Plant Physiol. (1984) 74, 272-2770032-0889/84/74/0272/06/$0 1.00/0

Is There a Role for the Apex in Shoot Geotropism?Received for publication March 28, 1983 and in revised form August 2, 1983

JAMES W. HART AND IAN R. MACDONALD*Department ofBotany, University ofAberdeen, Aberdeen AB9 2UD, Scotland (J. W.H.) and The MacaulayInstitutefor Soil Research, Craigiebuckler, Aberdeen AB9 2QJ, Scotland (I.R.M.)

ABSTRACI

Experiments with horizontal etiolated sunflower (Helianthus annuusL.) seedlings supported centrally such that both apical and basal endsare free to react to geostimulus, revealed that the apical end commencescurvature I to 2 hours earlier than the basal end. The later curvature inthe basal region is a consequence of the absence of growth in the initialperiod rather than merely slower growth. A comparison of zonal growthrates in a vertical and a horizontal seedling confirmed that geostimulusinduces a renewal of growth in a region where growth had ceased.Removing the apical half of the hypocotyl showed that the curvatureresulting from this growth initiation in the basal region is dependent onattachment to the apical region. Evidence that this dependence is unlikelyto be due to energy deficiency is adduced. The prior response of the apicalend to geostimulus and the apically dependent later initiation of newgrowth in the basal region are compatible with the delay inherent inmessage transport from apex to base and are considered as evidence forapical involvement in the totality of the seedling's georesponse.

cussing geotropism in the second edition (1978) oftheir textbook(15) said 'there is transmission ofa growth-regulating factor fromthe region of perception to the region of response,' in the thirdedition (1981) they wrote 'gravitropism in shoots does not in-volve the longitudinal signal transduction mechanism which formany years has been frequently assumed to occur' (16).While in many respects the emphases of Digby and Firn

constitute a helpful corrective to the too ready acceptance ofassumptions implicit in the Cholodny-Went theory, we feel thereis now the danger ofsome valid elements in the older work beingdiscarded. Specifically, the claim that the apex has no specialrole (as distinct from its normal growth-sustaining role) in geo-response, involves the repudiation of the concept of apical co-ordination of directional growth.Our previous work (13, 14) has emphasized that curvature

involves growth stimulation as well as inhibition. Moreover, thepolarity which we have discerned in the responses of the hypo-cotyl to light (13) and to gravity (14), suggests a contribution ofthe apical region to directional growth. In this paper, we re-examine the question of apical involvement in hypocotyl geo-tropism.

The concept of a hormonal flow from the apex as the basis ofgrowth regulation derives from the classical experiments ofCharles Darwin who postulated the transmission ofan 'influence'to explain his finding that the region of perception in grasscoleoptiles was separable from the region of response to a direc-tional stimulus (2). Further refinements of this concept of atransmissible influence led eventually to the Cholodny-Wenthypothesis which explained growth curvature in terms of theformation of a lateral gradient of auxin (17).

This hypothesis has now been seriously challenged on severalgrounds by Digby and Firn. First, they have argued (3, 9) thatnot only is there no convincing experimental evidence for lateralauxin transmission sufficient to account for differential growthin geocurvature, but additionally, since curvature arises fromdifferential growth of upper and lower peripheral cell layers, as,for example, in a semicylindrical section, there is no need topostulate auxin transmission across the hypocotyl (5). Second,they showed (4) that the feature common to all geostimulatedhypocotyls examined was a cessation of growth on the uppersurface, a finding which they argued eliminates the need for aflow of a growth promotor. They subsequently argued thatbecause not only georesponse, but also geoperception, is evidentalong the entire length of the growing axis, there is neitherevidence for, nor need of, a longitudinal transmission of ahormonal message (7). Finally the observation that the timecourse of geocurvature in normal and decapitated seedlings issimilar, led them to assert that 'the apex plays no special role ingeotropism' (8).

This sustained attack on the traditional view has not beenwithout effect. For instance, whereas Wareing and Phillips dis-

MATERIALS AND METHODS

Sunflower seeds (Helianthus annuus L.) were germinated atroom temperature (22°C) in the dark on moist filter paper andafter 3 d, by which time the radical had emerged to a length of5 mm, individual seedlings were transferred to disposable cu-vettes (1 x 1 x 2.5 cm) containing 1% agar. Two d later, theplumular hooks were well formed and the hypocotyls had at-tained a length of 1.5 cm. Etiolated seedlings ranging in lengthfrom 1.5 (5 d) to 3 cm (7 d) were selected for experiments. Theseedlings were kept in the dark, all necessary manipulationsbeing undertaken with green safe-light irradiation from an 8-wAtlas warm white fluorescent tube filtered through three layersof Rank-Strand Cinemoid film (No. 39).The growth of the seedlings was followed by time lapse pho-

tography as previously described (12). The seedlings were grownin the dark, photographs being taken at 5-min intervals using aflash positioned 2.5 cm from the growth chamber window (12)located above the seedlings. Both this window and the flashincorporated a double layer ofRank-Strand green Cinemoid film(No. 39). Seedlings given extended exposure to this irradiationlevel gave no evidence of phototropic curvature. The growth rateof different regions of the hypocotyl as a function of time wasfollowed by the use of resin beads to mark a series of approxi-mately 1.5-mm zones down the hypocotyl (12). The beads wereAmberlite Resin IR-105(H) (Rohm and Haas Co., Philadelphia)sieved through 25 to 35 mesh (400-500 ,m), the H having beenexchanged with Ca. White adhesive tape was attached to the seedcoat where necessary in order to provide a suitable backgroundto distinguish the beads.

In order to compare the curvature of apical and basal ends ofhorizontal hypocotyls, seedlings were removed with the agar

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THE APEX AND SHOOT GEOTROPISM

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FIG. 1. Time lapse sequences of photographs of etiolated sunflower seedlings undergoing geotropic curvature in the dark. The seedlings weresupported near the center of the hyocotyl and were placed horizontal at 10.10 (top lefthand frame). Subsequent frames show curvature extendingover a 22-h period. The grid lines in the background are 1 cm apart.

Table I. Time at Which the First Visible Curvature Response Occurredat the Apical and Basal Ends ofEtiolated Sunflower Hypocotyls

Centrally Pegged in a Horizontal PlaneThe seedlings are those for which photographs illustrative of the

curvature are shown in Figure 1. Photographs covering the time intervalsshown in this table are to be found in Figure 1 (zero time: 10.10, 11.30,and 12.30).

Time When First Visible Upward CurvatureSeedling Horizontal Apical end Basal end

Uppermost 10.10 10.35 12.25Middle 10.10 10.30 11.15Lowermost 10.10 10.35 11.35

block, from the cuvettes. The agar around the root was trimmedto leave a minimal amount to maintain the moisture content ofthe seedling. Three seedlings were then positioned, in slits acrossthe narrow edge of a wedge-shaped piece of expanded polysty-rene. Each slit opened into a circular hole of diameter commen-surate with that of the seedling hypocotyl. The seedlings werecarefully eased into these holes and adjusted to ensure that theywere held at the desired point of the hypocotyl. Resin beads wereplaced at intervals on the prospective lower side of the hypocotylto allow more exact observations to be made on the commence-ment of upward curvature. The polystyrene wedge was placed in

the growth chamber and the seedlings kept vertical until zerotime.

Although the data reported here refer only to the experimentsillustrated in this paper, the experiments themselves have beenrepeated many times and the results confirmed.

RESULTSA Comparison of the Commencement of Curvature at the

Apical and Basal Ends of the Sunflower Hypocotyl. Figure 1illustrates the time course of geocurvature in three randomlyselected seedlings. It is evident that pegging the hypocotyl in thecenter region resulted in a georesponse being exhibited at bothends ofthe hypocotyl so that eventually the hypocotyl took on aU shape. Clearly, the apical end achieved the vertical soonerthan the basal end. This is a consequence not just of the fastergrowth of the apical end but also of the earlier commencementof response at the apical end. This latter point is brought out inTable I which gives the time (to the nearest 5 min) at which thefirst visible movement occurred at each end of the hypocotylsshown in Figure 1. The apical end of each of the plants com-menced curvature about 20 min (+5 min) after being put hori-zontal, whereas the basal end showed no movement until 1 to 2h later.The Effect of Geostimulus on the Growth and Curvature of

Apical and Basal Regions of the Sunflower Hypocotyl. Two

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HART AND MACDONALD

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(f) are following 2, 3, 5, 8, and 11I h of growth, respectively. Resin beads have been placed on the seedlings to mark growth zones. The grid lines in

the background are cm apart.

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THE APEX AND SHOOT GEOTROPISM

Table II. A Comparison ofGrowth Rates ofSuccessive ZonesfromApex to Base ofa Vertical and a Horizontal Etiolated Sunflower

SeedlingGrowth rate is expressed as um/h calculated from the overall growth

during the II-h period illustrated in Figure 2.

Vertical seedling Horizontal seedlingZone

Left side Right side Lower side Upper side

Mm/hI (apical) 148 168 124 1162 148 124 117 1503 124 128 140 1604 80 96 144 1725 92 92 132 1526 88 68 110 1217 64 60 136 1218 36 40 112 1169 12 12 100 10410 a a 104 7611 a a 84 6012 a a 88 4413 a a 100 a14 a a 84 a15 a a 76 a16 a a 52 a17 a a a a18 (basal) a a a aTotal 792 788 1703 1392a No measurable growth.

matching sunflower seedlings were selected and the hypocotylsdelimited into an equal number of zones with resin beads. Oneseedling was placed horizontal and the other left vertical. Growthwas filmed over the following 11 h and selected frames are shownin Figure 2. A comparison of the growth of the two seedlings

shows that growth and curvature in a seedling experiencinggeostimulus extends to include a region which has ceased elon-gating in a vertical seedling. From the growth rate data given inTable II, it can be seen that growth is confined to the upper halfof the vertical seedling. In the horizontal seedling, all but the twomost basal zones resume growth on the lower side and the upperside also shows a resumption of growth at a more basal level.Although the mean growth rate of the upper side of a horizontalseedling during the 11 h period considered in Table II, shows astimulation over the vertical growth rate, sunflower hypocotylshave similar growth kinetics to that of cress and cucumber (14)in that inhibition ofgrowth occurs along the upper surface duringthe first 1 to 2 h of being horizontal and thereafter the inhibitionis progressively replaced by a stimulation of growth movingbasipetally along the hypocotyl.The Dependence of the Basal Region of the Sunflower Hypo-

cotyl on the Apical Region for Georesponse. Uniform sunflowerseedlings were selected and some decapitated at the midpoint ofthe hypocotyl before being placed horizontal for 24 h. Figure 3shows a typical result of such an experiment with two matchedseedlings before and after geocurvature. Removing the apicalhalf of the hypocotyl eliminated a georesponse from a regionwhich achieves a 900 curvature when in contact with the apicalhalf. Clearly, the basal region is dependent on the apical regionfor georesponse. That it is not a dependence on energy supplyfrom the cotyledons can be deduced from Figure 4 which illus-trates a sunflower hypocotyl severed at the base ofthe cotyledonsand at the hypocotyl base and then sectioned into thirds. Thesesections were inserted through holes ofappropriate diameter intoa polystyrene dish containing agar and left horizontal for 24 h.It can be seen from Figure 4 that the top third of the hypocotylwhen detached from the whole plant can undergo geocurvatureas can the middle third, although to a significantly lesser extent.No curvature is evident in the lower third.

DISCUSSIONAlthough seedling growth is normally dependent on the pres-

ence of the cotyledons and apex, the growth of a sunflower

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FIG. 3. A comparison of geocurvature in an intact etiolated sunflower seedling and one decapitated at the midpoint of the hypocotyl. Twomatching seedlings before (a) and following (b) decapitation of one. A resin bead marks the midpoint of each seedling at zero time. (c) The twoseedlings 24 h later. Note the extent of curvature in the basal half of the whole seedling. The arrow indicates the bead placed at the midpoint of thecontrol plant at zero time.

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HART AND MACDONALD

FIG. 4. A comparison of geocurvature in an etiolated sunflower hy-pocotyl sectioned into three equal segments. The sections were insertedinto holes in a polystyrene dish containing agar and left horizontal for24 h. The upper segment is the apical third and the lower segment thebasal third. The grid lines in the background are I cm apart.

Table III. A Comparison ofthe Increase in Length ofFour EqualZones ofthe Hypocotyl ofa Vertical and Horizontal Sunflower Seedling

over 11 HoursThe data relate to the seedlings illustrated in Figure 2.

Vertical Seedling Horizontal SeedlingQuarter Zero After Zero After

time I Ih Increase time ll h Increase

length in mm % length in mm %Apical 8.4 15.1 80 8.5 15.8 86Second 8.4 12.1 44 8.5 15.8 86Third 8.4 8.6 2 8.5 14.0 65Basal 8.4 8.4 0 8.5 9.2 8

Total 33.6 44.2 32 34.0 54.8 61

hypocotyl is not immediately affected by severance of the coty-ledons. Firn et al. (8) have shown that removing the cotyledonsis apparently without effect on geocurvature of the decapitatedseedling and, since this curvature occurs in 2 h, energy supplyfrom the cotyledons would not be expected to be critical. In thissense geotropism is unaffected by decapitation. Whether or notthe basal regions of the hypocotyl are independent of the apical-subapical region in their response to geostimulus is more difficultto decide.

In seeking to establish whether or not the apical region has aspecial role in the regulation of geocurvature throughout the

hypocotyl, two questions may be asked. First, is there a differencein the time at which tropic responses first appear in the apicaland basal regions of the hypocotyl? And, second, if the basalregion shows a later response, is this simply a consequence ofthe slower growth of the region or is it a consequence of thedelay inherent in the transport of a message from the apex? It isthis concept of message transport which recently has receivedsuch emphatic rejection (7-9). Cited as evidence against messagetransport have been reports (9) that the differential growth re-sponsible for curvature begins simultaneously and uniformlyalong the whole growing length of the organ: such claims havebeen based on measurements of segmental growth rates (4, 9)and on experiments in which a horizontal hypocotyl restrainedby some means 'about halfway along its growing axis, curvesupwards at both the apical and basal ends' (8).

Experiments already reported (14) bearing on the first of thesequestions indicate a distinct polarity from apex to base in theattainment of maximum differential growth during geocurvaturein cucumber hypocotyl, a feature which, although not conclusive,is at least suggestive of prior events in the apical region. Theresults now reported from the centrally pivoted sunflower hy-pocotyls confirm and extend that finding in that they not onlyshow that the angle of curvature subtended by the basal half ofthe seedlings increases more rapidly in the second 2-h period ofcurvature compared with the earlier period (a consequence ofincreasing differential growth) but, more importantly, they showthat the apical end starts bending before the basal end andcurvature continues at the apical end for a considerable period(1-2 h depending on the position of the medial restraint) beforeany movement ofthe basal end is apparent. This strongly suggeststhat such behavior is not to be attributed merely to the fastergrowth rate of the apical end, but has to be seen as representinga real difference in the time of initiation of events leading tocurvature at different positions along the hypocotyl. The positionof medial restraint is obviously critical. Ifthe restraint is imposedtoo near the basal end, growth may fail to be established to anextent sufficient to raise the end significantly. Conversely, whenplaced slightly above the critical point (i.e., 'about halfway alongits growing axis'?), the two ends do indeed show simultaneousmovement albeit with different rates of bending. But the factthat a position can be determined at which the behavior of thetwo ends is markedly distinct must indicate temporal separationof events along the hypocotyl.The critical nature ofthe point at which the seedling is pivoted

is evident from the consideration that, ifpivoted at a point wheredifferential growth is occurring both to the left and to the right,each end must show some movement. The fact that the basalend shows no movement for a longer or shorter period indicatesthat there is a renewal of growth in a nongrowing region. Thatthis is a new event induced by the geostimulus is made clear inTable II in which it can be seen that substantial growth is initiatedon the lower side of the nongrowing region (zones 9-16) andeven extends to include some zones (zones 9-12) on the upperside. Equivalent zones in plants kept vertical show no renewal ofgrowth, strongly suggesting that the growth is a consequence ofthe geostimulus experienced by the hypocotyl. This point is mademore explicit in Table III which gives the growth increase over11 h of the hypocotyl (considered as four equal sections) of thevertical and horizontal seedlings ofFigure 2. Although a resump-tion of growth following geostimulation has already been re-ported in the case of nodes of grass stems (1), it has not hithertobeen reported for dicotyledonous stems.The other question central to the debate is whether the later

response in the basal region is a consequence of slower growthor ofthe delay inherent in message transport and here two thingsmay be said. First, the evidence indicates a total absence ofgrowth initially in the region under discussion. Second, the lag

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THE APEX AND SHOOT GEOTROPISM

time of 1 to 2 h involved in achieving the growth responsesuggests dependence of this region on a compound flowing fromthe apex. If it is assumed that the necessary growth factors arecontinually flowing from the apex so that the basal cells are in aconstant state of readiness to resume growth in response to a re-orientation signal experienced directly by these cells, it wouldnot be expected that a lag time would be interposed. That thereis such a lag is compatible with apical involvement.However it could be argued that a nongrowing region, although

entirely self-sufficient in respect to geoperception and geore-sponse, nevertheless requires a longer period of time to giveexpression to its responsiveness, the resumption ofgrowth takinglonger than the acceleration of growth. But if such were the case,the basal half ofthe hypocotyl should ultimately show significantcurvature in the absence of the apical half and its failure to doso (Fig. 3) is clear evidence of dependence on a contributionfrom the apical region. That this contribution is not likely to beenergy supply is indicated by the fact that regions other than thebasal one can still undergo curvature when isolated from thecotyledons or any other part of the seedling (Fig. 4). Furtherevidence that the limiting factor is unlikely to be energy supplywas obtained from an analysis of sugars present in 4-mm zonesfrom the apex of the hypocotyl downwards. This showed totalethanol-soluble sugars to be present in all zones at a concentra-tion of approximately 26 mg/g fresh weight of tissue, confirmingthe nonexistence of a gradient of easily utilizable carbohydratedown the hypocotyl.

In the explanation of tropic movements, major emphasis hasrecently been placed upon the growth inhibition which is quickly,perhaps even uniformly, imposed along the length ofthe concaveside of the curving organ (4, 10). However, we have alreadysuggested that 'the' georesponse involves two distinct and argu-ably separate, types of growth reaction, viz. inhibition and stim-ulation (14). These phenomena ofgrowth stimulation and growthinhibition, while obviously co-ordinated in the sense of bothcontributing to the overall orientation process, need not representopposite actions ofa single mechanism, e.g. presence and absenceof a growth promoter. Indeed, inhibition and stimulation duringcurvature do seem to differ in their timings and in their locationsalong the hypocotyl, with growth inhibition being the moreubiquitous response (4). Thus, in agreement with the conceptsofFim et al. (8) and Franssen et al. (I 1), cells in growing regionsofan organ may themselves perceive a change in orientation andmay respond by ceasing growth. It could be further conjecturedthat a general response of growing cells to substantial environ-mental change, whether of orientation, light regime, or evenphysical perturbation, may be a (temporary) cessation ofgrowth.Since this is a response of any growing cell, this stage of tropiccurvature need not involve a transmitted message. However, thisaspect of perception and response is only one part of the overall

re-orientation process. The evidence presented in this reportindicates that growth stimulation, originating in the apical/sub-apical region and developing subsequently in the more basalregions, is an integral part of the response.

This of course is not to be taken as implying that the basalregion is not itself involved, to some degree, in geoperceptionand response. If, as the evidence of this paper suggests, the basalregion is ultimately dependent on the transmission of a growthfactor from the apical region, that transport may itself be trig-gered by a signal transmitted by the basal region. The point westress here is not so much the interdependence of one regionwith another as the functional dependence ofthe basal region onthe apical region in its overall response to gravity. The fact thata polarity, indicative of an apical role, is observable (6, 14) inautotropism-another manifestation of directional growth-it-self suggests that geotropic growth will be subject to similarregulation.

Acknowledgment-We acknowledge the assistance of the Photographic Unit ofthe Macaulay Institute.

LITERATURE CITED

1. BRIDGES IG, MB WILKINS 1973 Growth initiation in the geotropic response ofthe wheat node. Planta 112: 191-200

2. DARWIN C 1880 The Power of Movement in Plants. John Murray, London3. DIGBY J, RD FIRN 1976 A critical assessment of the Cholodny-Went theory of

shoot geotropism. Curr Adv Plant Sci 8: 953-9604. DIGBY J, RD FIRN 1979 An analysis of the changes in growth rate occurring

during the initial stages ofgeocurvature in shoots. Plant Cell Environ 2: 145-148

5. FIRN RD, J DIGBY 1977 The role of the peripheral cell layers in the geotropiccurvature of sunflower hypocotyls: a new model of shoot geotropism. AustJ Plant Physiol 4: 337-347

6. FIRN RD, J DIGBY 1979 A study of the autotropic straightening reaction of ashoot previously curved during geotropism. Plant Cell Environ 2: 149-154

7. FIRN RD, J DIGBY 1980 The establishment of tropic curvatures in plants.Annu Rev Plant Physiol 31: 13 1-148

8. FIRN RD, J DIGBY, A HALL 1981 The role of the shoot apex in geotropism.Plant Cell Environ 4: 125-129

9. FIRN RD, J DIGBY, H RILEY 1978 Shoot geotropic curvature-the location,magnitude and kinetics of the gravity-induced differential growth in horizon-tal sunflower hypocotyls. Ann Bot 42: 465-468

10. FRANSSEN JM, SA COOKE, J DIGBY, RD FIRN 1981 Measurements of differ-ential growth causing phototropic curvature of coleoptiles and hypocotyls. ZPflanzenphysiol Bodenk 103: 207-216

1 1. FRANSSEN JM, RD FIRN, J DIGBY 1982 The role of the apex in the phototropiccurvature of Avena coleoptiles: positive curvature under conditions of con-tinuous illumination. Planta 155: 281-286

12. GORDON DC, IR MACDONALD, JW HART 1982 Regional growth patterns inthe hypocotyls of etiolated and green cress seedlings in light and darkness.Plant Cell Environ 5: 347-353

13. HART JW, DC GORDON, IR MACDONALD 1982 Analysis of growth duringphototropic curvature of cress hypocotyls. Plant Cell Environ 5: 361-366

14. MACDONALD IR, JW HART, DC GORDON 1983 Analysis of growth duringgeotropic curvature in seedling hypocotyls. Plant Cell Environ 6: 401-406

15. WAREING PF, IDJ PHILLIPS 1978 The Control of Growth and Differentiationin Plants, 2nd ed. Pergamon Press, Oxford, pp 174-181

16. WAREING PF, IDJ PHILLIPS 1981 Growth and Differentiation in Plants, 3rded. Pergamon Press, Oxford, pp 189-200

17. WENT FW, KV THIMANN 1937 Phytohormones. Macmillan, New York

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