Physiological Studies on Pea Tendrils. I. Growth and Coiling ...

Plant Plhysiol. (1966 ) 41, 1014-1025

Physiological Studies on Pea Tendrils.I. Growth and Coiling Following Mechanical Stimulation

M. J. Jaffe and A. W. GalstonDepartment of Biology, Yale University, New Haven, Connecticut

Received February 4, 1966.

Summary. Unbranched tend(rils arising from the fifth node of light growni Alaskapea plants were found to reach mlaturity at the age of 10 days. Such tendrils, whenstroked, coiledI rapidly. They remained maximally irrital)le for at least 3 days.

Coiling was separable into 2 components, curvature (measured in (legrees) and elon-gation. Coiling reached a maxinium from one-half to 3 hoturs after tactile stimulation.This maxi,mum is followed by a decrease, then by a further increase up to 48 houirs.

The optimlum temnperature ranige for curvature was 16 to 300 after 2 hours and 16to 20° after 20 hours. Curvature was minimal anid elongation optimal at a pH of 6.6.Both curvature and elongation wN-ere greater in white light than in (larkness.

Severiiig the dorsal vascular bDundles decreased coiling, wthereas severing the ventralb)undles had no effect. Amputation of the tip of an excised tendril increased curvaturebut decreased elongation. In some cases, coiling of tendrils in situ was followed bycurvature of the subjacent stemii.

Various growth substances produce effects oIn elongationi and( cturvature of tendrilsin vitro, in ligh-t and dark. CCC and GA decrease curvatuire in the light and increasecurva,ture in the dark and eloi'gation in the light and(l in the dark. Kinetin decreasescurvature in the light, increases it slightly in the (lark and has Ino effect on elongation.IAA increases elongation in the light and in the dark at concentrationcs above 10-' MI.At ltower concentrations, curvatture was increase(l in the light and decreased in the (lark.

These data are discussed wTith a view towNards explaining the coiling event.

Plants with flexible stems have evolved variousimeans of supportinig their leaves albove those of theircompetitors. Among these (levices, and(i ustiallvclassed as their most highly evolved formii (4), aretendrils. Tend(rils are flexible, filiform organs, utsuI-ally modiofications of leaves or flower pedtlncles,which are thigmotropically sensitive and(I capalble ofcoiling around a support. The tendrils of moost spe-cies are, as they mature, capalble of circutimnulttation,ofteni in a direction inde,pendent of that of the stemii(4). This sweeping through space gives the tendrila better chance to contact a suupport. \N'henl a tenldrildoes totich the support. coitact coiling occurs?, duringwlhich the tendlril wraps itself arotluin( the suipport.It is this type of coiling which will be examinede inthis paper. If the support is not with(drawiv, and thetendril has coiled arouIl(l it. tendrils of mlany speciesthen form what MacDougal (13) calledI free coils,Free coiliiig is the formatioin of helical coils alongthe axis of the tendlril betwemn its attachmenlt to theplant andl its terminial, contact-coiled end. Free coil-ing always occurs after the term-inal part of the ten-dril has coiled, whether in response to contact ordtue to seniescence (16).

The Alaska pea (Pisiim sati'z,mii L.) producesboth bralnched and unbranched tendrils wlhich are notcapable of free coiling. For this simnplifying reason,

ams Nell as for the fact that plants of tendril bearn-age can be quicklI\ andl easly eroxvn, they were choseuas experiwental. material. The reoort preselte(l be-low considers the habital growth of tend(rils, the phv-sical parameters involved in contact coiling anid theeffeAts of growth sulstances onl contact coiling.

Materials and Methods

Pl(7ant Material. '1'ein(Ir;ls from light grownAlaska peas N-ere tise(l. The p)lants wvere grown invermiculite in plastic boxes which were automati'allvirrigated every 12 hours wxith a (lilute mineral solu-tion (Hvponex) (7). They were kept in trrowthroonmis at aboout 23' tin(ler a constanlt whiLe light illui-minance of about 450 ft-c provi(led by equtial numbers.of cool white and warm white flulorescent lamps withsup))lementary incan(lescenit lihting. Son-e exo)eri-irents wvere also perforn-ed with Progress No. 0, adwarf varietv of pea.

.11orphological Ob.srvations. Tendril forms oc-

cuirring at different niodes were observed. The uin-branched tendril arisinig at the fifth node was tise(dw-,hen the plant was between 10 and 12 days ol(l.\When (lark conditions vxere desired, plants were tran-sferred to a darkenle(d chamber eqluil)ped with .-Afe

101 4

JAFFE AND GALSTON-GROWTH AND COILING OF PEA TENDRILS

lights consisting of green fluorescent lamps wrappedwith 3 layers each of green and amber cellophane.The dark room temperature varied between 20 to 270,though usually from 26 to 270. For anatomicalstudies, tendrils were cooled in situ for 30 minutesat 50 and then placed in a fixative composed of 1-propanol: chloroform: acetic acid (6 :3 :1). 'hisprocedure killed and fixed the tendrils with minimumcoiling or change in dimnensions. Whole mounts andsquash mounts were observed in the fixative withthe light microscope. Standard methods of embed-ding and sectioning were not successful, due to theextrenme fragility of the organs.

Estimtiations of Growth and Curzvature. Observa-tions were made both on excised and on in situ ten-drils. For the latter procedure, all unneeded planitswere removed from the box. Five procedures wereemployed for measturement of growth and curvatureof in situ tendrils. A) Tendrils were observed andthe curvature estima,ted by eye at various times. B)Sample tendrils were removed at time zero and meas-ured; additional samples were removed and measturedwheni desired. C) Shadowgraphs were made of asingle tendril. A microscope lamp was used as alight source and the image was focused on a papercovered glass plate by an appropriate lens. D)Photos were taken of a single tendril at intervalstising a Polaroid Land Camera. E) Time lapse pho-tographs were made of coiling tendrils with a 16 mmBolex movie camera loaded with Kodak tri-X filmand controlled by a Samenco intervalometer.

In all cases, the tendril to be observed was strokedgently 5 times with a glass rod on the ventral sideat time zero. Curvature was always estimated byeye to the nearest 900. Length was measured witha millinmeter ruler. In the last 3 procedures, plani-meter mleasurements were made of enlarged tracingsof the pictures. Every treatment was replicated 3times, each replicate containing 10 tendrils.

Excised tendrils were obtained by gently pinchingthem off at their base. Curvature was estimated byeye and length was measured directly with a ruler.The tendril was then dropped into a bathing solutionin a petri dish and one of the following proceduresemployed. A) Petri dishes of 9 cm diameter con-taining 10 or 12 ml of solution were placed in shakersoscillating at about 60 cycles per minute. B) Petridishes of 5 cm diameter oontaining 5 nil of solutionwere used where shaking was impossible. The moreconfining space permitted greater contact among thetendrils and hence a greater chance for a thigmotropicresponse. In both cases, the bathing solution con-sisted of 0.5 % Tween-20 in 0.03 M potassium phos-phate buffer (pH 6.4). Except where otherwisementioned, the solution contained 0.1 M sucrose.Where the various test substances were used, thevwere added directly to the bathing solutions. Everytreatment was replicated 3 times, each replicate conl-taining 10 tendrils. In addition to the initial meas-urements, tendrils were normally measured at 2 hoturs,20 hours or both.

Topography of Coiling. In order to determinewhether length changes in the ventral or dorsal sideor both were responsible for coiling, tendrils wereexcised and a 2 to 4 mm length marked off on bothsides with fine lines of red nail polish. Cameralucida drawings were made followed by planimetermeasurements of these upper and lower lengths atzero, one-half and 2 hours after stimulation. Totest the e,ffects of growth substances on dorsal andventral elongation, similar measurements were madeon tendrils incubated in the presence of 10-4 M gib-berellin A3 (GA3) and 5 X 10-7 M indole-3-aceticacid (IAA).

In order to determine the effect of decapitationon coiling and location of areas of greatest thigmo-tropic sensitivity, 2 types of experiments were per-formed. In the first, tendrils were excised andmarked off into 4 equal zones with spots of red nailpolish. They were prepared in 3 lots having two-thirds, one-third or none of the terminal zone cut off.Length anld curvature measurements were made ofeach zone. The second experiment was performe insiitu. Tendrils were stroked on the ventral or on thedorsal side, on both sides at the same time, or on theventral side and then on the dorsal side after 10minutes. To observe the effect of vascular transporton coiling, cuts were made about 1 cm behind thetip on the ventral side, on the dorsal side or on bothsides. Each cut severed one of the vascular bundlesrunning the length of the tendril. Curvature meas-urements were made at time zero and at one-halfhour after stroking.

Effects of Ambient Factors oni Coiling. Excisedtendrils incubated in petri dishes in the light wereexposed to A) a range of pH values from 5.6 to 8.0B) sucrose and mannitol molarities from 0.1 to 0.5C) a range of temperatures from 3 to 520 and D)varlous concentrations of growth substances incluidingthe auxins IAA, 2,4-dichlorophenoxyacetic acid(2,4-D) and isatin (8) GA,, kinetin, and (2-chloro-ethyl) trimethyl ammonium chloride (CCC). Theeffects of most of the growth substances were alsotested in the dark.

Results

Tendrils of normal Alaska peas and dwarf Prog-ress No. 9 peas were equally capable of contact coil-ing. In a representative test in situ the formnercurved 1030 and the latter 145° in response to similarstimulations. All subsequent experiments were per-formed with the Alaska variety.

In order to assure uniform experimental material,unharanched tendrils from node No. 5 were used (fig1). The tendrils arising from the first 3 nodes wereless thigmotropically sensitive, while those found atthe sixth and following nodes were always branched.Figure 2 shows that elongation in situ (the sum ofthe length of all branches on a tendril) of tendrilsarising from the fourth and fifth internodes, respec-tively, levels off on the ninth an-d tenth days after

1015

PLAN-T PHYSIOLOGY

C)OKDOJKD C~1 2 3 A S 6

COC)DC)DO>C) C7 8 9 10 11 12

1.FIG. 1. Appearance of leaves alnd tendrils arising

from the (lifferent no(les of light grown Alaska pea

plants.

E

>

0

z

1-

Node at whichtendril arises

4

5

6

9 10 11

Age of plants (days)

sowing. The tendrils at the sixth initernode werebranched and their elonigation had not leveled off l)ythe thirteenth day. Tendrils arising from the fourthnode were neither as large nor as sensitive to touchas those at the fifth node; henice the latter tenidrilswere al,ways selected froil)plaIlts 10 to 13 (lays ofage, as these also showed maximuim irritability.

Selection of uniiform tendrils was coml)plicated bythe fact that snmall differenices i,n light intensity cauisedbig differences in the rate of growth of the plallts.Thus, plants irra(liated uvith 121 K ergs Cm1-2 sec-1of white light pro(luced the branched-tendril bearingnode No. 6 about a day sooner than those irradiate(dwith 96 K ergs cm-2 sec-1 (table I). Once the ten-drils had reached maturity (10 days after planting)there was little change in their thWgmotropic sensi-tivity. Table II shows that only the shorter (younger)tendrils had a significantlv retarded curvature )oten-tial, although their ability to elongate was niot af-fected. It should be notel 'that the in situ tend(lril isperfectly straight until ahbout the ninth day. Afterthat, a hook begins to develop at the tip. By aboutthe tenth day the tendclril bAegins to arch midway, alon'gits length, in a directioni opposite to that of the lhook.For convenience, we propose to call this habitaldevelopm.ent as contrasted with thigmotropic (level-opi-ent.

Table II. Correla ihon Coefficicnts of Nct Chianges inCurvature or Elongation Comiputed as a Function of

the¢ Initil! Tendril Lcngtlh

Coefficient of correlation (r) x itlh theinitial leingth of the tenidril

Parametermeasured Light Dar'k

chanige in length(mim per tendril)

Change in curvature(degrees per tend(ri')

- 0.24*

-1 o.,; 7

+ 0.24-

+ 0.20*

2.FIG. 2. The kinetics of habital tendril elongation on

the planit. For explaniation see text.

Table I. Effect (of Intens-ity of llWhite Lighlt on tileRelative Occurrcncc of Diffcrcnit Branching Patterns

of the Y'oungcst Maturc Tcndr-il

Fractioni of

tendlrils

Incideent energy inkXiloergs X cmn2 X sec-'*

96 102 108 114 121

\'ith] no braniches 0.78 0.78 0.69 0.62 0.62

With 2 brainclhes 0.06 0.10 0.11 0.08 0.11

WAitlh 3 branches 0.17 0.20 0.23 0.28 0.37

* Numl ers Inot consecutively uinderlined are significantlydifferent fromii oIne anotlher.

* Not sif-nificart.** Significanit at 1 % level.

In searching for ani acceptable fixative for ana-

tomical studies, we triedl various alcohols, both withand without acetic acid, but fotunid that they ind(utice(dcurvatuire dutiring fixatioin. Sections of tisstues treatedlwxith these reagenits were n,ot miiade. Table III showxs

thcat the loniger the carbon chain in the alcohol, thegreater is the curvature induce(d and the sloxver is thereaction to the alcohol. Acetic acidl, frequently u.iedin fixatives, reversed both these effects. Ill adclitionto the concentraticn of acetic acid shown in the table.several initernme(liate concelntraitions were also trie(l.In general, their effects fell between the 2 extremesshown in the table.

Anatomically there is little to dlistiniguish the col-cave from the colnvex si(le of the tend(ril w-heln viewvedwith the light microscoipe (fig 3 A). A\ notable

1016

1017JAFFE AND GALSTON-GROWTH AND COILING OF PEA TENDRILS

a

d

FIG. 3. Photomicrographs of killed anid fixed coiledtendrils. a) Wlhole mounit at 84 magnifications; b)squash mount at 240 magnifications, showing "clump"cells (cc) on ventral side; c) squash mount of terminalportion at 240 magnifications; d) whole niouit at 1080magnifications showiing closed (left) anid open (right)stomata; e) wN-hole mount at 1080 magnifications showingtendril tip.

JAFFE AND GALSTON-GROWTH AND COILING OF PEA TENDRILS

Table III. Effect of Acetic Acid and Chain Length of Alcohol on Curvature of Excised Tendrils

No of carbonsin normal Max change Sec to max

Treatment alcohol (Degrees curvature) change

0 acetic acid,pH 7.37 1 18 240

2 19 1803 27 2404 108 2705 135 300

32 % acetic acid, 1 351 300pH 1.88 2 261 278

3 108 604 45 385 72 30

exception is a line of cells running just beneath theconcave epidermis. These cells (fig 3 B) seem tohave their contents clumped at the end proximal tothe base of the tendiril. Vascular bundles run throuighalmost the entire length of the organ, terminating justshort of the tip (fig 3 C). The tendril, a modifiedleaflet, has functional stomata of a relatively simpletype (fig 3 D). The cells at the tip are exceptionallylong and pointed and appear extruded (fig 3 E).

Coiling of tendrils in situ in response to strokingis a rapid reaction. Figure 4 shows tracings made ofa series of still photographs and illustrates a typicalresponse pattern. Although the coil follows the formof a spiral rather than a circle, 1 complete revolutionwas estimated as 3600, and 900 increments could beeasily estimated. In Figure 4, increments of 450were estimated. Such estimations, made indepen-dently by different observers, invariably agreed towithin 450. It can be seen tha,t the tendril had curvetdappreciably within 4 minutes after stroking. Themaximum curvature was reached by 32 minutes andshowed some decrease at 64 minutes, due to continu-ing symmetrical straight growth. A maximum oflength was reached after 16 minutes, with a decreaseat 32 minutes and a further increase at 64 minutes.

Minutesoftercontact 0 2 4 8 16 32 64

Curvature (-) 225 270 315 450 495 540 495

Length (mm) 66 66 71 71 74 68 76

4.FIG. 4. Appearance of a tendril at various times after

rubbing. Arrows indicate position of stimulation by 5strokes with a glass rod. The curvature is estimated to450 and the length measured with a calibrated planimeter.

FIG. 5. Kinetics oftendrils. Each point istendrils each.

Hours (Log Scale)

5.curvature of excised and in situthe mean of 3 replications of 10

If excised or in situ tendrils were allowed to coilfor a long period of time, curva,ture reached an in-itial maximum at anywhere from 0.5 to 3 hours,decreased, and then steadily increased (fig 5).Changes in fresh weight and elongation parallel thesekinetics.

Both the dry weight and fresh weight of tendrilsin repose decrease towards the tip. The fresh weightof coiled tendrils shows a similar trend, but theirdry weight increases at the tip. When dry weight isexpressed as percent of fresh weight, there is littledifference in trend between the 2 types of tendril,but coiled tendrils have a significantly higher valuethrouighout (table IV). Because of these patterns,tendrils were routtinely assayed at 2 houirs, 20 hoursor both.An interesting phenomenon was observed when the

coiling tendrils were photographed by time lapse cine-matography. About 18 minutes after the tendrilswere stroked, and about 14 minutes after they hadstarted to coil, the stems below the node at which the

1019

Table IV. Changes itn Dry I![cight antd Freshi Weight in Various Portions of !he Tendril as a Result of CoilingEachl figure represents 3 replicates, each of which conitained 10 tenidrils.

Condition Quarter of the tendril Enitireof tendril Basal Second Third Apical SE** Tendril

Fr wt (nmg,0)

Dry vt (mg)

Dry xA-t (as % offr wt)

In reposeCoiledIn reposeCoiledIn reposeCoiled

22.725.01.82.88.111.4

15.715.71.51.99.5

12.2

11.314.71.52.112.815.4

9.715.01.62.9

17.219.4

3.22.5)0.10.22.22.0

59.669.66.49.6

47.(458.4

Chanige in weighthsd* (Iper (juarter)

4.314.8

- 0.072.9 -J 0.0+3.0+2.7

* Honestly signiificant difference at 5 %. (Steel, R. G. D. anid J. H. Torrie. 1960. Principles aind Proceduresof Statistics. McGraw--Hill Book Company. News York. 481 pp.)

** Stanidard Error.

6.FIG. 6. Appearance of a tendril and stem at approxi-

mately 2-minute intervals after rubbing the ventral sur-

face of the tendril. Tracings are abstracted from timelapse cinematographs. The horizontal mark indicatesthe node at which the tendril-bearing leaf is attachled.

tendrils were borne also started to bend, in the same

direction as the coiling. This is shown graphicallyini figure 6. It indicates rapid transmission of a

stimuluis from the tendril to the subiacent stem.If the 2 sides of the ten(dril are ruibbed separately,

the ventral (concave) side is about 3 times as thig--.otropically sensitive as the dorsal side (fig 7).

If both sides are rubbed at the same time, about one-

half of the response to ruibbing the ventral side is

lost. If, hoxwever, there is an interval of 10 minutesbetween stimuilationi of ventral aned dorsal sturfaces,none of the response is lost.

Measurement of indivi(lual sides of a curvinigtendril (table V), revealed that there was a constantexpansion of the dorsal side. At one-half hour, theventral side hadl contracted. By 2 hours it had be-guin to expand, although more slowly than the lorsalsi(le. Thus, the initial reaction involves a contrac-

tionl of the ventral surface. Subsequent coiling.however, seems to be dtle to greater expansion of theconvex dorsal side than of the concave ventral side.

The ability of a tendril to curve or elongate de-creases as the distance from the tip increases (fig 8).The results obtained fromii the third quarter are moreaccurately evaluated than those of the tip because of

300

C

0I

0)

4'~~~~~~~~~~~~~~~~J

> 100 -

'.E

c

4'

A B C D E

7.FI(;. 7. Thigmotropic sensitivity of dorsal and x-eni-

tral surfaces. Vertical lines indicate the magnitude of thestanidard error of the meani. A) unrubbed control; B)rubbed on ventral side only; C) rubbed on dorsal sideonly; D) dorsal and ventral sides rubbed simultaneously;E) ruibbed on dorsal side 10 illilintes after ruibbillg onventral side.

1020 PLANT PHYSIOLOGY

JAFFE AND GALSTON-GROWTHI AND COILING OF PEA TENDRILS

Table V. Changcs in Length of D)rsal,nd Vcntral Sides of C(;iltng TendrilsEach (latuIml is folloxN ed by its standard error.

Mea.,urmeient

Chanige in total cur'vature ot

te:nril (ceg i-ees)Change in leiigthi (mm) of:

Venitral sideDorsal sidle

Difference (mm) between ventraland (lorsal sides

0-'2 hrs

176 + 24

-0.12 -+- 0.100.18 ± 0.15

0.30 + 0.12

base 2d 3d tip ba

Quarter of tendril

8.Fi(;. 8. The effect of decapitation of various lengths

oni cuirvatulre and elongation during coiling of excisedtendlrils. For explaniation see text.

300

41

E0

410

.;_

0c

c

i-o

0

U)

the physical effect of amputationi. As the length ofthe amputated regioni increases, the ability to elon-gate decreases. However, tendrils that had onle-thirdof the tip removed curvedl more thani intact ones.

Those that had two-thir;ds of the tip remove(d occu-

pied an intermediate position. This effect of almpu-tation was so pronounced that even in the ternminalquarter, the tendrils which had had onie-third of thetip removed curved about 25 % more than the intactones.

Notching was employed in an attempt to sever

vascular bundles on one or 1both sides of the tendril(fig 9). Notching both sides caused a severe de-crease in curvature, probably due to the traumaticeffect of severe imiutilationi. Notching the venitral

410

0

41

0

0)

->

u

._

c

A B C D

9.FIG. 9. The effect of dorsal and/or ventral notching

oni curvature during coiling of tendrils in situ. A)notclhed conitrol; B) ventral side nlotched; C) dorsal

si(le notched; D) botlh sides notched.

RI,I,

I'

I

Il

Il

I

I

O-O 2 hours

O* -4 20 hours

0 3 10 16 20 30 37 52

Degrees Centigrade

10.FIG. 10. The effect Cf temp)erature oni curvatuire dur-

ing coiling of excised tendrils.

1021

Period0-2 hrs

324 -+ 42

0.17 -+ 0.040.57 --_ 0.08

0.40 -+- 0.09

V2-2 hrs148 -± 51

0.29 ± 0.130.39 + 0.17

0.10 ± 0.07

1022 PLANT

side had no effect, but notching the dorsal side de-creased curvature by about 25 %. This indicatesdecreased transport of a growth stimulatory materialdown the dorsal side.

Both curvature and elongation are sensitive totemperature, especially the former. Figure 10 showsthat curvature has a rather narrow optimum rangeof about 15 to 25°. At 2 hours, the maximum wasat 300, but at 20 hours there was very little curvatureat that temperature. The Qlo from 10 to 200 wasbetween 5 and 6 at both 2 and 20 hours.

When the effect of pH was tested (figure 11), itwas found that elongation is maximal and curvatureminimal at pH 6.6. A study of the effects of variousconcentrations of sucrose and mannitol after 20

PHYSIOLOGY

hours incubation (fig 12) showed that the higherthe concentration of the solute, the less is the curva-ture and elongation. The experiments with the 2substances were performed at different times, ac-counting for the different control (0.0 m) data ob-

E4

800[

Di

10,

8

A6410

E=

_

200F 21

5

~~~~ %00~~~~~~~~1.%

00~~~~~~~1

*----. Elongation*- *-e Curvature

6 7 8

pH

11.

FIG. 11. The effect of pH on curvature and elonga-tion of excised tendrils after 20 hours.

0.0 0.2 0.4 0.0 0.2

M. Mannitol M. Sucrose

12.FIG. 12. The effects of sucrose

curvature and elongationl of excisedhotirs.

and mannitol on

tendrils after 20

3,

33

30

\ ~ 27

24

is

0.4

1400-

goo

iX4

1C04 IM. CCC

M. Kinetin

.4G,/

E

M. GA

M. IAA

F

j0 107 10,o * IO5 10-4 0 10-w 10-6 IO'S lo 4

M. 2,4-D M. Isatin

FIG. 13. Dosage responses of coiling and curvatureto selected growth substances incubated for 20 hours in

the light and in the dark. curvature;- elongation; Q, light; 0 dark.

WI 600

o 400

' °°r

PF

E

c

.C

JAFFE AND GALSTON-TGROWTH AND COILING OF PEA TENDRILS

Table VI. Effect of Growth Substancts on Dorsal and Ventral Lenqth Changes of Coiiing Tendrils

1023

Each datum is followed by its standard error.

Change from untreated control in I/2 hourMeasurement 5 X 10-7 Ai IAA 10-4 M GALength of ventral side (mm) + 0.27 + 0.10 + 0.00 ± 0.11Length of dorsal side (mm) + 0.23 + 0.14 + 0.21 ± 0.07

served between them. Similar trends were found formannitol at 2 hours.

Growth substances yielded complex results. CCCdecreased growth in the light and in the dark, espe-cially in the concentration range from 10-3 M to10-2 M (fig 13 A). 10-3 M was stimulatory in thedark but was part of a general decrease with increas-ing concentration in the light. Figure 13 B showsthat as the concentration of GA increased, elongationincreased in the light and in the dark, whereas curva-ture increased in the dark and decreased markedlyin the light. There was no effect of kinetin on elon-gation (fig 13 C) but in its presence curvatture de-creased in the light and increased slightly in thedark. Both curvature and elongation showed a gen-erally increasing trend in response to increasing levelsof IAA (fig 13 D). There was, however, one verypronounced anomalous effect at 5 X 10-7 M on curv-ature. In the dark, this was a minimum value whereasin the light it was a maximum. At the highest con-centrations of IAA, tendrils shaken in the light be-came very soft and plastic. Figures 13 E and 13 Fshow that 2,4-D generally increased coiling in thelight, whereas isatin had no effect on elongation butproduced an optimum curvature between 10-7 M and10-6 M.

IAA had no differential effect on elongation ofdorsal and ventral surfaces after one-half hour ofincutbation (table VI). GA, however, producedmore elongation on the dorsal side than on the ventralside. Both growth substances increased the totallength of the excised tendril.

Discussion

The work described above emphasizes the separa-bility of elongation and curvature of tendrils. Be-cause of this separability, it is possible to conceive ofcoiling as resulting from a change in the distributionof growth between dorsal and ventral surfaces. Inhabital development, the rates of expansion of thedorsal and ventral surfaces are approximately equal.In the early contraction stage of contact coiling,dorsal elongation proceeds, while the ventral sidecontracts. In the later phase of contact coiling, therate of elongation of the dorsal side is constantlygreater than that of the ventral side. It would beinteresting to know if the rate of dorsal elongationduring contact coiling is the same as that of bothsides during habital growth. The curvature itself isof 2 consecutive types (4, 5, 14). In the first, assuggested by Gray (10), the dorsal side elongates

but the ventral side contracts. This phase, whichwe propose to call the contraction phase, results inthe coiling of the tendril about the support. It isfollowed by what we may call the growth phase, inwhich both surfaces grow at a steady rate, with thatof the dorsal side exceeding that of the ventral side.Fitting (6) has shlown with tendrils of various spe-cies that acceleration of dorsal elongation is veryrapid, almost instantaneous, whereas acceleration ofventral elongation following contraction is slower.Similarly, deceleration of elongation is slower on theventral side than on the dorsal side. In agreementwith Fitting, the present data show that ventral con-traction is observable up to one-half hour after coil-ing has begun.

Although considerable attention has been paid tothe habital elongation 'of tendrils (6, 13), surprisinglylittle has been paid to the elongation 'parameter ofcontact coiling (e.g. thigmotropic elongation). Bothauthors mentioned above found that habital elonga-tion is largely confined to the basal portion of theorgan. If the elongation that occurs during contactcoiling is measured, however, it is found that mostof it occurs toward the apical part of the tendril (9).Zeltner (20) found that elongation decreases as therate of coiling decreases. We intend to study thisfurther.

Exogenous sucrose does not seem to be necessaryfor either curvature or elongation; indeed, the addi-tion of sucrose even decreases coiling. This decreaseseenms to 'be osmotic; this may be inferred by niotingthat mannitol, a substance which is metabolicallyinert in many planits (17), produces much the sameeffect.

Sixteen to 200 are adequate temperatures for curv-ature. Thirty degrees is the optimal temperaturefor curvature at 2 hours but is debilitating after 20hours. This may possibly be explained by postu-lating a temperature-sensitive mechanism, possiblyan enzyme system, which is responsible for curvature.This mechanism, is brought to maximuim activity in ashort time at 30° but is inactivated by prolongedexposture to that temperatuire. Such a mechanism is,at present, only hypothetical.

The notching experiment indicates that a sub-stance involved in coiling is translocated along thedorsal side shortly after rubbing. From the resultsof the decapitation experiments, this undefined prin-ciple appears to be produced in the apical sixth of thetendril. The symmetrical exogenous application ofauxin in our experiments yielded no increase in curv-ature, but did increase elongation. Thus, if auxin is

1024

causally involved in curvatuire, it mutst b)e asymmet-

rically (Iistril)tuted in a ten(lril following stimuilation.Boresch (2) qtioted by B1rgstrom (3), reporte(d thatunilateral dorsal application of IAA caused coiling.This would indicate that transport of IAA across thetendril occurs only slowly; thus the failure of syvi-nmetrically applied IAA to elicit curvature is utnder-standable exven if unilateral auxin accumulation in thestimulate(l tenlclril normiially occurs. GA, on the otherhandl, does produce a differential enhanicemenit ofdorsal andl ventral elongationi. Thus exogenous GAcanl incluce curvature, even when alpplied symiimetrica-ally (table V'I). From these resulits it would appearthat it nmaylbe mnore directly relatedl to coiling thanIAA.

Further evidence for the existence of a translo-

cate(l principle is seen in the bendinig of the steilm 20minutes or so after the tendrils were rubl)edl. In thiscase, the principle might be the samie as that involvedin teli(Iril coiling. If so, its rate of translocation to

the stem is slowved (lowil, possibly at the nodle. It

might also be a seconcl principle, wllose production or

movenent is triggere(l by the first.Altlloigh rlibbing the dorsal sidle does not in itself

cause coilillg, and rubbing the ventral side does, theformer treatimlent does prevent coiling. A simlilarresult was obtained by Fitting (6) with teindrils of4 species. He rubbed the dorsal surface for a shortdistance aild the veltral surface over a long distance,overlapping the other. He found that the portionof the tendr-il rubbed on both sitles remained straight,whereas coiling occuirred C)I both sides of it. Theseobservations indicate that both surfaces of the ten-dril are capable of receiving the touch stimtilts, butthat the events following this reception are different,depending on which side is stroked. The nature ofthis difference should be examined.

The physical receptors of the touch stiimulus are

unknown. We have as yet been unable to demiion-strate specialized epidermal cells such as those foundin Eccrcmtocarpus sc(abcr (18), the tactile pits ofBrvonia (lioica (1, 19), or even differential dorsi-ventral surface corrugation as noted by Dastur anidKanga ( 12) in Vitis zvinifcr(a. That there is an histo-logical (lifference between the dorsal andl venitralsidles, however, is evidenced by the presence of the"clump" cells occurring along the irritail)le portionlsof the ventral sulbepidermal tissue. It is unlikelythat these cells are initial thigmloreceptors, becauseof their sulbepidermal locaition. It is possible thatthese cells serve as a "sink" for water leaving theventral epidermal cells during the contraction lihase.That coilinig results froml changes in ventral cell sizehas bleen claime(l by MacDotigal (14). This wouldinl)ly differential dorsi-veiltral membrane character-istics, a possibility which is reinforced by the resultsof our experiments with alcohols of various chainlengths. At pH 7.37, the time necessary to achieveillaximulli curvature increased somewhat as the niole-cules increased in lengtVh. This can be interpretedas a consequence of iilcreasillg pelletration time. Atthis pH, the larger mlolecules causedl more curvature.

PH YSIOLOGY

Both parameters were reversedl at a lower pH. Thereasons for these l)henomlena are obscure at l)resenlt,but the decrease in curvature with increasiig p)Hseemus (lefinitely to he a functioin of pH rather thai-iacetic aci(l per se (fig 11) This is so because in-creasing the pH in aquleous btiffer solutionl providesthe saime effect. A possible interpretatioll of thi.spIhenomllenon miay lbe that increasing the )1 I ten(lsto lirect growth activity towar(l on-ation r-ltherthan toward curvature.

The fact that 6.6 is the critical pH -fcor 1oth eloni-gation and c,turvature may indicate that b1th are con-sequenices of the same mechanism, which n-,ay actalong 2 different paths. Varying the pH inicreasesone of these paths at the expense of the other. Suicha "mirror-imiiage" phenomenon can also be obYervedif GA, CCC, or kinetini concentrationi is Xvar-ied inthe light.

Galun (9) has treated cucumlniber ten(lrils wxith G-Xan(l IAA and founcl that over a relatively narrowxrange IAA calise(l coiling btit Ino elongationi, wxhereasGA catlse(l elongation buit no coiling. Pea teindril(lo not show this (lifferential response.

The effects of the growth substances are nioteasily sortedl out. In addition to the one miienltionie(dabove, 2 others stand( out. The first involves IAA.Elongation increases in the light and in the (lark asthe dosage increases, but curvature reaches a maxi-mum in the light anid a minimumni in the d(ark at 5 X10-7 1. \With the tendrils incutbated in the light.curvature decreases at 10 - MI anld thenl reaches alnoptimumll againi at 5 X 10- M. Such dual miiaximla,while not co,mmon, have been nioted p)reviously (A.NV. Galston, unpu,blished (lata). It renmains to heseen whether or inot they represent activity at 2 sites.

\Ve have note(d that at higher IAA conceintrationis,tendrils incurbated in the light became extremvelV plas-tic. This effect did not occur upon treatmenit xvitheither isatin or 2,4-D, but was so strikinig in the caseof IAA that the tendril system seems to be an excel-lent source of material for investigating the effectsof auixin on cell wall extensibility.

Another effect of special interest is notel1 wNithGA and CCC. Except for the highest (and perhal)ptoxic) concentrationi of CCC. both of these comii-pounds show decreasing curvature in the liglht andlincreasing curvature in the dark. They 1)oth showgenerally inocreasing elongationi in both light and (lark-ness. It is unexpected that CCC, a compound(i sup-posedly able to stop the biosynithesis of GA (15),should produce the same sort of coilinig patterns as

does GA. This is interesting in light of the fact thatl-(linmethyl amino succinamic acid, a compound xvithactivity similar to that of CCC, is ab,le to conitroltendril formation in cucumbers (11). It is also ofinterest that GA decreases curvature in the light, butincreases it in the darkness.

In figuire 13, it can be seen that where I10 a(ldeIndaNwere present, 1)oth l)arameters of coiling vere greaterin light inculbated than in' dark incubate(l tend(Irils.When these factors alonie were comllpared. it \\asfound that curvature in the light was X730( a

JAFFE AND GALSTON-GROW'TH AND COILING OF PEA TENDRILS

stan(lard error of 44° and in the dark was 5160750. Elongation in the light was 13.0 mmn+ 0.6 mmanid was 11.0 0.8 nmm in the dark. Thus whiteliglht definitely accelerates coiling. The mechanismof this effect will be exploreed in another paper inthis series.

Acknowledgments

We acknowledge the generous support of the NationalScience Foundation through a grant to the second author.We are grateful to Dr. Raphael Goren for numerousstimulating discussions, and to Miss Sally Seiael forexl)ert technical assistance during her tenure of a NationalScience Fouindation summer research grant.

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