxy-uridine was shown to block DNA synthesis in the culturedstem segments. When the tissues were isolated on mediacontaining 10-. M 5-fluorodeoxyuridine, wound vessel mem-ber differentiation was inhibited by approximately 80%, inboth the presence and absence of auxin. Thymidine at 104 Mcompletely overcame the 5-fluorodeoxyuridine inhibition ofwound vessel member formation. 5-Fluorodeoxyuridine waseffective in blocking xylogenesis only when this substancewas supplied to the tissues during the early part of theculture period. 5-Fluorodeoxyuridine had no effect on xylemdifferentiation when it was applied after the 3rd day ofculture.

Although auxin has been shown repeatedly to act as a limitingfactor for xylem differentiation (7, 12-14, 24, 25), we know verylittle about the nature of this regulation. Ultimately one wouldhope to elucidate the biochemical mechanism(s) by which auxin

Cambridge, Massachusetts 02138

controls xylogenesis. To facilitate this achievement, it would beuseful first to discover where auxin acts during xylem elementformation. Is auxin directly involved in the initiation of xylemdifferentiation, or does it limit the rate of steps which occur aftera cell is committed to differentiation, such as secondary walldeposition or lignification? It is well established that the composi-tion of the secondary wall of tracheary elements in woody speciesis determined, in part, by the concentration of auxin in the tissues(3, 16). However, in this case auxin appears to regulate the pro-portions of cellulose, hemicellulose, and lignin in the wall, ratherthan to determine the rate at which these substances are de-posited. The simple study of the time course of xylem differentia-tion, as influenced by auxin, might be expected to tell us some-thing about the role of auxin in this process. If auxin limitedxylogenesis by controlling the rate of secondary wall deposition,one might expect auxin to accelerate the rate at which xylem ele-ments appeared, but to have relatively little effect upon the totalnumber of elements that were formed in a given experimentalsystem.Another process, apparently regulated by auxin, which has

been shown to be an integral part of the ontogeny of xylem ele-ments, is DNA synthesis. DNA synthesis appears to be involvedin xylogenesis in at least two different ways. First, DNA synthesiswould be expected to accompany the mitotic activity which pre-ceeds and is necessary for the initiation of xylem differentiation(6, 8, 14). Secondly, in some species an increase in nuclear DNAcontent accompanies the maturation of xylem elements. List (15)observed that the nuclei of differentiating metaxylem elements inthe roots of Zea frequently attained amounts of DNA up to 32times the normal diploid level. Although there is little direct evi-dence for or against the hypothesis that auxin regulates xylogene-sis by controlling DNA synthesis, a number of studies have shownthat cell proliferation can be stimulated in intact or woundedplants by auxin application (1, 9, 11, 17, 20-22). Also, the work ofSkoog and co-workers (4, 18) suggests that auxin may be directlyinvolved in the regulation ofDNA synthesis. They found that thetreatment of tobacco pith tissue with auxin alone brought aboutDNA synthesis in a significant proportion of the cells, althoughrelatively few of these cells went on to divide. As is well known,both auxin and cytokinin were required for the complete divisioncycle in this tissue.

In the present investigation, the time course of xylem differen-tiation and DNA synthetic activity was determined in culturedColeus stem segments, and the effect of auxin upon these processeswas examined. Additional evidence is presented that DNA syn-thesis is necessary for xylogenesis, but the data do not support theidea that auxin regulates xylem differentiation through its effectupon DNA synthesis, or through possible effects upon the rate ofsecondary wall deposition.

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TIME COURSE OF XYLEM DIFFERENTIATION

MATERIALS AND METHODS

A clone of Coleus blumei Benth., c.v. Beefsteak, was propagatedin the greenhouse with 16 hr of supplemental incandescent lightper day. Prior to stem segment isolation, the apical bud, lateralbuds, and the first three pairs of expanded leaves were removedfrom selected shoots. Two days later, the second internodes wereexcised, surface-sterilized by means of a 15-min treatment with10% Clorox, and passed through three changes of sterile distilledwater. The most apical 3 mm of each second internode was re-moved and discarded. Two stem segments were prepared from theremaining internodal tissue by means of two additional transversecuts, approximately 2 mm apart. As the stem segments wereexcised, they were placed with their morphological apical ends upin a separate dish containing sterile distilled water. After one addi-tional rinse with sterile distilled water, the stem segments werecultured with their morphological apical ends up on the surface ofagar-solidified media in 10-cm diameter glass Petri dishes. Thestem segments in Petri dishes were placed in a controlled environ-ment chamber where they received a 12-hr daily photoperiod ofcombined fluorescent and incandescent light at an intensity ofapproximately 150 ft-c, and a constant temperature of 23 + 2 C.The stem segments were isolated at such a time of day that theculture period was begun with a 12-hr light cycle.Counts of differentiated wound xylem elements were made after

clearing and squashing the stem segments. The segments werecleared by sequential treatment with 1 N NaOH and 84% chloralhydrate. The cleared segments were stained briefly with diluteaqueous safranin, washed thoroughly with tap water to removethe excess stain, and squashed onto glass slides in glycerin jelly.

Nucleic acids were extracted by means of a phenol-sodiumdodecyl sulfate procedure. Twenty stem segments (approximately0.5 g fresh weight of tissue) were homogenized by hand with an

all glass tissue grinder in 3 ml of 80% phenol and 2 ml of 0.05 Mtris buffer, pH 7.4, containing 4% sodium dodecyl sulfate and0.1% 8-hydroxyquinoline. After centrifugation at 10,000 g for 30min, the aqueous layer was carefully removed. Two volumes of100%/ ethanol were added to the aqueous layer, and this mixturewas allowed to stand for 24 hr at -20 C, in order to precipitatethe nucleic acids. The precipitated nucleic acids were collected bycentrifugation at 0 C (10,000 g for 20 min), and washed twice withice-cold 50% ethanol containing 10-4 M thymidine or 10-4 Mdeoxycytidine.

In experiments to determine the rate of 3H-thymidine in-corporation into DNA, segments incubated for 12 hr on amedium containing 2 ,uc/ml methyl 3H-thymidine (New EnglandNuclear, specific radioactivity 6.7 c/mmole) were extracted asdescribed above. After ethanol precipitation, the washed nucleicacids were redissolved in 2 ml ofhot 1 M perchloric acid (70 C for20 min) and 0.5-ml aliquots of the resultant solution were takenfor scintillation counting and DNA determinations.The effect of 5-fluorodeoxyuridine on DNA synthesis was in-

vestigated by following the incorporation of 3H-deoxycytidine.Control of FUdR-treated1 stem segments were incubated on amedium containing 1 ,uc/ml 3H-deoxycytidine (Schwarz Bio-Research, specific radioactivity 26.2 c/mmole), and nucleic acidswere extracted as described above. After ethanol precipitation,the nucleic acids were washed with 50% ethanol containing 10-4M unlabeled deoxycytidine. The radioactivity of the supernatantand washings was determined by scintillation counting. The pre-

cipitated nucleic acids were suspended in 1 ml of 0.1 N KOH andheated to 70 C for 20 min. After chilling in an ice bucket for 30min, the solution was made 1 M in perchloric acid. This suspen-

1 Abbreviations: FUdR: 5-fluorodeoxyuridine; SA: a medium con-

taining 2% sucrose and 1% agar; SAIAA: SA medium supplementedwith 50 gg/liter indoleacetic acid; WVM: wound vessel member.

sion was centrifuged at 10,000 g for 20 min, and the supernatantwas decanted. The precipitate was washed with 0.5 ml of cold 0.2M perchloric acid, and the washing was combined with the super-natant from the previous step. This constituted the RNA frac-tion. The precipitate which remained represented the DNA frac-tion. The DNA was dissolved in 2 ml of hot 1 N perchloric acid(70 C for 20 min). Aliquots of the RNA and DNA fractions weretaken for quantitative determinations of the respective nucleicacid and for scintillation counting. Amounts of RNA were esti-mated from the absorbance of the solution at 260 nm. DNA wasdetermined by the diphenylamine reaction (2), with salmon spermDNA as a standard.

RESUTLTS

Time Course ofXylem Differentiation. The time course of xylemdifferentiation was determined in stem segments cultured on amedium containing 2% sucrose and 1% agar and in segments cul-tured on the same medium supplemented with 50 j,g/liter IAA.When the number of wound vessel members observed on anygiven day was plotted against time in culture, the curves shown inFigure 1 were obtained. Tissues cultured on the SAIAA mediumproduced approximately four times as many WVMs as the SA-grown stem segments. Nevertheless, xylem differentiation oc-curred with the same general time course under the two culturalconditions. The presence of auxin in the media did not shortenthe lag period. No WVMs were observed until the 3rd day aftersegment isolation in either SA- or SAIAA-grown tissues. Thelargest increases in WVMs occurred between the 3rd and 4th days

(Q)

Q-14

a

%2qI)12

X

10 B%u(I)

8X

106 2

4K

2

0 2 3 4 5 6 7

Days After IsolationFIG. 1.The time course of DNA synthesis and xylem differentiation

in Coleus stem segments cultured in vitro. Counts of differentiatedwound xylem elements were made at daily intervals in stem segmentscultured on SA medium or on SAIAA medium. The mean number ofwound vessel members per segment for each daily sample is given.Daily rates of DNA synthesis were estimated from the amount of3H-thymidine incorporated into DNA within a 12-hr labeling period.DNA was extracted and analyzed as described in "Materials andMethods." The observed incorporation of 3H-thymidine in SAIAA-grown stem segments is indicated by the vertical bars, the width ofwhich represents the duration of the labeling period.

65Plant Physiol - Vol - 46, 1970

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Plant Physiol. Vol. 46, 1970

Table I. Effect ofFUdR on 3H-Deoxycytidine Uptake andIncorporation into Nucleic Acids

After 3 days in culture on SA medium, stem segments weretransferred to SAIAA medium, with or without 10-6 M FUdR and105 M uridine. Segments were incubated on the above medium,containing 1 Mc/ml 3H-deoxycytidine, for the period indicated be-low. Nucleic acids were extracted and separated as described in"Materials and Methods."

Time (hr.) FUdR Ethanol-soluble Ethanol-precipitable

cpm/segment cpm/pg RNA cpm/Mg DNA

0-4 _ 5278 104.15 36.440-4 + 6714 112.36 21.380-8 _ 10246 211.16 125.160-8 + 13079 212.45 73.260-12 - 15915 296.23 168.180-12 + 18019 323.97 72.7812-24 - 15833 275.25 194.6012-24 + 17735 285.46 94.22

of the culture period. With the SA-grown tissues, 42% of theWVMs appeared between the 3rd and 4th days of culture, andanother 23% of the total WVMs appeared between the 4th and5th days. An even higher proportion of the total WVMs ap-

peared in these time periods in segments cultured in the presenceof auxins. Between days 3 and 4, 46%, and between days 4 and 5,35% of the total WVMs appeared in SAIAA-grown segments.Thus, 90% of the total number of WVMs had appeared by the5th day in SAIAA-grown segments, while 75% of the WVMsformed in SA-grown tissues appeared in the same time period.Time Course of DNA Replication. The rate of 3H-thymidine

incorporation into DNA was determined in order to estimate thedaily DNA synthetic activity of the cultured stem segments. Seg-ments initially cultured on SA or SAIAA medium were trans-ferred to freshly prepared medium containing 2 .c/ml 3H-thymi-dine at the start of the daily dark period. The segments were har-vested for analysis at the end of the 12-hr dark period. Theamount of 3H-thymidine taken up by the tissues from the mediumduring each 12-hr labeling period was approximately the same.

Therefore, the specific radioactivity of the DNA may be con-

sidered to reflect the amount of DNA synthesis which occurredduring each labeling period. As is shown by the data in Figure 1,comparatively little 3H-thymidine was incorporated into DNAduring the 1st day of culture. The greatest amount of DNA syn-thesis occurred during the 2nd day of culture, after which a pro-gressive decline in DNA synthetic activity was observed. By the5th day of culture the amount of 3H-thymidine incorporation was

only slightly greater than that observed on the 1st day.A comparison of the amount of 3H-thymidine incorporation in

segments cultured in the presence and absence of auxin demon-strated that 50 ,Ag/liter IAA had a relatively small effect on DNAsynthesis. The general pattern of DNA synthetic activity was thesame in SA- and SAIAA-grown segments, with peak activitiesoccurring on the 2nd day under both cultural conditions.

Relationship between DNA Synthesis and Xylogenesis. Thepossible relationship between DNA synthesis and xylem differ-entiation was investigated through the use of an inhibitor ofDNA replication, 5-fluorodeoxyuridine. The effect of FUdR on

3H-deoxycytidine uptake and incorporation into nucleic acidswas examined. Stem segnents cultured for 3 days on SA mediumwere transferred to SAIAA medium containing 1 ,c/ml 3H-deoxycytidine, in the presence and absence of 10o6 M FUdR and10-5 M uridine. Segments were harvested at 4-hr intervals foranalysis. FUdR reduced the amount of 3H-deoxycytidine incor-

porated into DNA within 4 hr after the start of treatment (Table

Table II. Effect of FUdR oni Wound Xylem Differentiation inCultured Coleus Stem Segments

Segments were isolated on SA medium, SAIAA medium, orthese same media supplemented with 10-6 M FUdR and 10-5 Muridine. After 7 days in culture, segments were cleared andsquashed for counts of differentiated wound vessel members. Themeans are given together with the standard deviation of the means(x i t.o5 si).

Medium No. of WVMs Inhibition

SA 514 i 44 ..

SA + 10-6M FUdR 106 ± 23 80SA + 10-6 M FUdR + 10-5 M thymidine 489 i 31 5SAIAA 1877 i 147SAIAA + 10-6 M FUdR 382 i 32 79SAIAA + 10-6 M FUdR + 10-5 M 1746 4 97 7thymidine

I). A progressively greater degree of inhibition of DNA synthesisby FUdR was observed with increasing exposure time. After 12hr of incubation, FUdR had inhibited 3H-deoxycytidine incor-poration into DNA by 56%7,. FUdR did not inhibit the uptake of3H-deoxycytidine or its incorporation into RNA during this timeperiod.The addition of 10-6 M FUdR and 10' M uridine to SA or

SAIAA medium greatly reduced the numbers of WVMs formedduring the 7-day culture period. The degree of inhibition of xylemdifferentiation by FUdR was the same (approximately 80%) inboth SA- and SAIAA-grown stem segments (Table II). Whenstem segments were isolated on a culture medium containing 10-5M thymidine, as well as 10O' FUdR and 10-5 M uridine, no statisti-cally significant inhibition of xylem differentiation was observed.Thymidine completely overcame the inhibition of xylem differ-entiation due to FUdR.Time Course of Auxin Action. A lag period of 2 to 3 days was

observed before the appearance of WVMs in the cultured stemsegments. Furthermore, adding auxin to the culture medium didnot shorten the lag period. Must auxin be present during the lagperiod for the subsequent formation of WVMs? In order to deter-mine more carefully the time course of auxin action in this sys-tem, 50 j,g/liter IAA was supplied to the tissues at different timesafter the start of the culture period. Stem segments were isolatedon SA medium, and some of the segments were transferred toSAIAA medium at daily intervals thereafter. All segments wereharvested after 7 days in culture. Approximately the same num-bers of WVMs were formed in segments cultured continuously onSAIAA medium and in segments transferred to SAIAA after 1 or2 days on SA medium (Table III). In all three cases, approxi-mately five times as many WVMs were formed in the auxin-treated tissues as in the SA-grown controls. When auxin wasgiven to segments after 3 or 4 days of culture on the SA medium,its effect was less pronounced. In particular, supplying the tissueswith auxin after the 4th day of culture produced only a 2-foldincrease in WVMs. Additional time in culture, up to a total of 9days, did not significantly increase the number ofWVMs formedwhen tissues were transferred to SAIAA after 4 days in culture onthe SA medium.Although these results clearly show that auxin was most effec-

tive in stimulating xylogenesis when it was supplied during theearly part of the culture period, it is also apparent that a signifi-cant stimulation of xylem differentiation occurred when auxinwas given to the tissues after the 3rd or 4th day of culture. Thesetwo results would not be inconsistent if supplying the tissues withfresh auxin after 3 or 4 days in culture led to the formation of a

66 FOSKET

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TIME COURSE OF XYLEM DIFFERENTIATION

Table III. Effect of Auxin, When Supplied at Different Times afterTissue Isolation, upon Wound Xylem Differentiation

Stem segments were isolated on SA medium. At daily intervals,some tissues were transferred to SAIAA medium. All tissues wereharvested after a total of 7 days in culture, cleared and squashedfor counts of differentiated wound vessel members (WVM). Themeans are given together with the standard deviation of the means(i ± t.05 st).

Time in Culture on the Indicated MediumNo. of WVMs

SA SAIAA

days

0 7 2166 i 1521 6 2387 4 1582 5 2084 1133 4 1393 1314 3 843 1507 0 385± 28

18

Q)

-q)-I)Ct)

I...

6

14

12

10

8

6-

4

2

0

2 3 4 5 6 7

Days After IsolationFIG. 2. The time course of xylem differentiation in stem segments

treated with auxin after 3 days in culture on a medium lacking auxin.Segments were isolated on SA medium. At the end of the 3rd day ofculture, half of the segments were transferred to SAIAA medium. Atdaily intervals, 10 segments from each medium were harvested, cleared,and squashed for counts of differentiated wound vessel members.The mean number of wound vessel members per segment for eachdaily sample is given, together with the standard deviation of the mean(x tvors)).

new population of wound xylem elements. This possibility wastested. As is shown by the data of Figure 2, the time course ofxylem differentiation was altered when stem segments were givenauxin after 3 days in culture on SA medium. The total number ofWVMs formed in 7 days was over four times that of the SA con-trols. However, an auxin-induced increase in the number ofWMVs did not begin to be apparent until the 5th day of culture, 2

67

days after transfer to the auxin-containing medium. As previouslystated, 75% of the WVMs formed by the SA-grown tissues, and90% of the WVMs formed by the SAIAA-grown tissues, willhave appeared by the 5th day of culture. By contrast, only 34%of the total WVMs had appeared by the 5th day in segments trans-ferred to SAIAA after 3 days of culture on SA medium. Thelargest increase in the number of WVMs occurred between the5th and the 6th days of culture, 3 days after transfer to the LAA-containing medium, when 52% of the total WVM populationappeared.

Thus, giving segments auxin after 3 days of culture on amedium lacking auxin appeared to bring about the formation ofa new population of WVMs. This conclusion is supported by theresponse of these segments to FUdR. The data of Table IV showthat the addition of 106 M FUdR to segments cultured for 3 dayson either SA or SAIAA media did not reduce significantly thenumber of WVMs formed in 6 days. The addition of auxin toSA-grown segments after 3 days in culture resulted in a 3-foldincrease in the number ofWVMs formed during the 6-day cultureperiod. Whereas FUdR treatment at day 3 was ineffective inblocking WVM differentiation in segments cultured continuouslyon SA or SAIAA medium, the increment in WVMs, due to thetransfer of 3-day-old, SA-grown segments to SAIAA medium,was blocked by the simultaneous application of FUdR.While it is clear that exogenous auxin, applied at the time of

tissue isolation, had relatively little effect on DNA synthesis, it ispossible that auxin might be limiting for DNA synthesis by the3rd day of culture. In order to investigate this possibility, changesin 3H-thymidine incorporation into DNA were studied upon the

Table IV. Effect of Auxin (50 ,g/liter IAA) and FUdR (106 M)When Given 3 Days after Stem Segment Isolation

In each case, stem segments were sampled after being culturedfor a total of 6 days, either continuously on the same medium, orsequentially on different media. The means are given togetherwith the standard error of the mean (x ± t.05 Si).

Treatment No. of WVMs

SA, 6 days 390 ± 34SAIAA, 6 days 2059 ± 155SA, 3 days, -- SA + FUdR 413 ± 84SAIAA, 3 days, -* SAIAA + FUdR 1845 ± 159SA, 3 days, SAIAA 1360 ± 104SA, 3 days, SAIAA + FUdR 499 ± 31

Table V. Incorporation of 3H-Thymidine into DNA in StemSegments Transferred to an Auxin-containing Medium,after 3 Days in Culture on a Medium Lacking Auxini

Coleus stem segments were isolated and placed on SA medium.After 3 days in culture (72 hr), the segments were either transferredto SAIAA medium or continued on SA medium. For the labelingperiods indicated, the segments were incubated on a medium ofthe same composition, supplemented with 2 suc/ml 3H-thymidine.At the end of each labeling period, nucleic acids were extractedand analyzed as described in "Materials and Methods."

IncorporationLabeling Period Increase

SA medium SAIAA medium

hr after. isolaton cpmlsg DNA %

84-96 (4th day) 1722.95 1985.83 15.2108-120 (5th day) 660.94 885.41 33.9132-144 (6 h day) 364.56 532.56 46.1

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Plant Physiol. Vol. 46, 1970

transfer of SA-grown segments to SAIAA medium after the 3rdday of culture. Again, the general pattern of DNA synthetic ac-tivity was not altered by the auxin treatment. With or withoutauxin, the amount of 3H-thymidine incorporated into DNA de-clined during each subsequent labeling period (Table V). Auxintreatment did lead to increase 3H-thymidine incorporation intoDNA, however. Within 24 hr after applying IAA, the amount oflabel incorporated into DNA had increased by 15%, while themaximal difference in the amount of 3H-thymidine incorporatedoccurred 3 days after the initial application of auxin; i.e., 6 daysafter segment isolation.

DISCUSSION

The data of the present study suggest that auxin does not limitxylogenesis by regulating the rate of secondary wall deposition orlignification. In the light microscope, differentiated xylem ele-ments are recognized by the presence of a secondary wall. Oncesecondary wall deposition has begun, a cell will be counted as adifferentiated xylem element. Thus, the data of Figure 1 show thatsecondary wall deposition does not begin in some differentiatingWVMs until the 3rd day of culture, and not until the 4th or 5thday of culture in most of the differentiating WVMs. Evidencefrom both biochemical studies (19) and electron microscopy(10) has shown that lignification parallels secondary wall deposi-tion during WVM formation in cultured Coleus stem segments.Yet the data presented here clearly show that the major portionof the increase in WVMs, brought about by exogenous auxin,results from something auxin does during the early part of theculture period, before secondary wall deposition has begun. Thisis not to say that auxin cannot, or does not, regulate the rate ofsecondary wall deposition, but only that such an effect is unlikelyto account for the role of auxin as a limiting factor in xylem differ-entiation.The data of this study also do not support the hypothesis that

auxin regulates xylem differentiation by limiting DNA synthesis.It would appear that the effect of auxin on DNA synthesis is atleast an order of magnitude too small to account for the effect ofthis hormone on xylem differentiation. The addition of 50 jig/literLAA to the SA medium resulted in a 400 to 500%7, increase in thenumber of WVMs formed in 7 days. The observed increases in3H-thymidine incorporation into DNA in segments treated with50 ,g/liter LAA were comparatively small. Within the 1st 36 hrafter auxin application, these increases were on the order of 15 %.Although auxin does not appear to regulate xylogenesis

through its effect on DNA synthesis, DNA synthesis does seem tobe necessary for auxin action. In other species, FUdR has beenshown to block DNA replication by acting as a competitive inhib-itor of thymidylate synthetase (5). The fact that FUdR inhibitedxylogenesis when given to stem segments during the early part ofthe culture period and the fact that this inhibition was preventedby the simultaneous application of thymidine strongly suggestthat DNA synthesis is necessary for auxin-induced xylem differ-entiation. Since colchicine also has been shown to inhibit xylo-genesis when given to Coleus stem segments during the early partof the culture period (6), we may conclude that the completemitotic cycle, rather than DNA replication alone, is essential forthe initiation of xylem differentiation. These data would be con-

sistent with the hypothesis that cytodifferentiation is initiatedduring some phase of the mitotic cycle. The hormonal and nutri-tional environment in which division occurs would determine,according to this hypothesis, the type of specialization one or

both of the daughter cells would undertake upon the completionof mitosis (6, 23). The present study demonstrates that auxinregulates events which must occur quite early in xylogenesis. It isproposed that auxin, at a certain critical concentration, initiatesxylogenesis through an unknown effect on dividing cells.

Acknowledgments-I would like to thank Dr. J. G. Torrey, Harvard University,for the use of his facilities during the course of these investigations and for manystimulating discussions about xylogenesis.

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6. FosKET, D.E. 1968. Cell division and the diflerentiation of wound-vessel membersin cultured stem segments of Coleus. Proc. Nat. Acad. Sci. U.S.A. 59: 1089-1096. F

7. FOSKET, D. E. AND L. W. ROBERTS. 1964. Induction of wound vessel differentiationin isolated Coleus stem segments in vitro. Amer. J. Bot. 51: 19-25.

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16. MoREY, P. R. AND J. CRONSHAW. 1966. Developmental changes in the secondaryxylem of Acer rubrum induced by various auxins and 2,3,5-tri-iodobenzoicacid. Protoplasma 65: 287-313.

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