Interpreting Plant Responses to Clinostating · influence plant growth, it seems certain that mechanical stresses mustinfluence plant responses to clinostating. It seemsalmostas certain

Plant Physiol. (1981) 67, 677-6850032-0889/8 1/67/0677/09/$00.50/0

Interpreting Plant Responses to ClinostatingI. MECHANICAL STRESSES AND ETHYLENE'

Received for publication March 7, 1980 and in revised form September 15, 1980

FRANK B. SALISBURY AND RAYMOND M. WHEELERPlant Science Department UMC 48, Utah State University, Logan, Utah 84322

ABSTRACT

The severe epinasty and other symptoms developed by clinostated leafyplants could be responses to gravity compensation and/or the mechanicalstresses of leaf flopping. Epinasty in cocklebur (Xanthium stimariwn L.),tomato (Lycopersicon esculentum Mi.), and castor bean (Ricinus commnwisL.) is delayed by inhibitors of ethylene synthesis and action (aminoethoxy-vinylglycine and Ag+), confirming the role of ethylene in clinostat epinasty.To test the possibility that clinostat mechanical stresses (leaf flopping)cause ethylene production and, thus, epinasty, vertical plants were stressedwith constant, gentle, horizontal, or vertical shaking or with a quick, back-and-forth rotation (twisting). Clinostat leaf flopping was closely approxi-mated but with a minimum of gravity compensation, by turning plants sotheir stems were horizontal, rotating them quickly about the stem axis,and then returning them to the vertical, repeating the treatment every fourminutes (clinostat rotation time). None of these mechanical stresses pro-duced significant epinasties, but vigorous hand-shaking (120 seconds perday) generated minor epinasties, as did Ag+ applied daily (concentrationshigh enough to cause leaf browning). Plants gently inverted every 20minutes developed epinasty at about the same rate and to about the sameextent as clinostated plants, but plants inverted every 20 minutes andimmediately returned to the upright position did not become epinastic. Itis concluded that clinostat epinasty is probably caused by disturbances inthe gravity perception mechanism, rather than by leaf flopping.

Plant growth and development are obviously strongly modifiedby gravity, as illustrated by the sensitive gravitropic (geotropic)responses and the consequent plant shape and orientation. Plantsrespond to gravity because of their weight, which is a force causedby the pull of gravity on the plant. An object in orbit is in freefall, with the acceleration caused by gravity pulling toward thecenter of the orbited object (centripetal force) exactly counter-balanced by the tendency to move in a straight line (centrifugalforce). Modern physics says an object cannot distinguish betweenaccelerational forces caused by a gravitational field and inertialforces, so an object in an orbiting satellite laboratory is potentiallyweightless. Actually, unbalanced forces (e.g. caused by movementsof animals or machines) may provide low levels of acceleration(estimated to be l0-' to 10-3g, lg = 980 cm s-2).

Sachs, in 1873 (33), continuously changed the vector of gravi-tational force, so the accelerational forces summed to zero oversome brief interval, by rotating a plant around a horizontal axison a "clinostat" (his term; new Latin from the Greek meaning"slope made constant"). If rotation time approximates the plant's

'This work was supported by Utah State Agricultural ExperimentStation Project 2657 and by National Aeronautics and Space Administra-tion Grant NSG-7567.

response time to gravity, the plant could respond as though it wereweightless. This can be called "gravity compensation" (5).

Usually a plant is turned 90°, so its axis is horizontal, and thenit is rotated about its axis. Actually, initial orientation of the plantis immaterial as long as the axis of rotation is horizontal. New-combe noted in 1904 that "tumbling" works well (30) [Hoshizaki(14) gives several references to similar studies]. For a point locatedexactly on the axis of rotation, the speed of rotation is immaterial;thus, fast clinostats (e.g. 55 to 120 rpm) have been used for seedlingroots and shoots so small that all of their cross-sections lie close tothe axis of rotation (37). Leaves of mature plants are always atsignificant distances from the axis of rotation, so rotation ratesmust be slow enough for centripetal forces to be negligible (26,30). Rates of 0.25 to 4 rpm have often been used. If clinostatrotation is too slow (e.g. 0.03 rpm), plant parts will respond togravity, growing in a spiral as they follow the clinostat rotation(23, 30).Brown and co-workers (4-6) have shown that clinostated plants

do not behave exactly as if they were weightless. Indeed, condi-tions of a leafy plant on a clinostat might differ from those of aplant in the nominal weightlessness of a satellite in at least twoimportant respects. First, on a clinostat but not in a satellite,stresses and strains will be set up in the leaves, petioles, and stemsas the plant rotates (ie. leaves will flop; ref. 38). Second, undernominal weightlessness, a cellular organelle of different densitythan the medium in which it occurs will presumably come to restsuspended in the medium; several such organelles might be evenlydistributed throughout the cellular medium, although cytoplasmicstreaming could influence this. On a clinostat, the organelle willcontinually be settling as it is accelerated by gravity, tracing aroughly elliptical path during each rotation of the clinostat (10,36). If the medium is highly viscous, the density of the organelleis close to that of the medium, and the rate of rotation is ideal, theelliptical pathway will be extremely small, so small, perhaps, thatthere might be no significant difference between the conditions ofthe organelle on a clinostat and in a satellite. If the pathway islarger, we can think of organelle "stirring" or "mixing" on aclinostat as opposed to that in a satellite (34).

Plant experiments with clinostats have been legion, and manyresponses have been reported (14). Dedolph et al. (8) observedincreases in root growth rates, and Brown et al. (6) observed suchincreases in hypocotyl lengths. Hoshizaki et al. (15) observeddecreased rates of stem growth in Xanthium, this being reversedby gibberellin application (see also 6, 14). Several authors haveobserved increased sensitivity to gravistimulation or to photo-stimulation (e.g., 8, 11, 35), as well as increased curvature inresponse to applied auxins (9). Attenuation of flowering in Xan-thium by clinostating was reported by Hoshizaki and co-workers(15). Dedolph et al. (9, 10) measured increases in respiration rates.Sometimes clinostating causes stem circumnutations to stop; othertimes it does not (16, 20). By far the most obvious response of

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SALISBURY AND WHEELER

leafy plants is a marked epinasty (downward bending or curving)of leaves (Fig. 1). This is mentioned by virtually all who haveworked with such plants (e.g. refs. 4, 6, 14, 19, 23, 24, 26, 27, 32).

At present, two contrasting hypotheses could account for clinostatresponses (especially epinasty); a third hypothesis is a combinationof the first two.

FIG. 1. Epinastic responses of a tomato plant to clinostating. Upper left, plant when first placed on the clinostat at 17:00 h; Lower, plant in thehorizontal position on the clinostat at 11:00 h the next day after 18 h horizontal clinostating; Upper right, plant placed in the upright position tocompare with its condition at the beginning. Note extreme downward curvature of the leaves after clinostating.

678 Plant Physiol. Vol. 67, 1981

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CLINOSTAT STRESSES AND ETHYLENE

CLINOSTAT RESPONSE HYPOTHESES

First, Clinostat Responses Are Caused by Gravity Compensa-tion. The first step in any gravitropic response must be perceptionof the gravitational stimulus, which is then some way transduced,leading to observed gravitropic responses (14, 21, 41, 45). Thus,upsetting the gravity perception mechanism might, in some way,lead to epinasties and perhaps other responses. Leaves are nor-mally oriented with respect to gravity, and the epinasty might bethought of as an upset in this orientation. It is thought that thegravity perception mechanism in many plant organs involves thesettling or movement within perceptive plant cells (statocytes) ofstatoliths, which in many cases may be amyloplasts, each contain-ing two or more starch grains (21, 41), but our preliminary effortshave not yet detected such statoliths in the gravity-sensitive stemsand leaves of our green plants. The discussion here is independentof the mechanism of gravity perception, however.

Epinasties are commonly caused by ethylene and by treatments(e.g., with auxins) that cause a production of ethylene (see reviewsin refs. 1 and 22). Leather et al. (24; see also ref. 32) not onlymeasured increased ethylene from clinostated tomato plants butalso prevented epinasty with high CO2 concentrations, a treatmentknown to prevent ethylene responses (1). Hence, the first hypoth-esis as it relates to epinasties might be stated as follows: clinostatingupsets gravity perception (via organelle mixing?), leading to pro-duction of ethylene and, thus, leaf epinasties (and perhaps otherresponses as well).

Second, Clinostat Responses are Caused by MechanicalStresses (Le. Leaf Flopping). Leaves flop when plants are clinos-tated (Fig. 2; ref. 38). Furthermore, plants respond in various waysto mechanical stresses, and these responses often involve ethylene.By far the most striking response of plants to mechanical stresses(e.g. to touching, rubbing, shaking, bending, wind, etc.) is aninhibition ofstem growth (often about 40%), sometimes observablewithin 6 to 30 min after stimulation, with recovery after 2 or 3days (e.g. refs. 17, 18, 28, 29, and 43). Leaf growth, fruiting, etc.,are sometimes also inhibited. Stem dwarfing is often accompaniedby an increase in stem radial enlargement (40). Several authors(7, 13) have observed a release of ethylene in response to mechan-ical stresses, and Jaffe and Biro (18) showed that applied ethylenemimics mechanical stress responses. Inasmuch as mechanical stresscauses plants to release ethylene and ethylene causes leaf epinasty,it was postulated that clinostat epinasty was only a response tomechanical stresses (24, 32, 38). But we might ask whether leafflopping is a sufficient stress to cause epinasty. Actually, there arefew reports that mechanical stresses of any kind cause epinasty,although Mitchell et al. (29) report that rubbing tomato stemsinduces epinasty in nearby leaves.

Third, Clinostat Responses Are Caused by Both Gravity Com-pensation and by Mechanical Stresses. Because clinostating pro-duces mechanical stresses and mechanical stresses are known toinfluence plant growth, it seems certain that mechanical stressesmust influence plant responses to clinostating. It seems almost ascertain that gravity perception is also involved. We might ask:What is the relative importance of gravity perception versus me-chanical stress effects on observed responses (especially epinasty)of clinostated plants? If mechanical-stress is shown to account formore clinostat responses, then gravity compensation (difficult totest directly) becomes less significant; conversely, if mechanicalstress is shown to be less important, then gravity compensationbecomes more significant, the horizontal clinostat might be viewedas a good simulator of weightlessness, and we could expect plantsin an orbiting satellite to resemble plants on a clinostat. Hence, itis reasonable to assess the importance of mechanical stresses inclinostat responses. Two tests come to mind.

First, if epinasties are produced on a clinostat by mechanicalstresses (leaf flopping), then it should be possible to induce such

mating the mechanical stresses experienced by plants on a clinos-tat. We have subjected vertical plants to various forms of mechan-ical stresses that approximate those experienced by clinostatedplants and we have used "intermittent clinostating" to simulateleaf flopping closely, with minimal gravity compensation.

Second, if clinostat epinasty is caused only by the mechanicalstresses of leaf flopping, then it should be possible to reduce oreliminate the epinasty by eliminating the leaf flopping. We reportresults of such experiments.

In either case, ethylene might be the causal agent in clinostatepinasty. Thus, we further document the role of ethylene in theclinostat response, using ethylene inhibitors in addition to CO2 (1,24). Silver ions inhibit the action of ethylene (3), and AVG2inhibits ethylene synthesis (reviewed in ref. 25).

MATERIALS AND METHODS

Species and Growing Conditions. Critical parts of all experi-ments reported here have been repeated at least three times withvegetative cocklebur plants, Xanthium strumarium L. (Chicagostrain). Plants varied from 25 to 90 days old, although mostexperiments used younger plants (about 30 days old). Most exper-iments were also repeated at least once with tomatoes (Lycopersi-con esculentum Mill., var. Bonny Best) and castor beans (Ricinuscommunis L.). Bell peppers (Capsicum annuum L., var. YoloWonder) were also used in some experiments. Seeds (fruits ofcocklebur) were planted in flats of sand and transplanted into 10-cm, square plastic pots when about 3 to 5 cm high (I to 2 weeksold). Greenhouse soil was a mixture of dark loam:sand:peat moss(3:1:1, v/v). Mineral fertilizers were added at approximatelyweekly intervals. Plants were grown in a corrugated-fiberglassgreenhouse (diffuse light) with supplementary fluorescent light inthe morning and evening, providing an 18-h photoperiod tomaintain cocklebur plants in a vegetative condition. Older leaveswere usually removed from cocklebur plants so that only one tothree fully expanded leaves remained on the plant. Temperaturesin the greenhouse were usually maintained between 24 to 27 C,although temperatures sometimes dropped as low as 18 C at nightin winter and went above 27 C on summer days.

Equipment. Some experiments used a simple horizontal clinos-tat, rotated 0.25 rpm by a small synchronous motor (Fig. 1). Mostexperiments used a larger clinostat with six tumtables, all rotatedat the same speed (but two in opposite direction from the others)by a belt-drive system. The belt was tumed by a 1/25 horsepowermotor connected through a zero-max gear system that allowed awide range of tum-table velocities. Vibration was low. Rotationrate in experiments reported here was approximately 0.25 rpm.Tum-tables of both clinostats can be placed at any angle, but onlythe vertical or horizontal positions were used. Some experimentsused a shaker that moved with a forward and backward motionof 90 cycles/min (low speed) or 132 cycles/min (high speed), witha table displacement of 3.7 cm. The shaker was placed on end toprovide vertical instead of horizontal shaking motion. For someexperiments, the six-turntable clinostat was modified with a crankarm, so that the turn tables rotated back and forth about 1800,usually at 38 cycles/min. The result was a twisting motion appliedto vertical plants, roughly simulating (although at much higherfrequency) the leaf flopping experienced by horizontal plants onthe clinostat. If the tip of a leaf was 6 to 16 cm from the axis oftwisting, centripetal forces on that tip varied from about 0.03 to0.08g. Accelerational forces might be somewhat higher during themoment of reversal of direction (the moment when the leaf"flops," when forces are applied to the petiole).For inversion experiments, four plants were bolted together

between two boards, far enough apart so that their leaves did not

epinasties on vertical plants by duplicating or at least approxi-

679Plant Physiol. Vol. 67, 1981

2Abbreviation: AVG, aminoethoxyvinylglycine.




0

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FIG. 2. Leaf flopping on a clinostat. The four photographs were taken with the camera lens on the axis of rotation, the camera aimed horizontallyat the plant. They show the tomato plant of Figure 1 at I-min intervals during one rotation cycle (4 min), shortly after clinostating began. Eachphotograph was rotated 900 in relation to the adjacent photographs, so the plant appears to remain in the same relative position as it rotates, emphasizingthe changing leaf positions. Tracings of the large lower leaf of the photographs are shown at the left, with arrows pointing in the direction that was downat the time of the photograph.

touch (46.5 cm); thus, four plants could be inverted at a time,placing them upside down or rightside up in special racks builtfor the purpose.

Several experiments (most of those shown) were carried out inthe full light of the greenhouse, but some were performed in one

of two darkrooms, sometimes with a bank of cool-white fluores-cent tubes placed so that light rays were approximately parallel toeither horizontal or vertical plant stems (photosynthetic photonflux density, 75 to 200 ,AE s-1 m2, depending on distance fromlamps). Some experiments used a green safelight for plant manip-ulations (incandescent light filtered through blue and green Plex-iglas). Dark-room temperatures were controlled at 25 ± I C.

Anti-Ethylene Treatments. AVG, donated by Hoffmann-LaRoche, Inc., was applied as a simple solution, but silver ion was

complexed with thiosulphate (39) by pouring 2 mm AgNO3 intoan equal quantity of 8 mi Na2S203 (or 1 mm AgNO3 into 4 mMNa2S203). All solutions contained I drop Tween 20/100 ml. Plantswere dipped in these solutions 30 to 60 min before clinostating.Measurement of Epinasty. In the four species that we used,

epinasty is expressed as a rather even, downward curvature of theleaves, beginning at the axil and extending along the petiole andthe midrib to the tip of the leaf (Figs. I and 3). This is somewhatless true for the castor bean plants than for the other three, inwhich the curvature approximates that of a circle. Figure 3 indi-cates the three lengths (from axil to base of leaf blade and to leaftip, and from base of leaf blade to leaf tip) that were measured,using calipers or a ruler. These three values then could be used inthe equation shown in Figure 3 to give a radius of curvature

(reproducible within ± 1.0 mm), using a programmable handcalculator. The method fails for young tomato leaves that coilbeyond a circle to a small spiral, although different points couldbe used for the measurement. If epinasty were expressed only asa change in axillary angle, this method would fail to detect sucha change but, in our experience, changes in axillary angle werealways accompanied by changes in curvature from the axil to thetip of the blade. Indeed, curvature may occur with little change inaxillary angle (31). The data for epinasty are plotted as thereciprocal of radius of curvature (32), producing values thatincrease as epinasty increases and that intuitively seem propor-tional to severity of treatment. With this system, a straight leafhas a value of zero (but a radius of curvature of infmity). [Palmer(31) plotted the inverse of the radius by matching leaf curvaturesto templates with an accuracy of ±0.5 to ±2.0 cm radius.] Sinceabsolute values depend strongly on leaf size, initial values aresubtracted from all those for a given leaf or set of leaves, so thatinitial values for all plants are normalized to zero. We usuallyaverage values for two leaves on each plant. Young expandingleaves exhibit the most curvature. In a few experiments, othermeasurements of plant growth were also made, but these involvedordinary techniques.

RESULTS AND DISCUSSION

Confirming Role of Ethylene in Response of Plants to Clinos-tating. Figure 4 shows results of an experiment in which devel-opment of epinastic curvature of cockleburs on a clinostat isfollowed as a function of time for untreated plants and for plants


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FIG. 3. Measurement of leaf epinasty. The three distances a, b, and care measured and used in the formula (derived from the law of cosines) tocalculate the radius (R) of the circle that inscribes the triangle formed bythe lines between the three points. The geometric construction shows howthe center of the circle can be found by drawing perpendicular bisectorsfrom each side of the triangle. The reciprocal of the radius of curvature isdirectly proportional to the extent of epinasty and is used as the measureof epinasty in the following figures. The upper plant (cocklebur, X.strumarium) was traced from a photograph taken in the early morning(Feb. 23, 1980), when the leaves were in the daytime position, only slightlyepinastic. Under some conditions (so far unknown), cocklebur leavesbecome quite epinastic just before darkness, when the photograph wastaken from which the lower drawing was traced (Feb. 22, 1980; 2,045 h).Often, leaves straighten just before and after dark, becoming even lessepinastic for a few h than the upper plant. These are the daily, circadian,sleep movements noted in Figures 4 to 7.

treated with AVG and with Ag+. We have repeated such experi-ments several times (e.g. see Fig. 7, below), and with tomatoesand castor beans; the two ethylene inhibitors always retard thedevelopment ofepinasty, as in the upper part of Figure 4. Contraryto these results, however, AVG is usually somewhat more effectivethan Ag+. We have observed a delay of epinasty in response to 5%CO2 [confirming Leather et al. (24)], but development of epinastycould not be measured, because plants were inaccessible in clearplastic jars.The stationary, untreated, control plants of Figure 4 also devel-

oped a rather extreme epinasty during late afternoon and evening.This is part of the daily sleep movements, but it is not clear whythe epinasty can sometimes be extreme yet not appear at all atother times. The control plants of Figure 4 and the lower plant ofFigure 3 show extreme examples of sleep-movement epinasty.Control plants of the experiment of Figure 5 also became epinasticjust before dark, but not nearly as much as those of Figure 4.Controls of Figure 6 show virtually no predarkness epinasty butsome leaf straightening during the dark period, whereas those ofFigure 7 show no epinasty before dark but a marked leaf-straightening during the dark period. Various possible causes forthese differences come to mind: weather conditions, ethylenepollution in the greenhouse, etc. Observed epinasties do notcorrelate with weather, however, and we note that all cockleburplants in the greenhouse exhibited the same epinasty or lack of iton each occasion, although tomato plants, randomly dispersed

so0x0-

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FIG. 4. Upper, delay in the development of clinostat epinasty causedby 1.0 mm AVG and by silver ions (2.0 mm AgNO3 + 8.0 mm Na2S203).The experiment was carried out in the greenhouse (Feb. 26-27, 1980), andlight conditions and real times are indicated at the top of the figure. Eachpoint represents an average of two cocklebur leaves (one-half to three-quarters expanded) on two plants. Lower, stationary controls and station-ary plants treated with the AVG and Ag+ solutions. Clinostat controlcurve is given again for comparison. Note the marked epinasty ofuntreatedstationary controls at 21:00 h. AVG and especially Ag+ reduced this sleepmovement epinasty (compare Fig. 7).

throughout the greenhouse, showed no epinasty. (Tomatoes arenotoriously sensitive to ethylene (1).]

Clearly, inhibitors of ethylene action delay the development ofepinasty on clinostated plants and reduce the sleep movements ofFigures 4 and 7. As a rule, epinasty of treated plants eventuallyappears on the clinostat. Ethylene inhibitors typically reduce ordelay but do not prevent responses to ethylene (1).Measurement of ethylene evolution during clinostating has so

far depended upon excised tissues (24). We are currently measur-ing ethylene evolution of intact plants.

Attempts to Induce Epinasty by Applying Mechanical Stressesto Upright Plants. For the past three years, we have studiedmechanical stress effects on plants (43), but we have not observedobvious leaf epinasties in response to such stresses as shaking for10 to 120 s, bending the stems to some predetermined angle, orspraying plants for approximately 10 s with a fairly strong waterspray. We do observe the usual reduction in stem growth rates ofabout 40% (along with various other less obvious symptoms). It

Plant Physiol. Vol. 67, 1981 681




seemed likely that we failed to produce obvious epinasty becauseour treatments (sufficient to produce maximum growth inhibi-tions) were applied for too short a time.To produce mechanical stresses sustained over long time inter-

vals and of a magnitude comparable to that produced by leafflopping on clinostated plants, we left plants on a shaker forseveral hours to several days. The horizontal shaker produced agentle stem and petiole bending. Petiole and blade bending isaccentuated and stem bending is minimized by vertical shaking.Twisting even more closely simulates the leaf flopping experiencedon a clinostat, but at higher frequency. Figure 5 presents results oftwo combined experiments in which plants were shaken horizon-tally or vertically (low speed) or were twisted. High points at 12 hare the sleep-movement epinasties just discussed. In two otherexperiments (results not shown), plants on the shaker were placedunder a transparent jar with the thought that evolved ethylenemight accumulate, accentuating epinasty.

Table I summarizes results of a comprehensive experiment inwhich plants in the greenhouse were subjected for a week tohorizontal shaking; daily, manual, vigorous shaking for 120 s orclinostating. Half the plants in each treatment were treated withAg+. At daily and other intervals, measurements were taken ofepinasty, leaf sizes, stem heights, and stem diameters. Some plantswere measured only at the beginning and the end of the treatment,so measurement itself would not act as a significant source ofmechanical stress.

In no case, including plants under jars, have we been able toproduce extreme leaf epinasty compared with controls, except byclinostating plants (and by gently inverting them at regular inter-vals, see below). Slight epinasties were produced by vigoroushand-shaking and by repeated applications of silver ions, whichcause extensive browning of leaves (Table I). Thus, ethylene canapparently be evolved in response to extreme mechanical stressesand even to high silver salts (2, 42), even though silver ionsnormally prevent development of ethylene responses and delaydevelopment of clinostat epinasty.

Effects on stem growth (Table I) were somewhat unexpected.Hand shaking gave the usual inhibitions, but gentle shaking onthe shaker did not. The most inhibition of stem growth occurredon the clinostat (confirming ref. 15). Apparently, plants subjectedto sustained, rhythmical, relatively gentle mechanical stresses arenot inhibited as much in their stem growth as are plants shakensomewhat more vigorously for only a few seconds each day (aresult also obtained by C. Mitchell at Purdue University, personalcommunication). Inhibition of stem growth of plants on theclinostat might well be a response to the mechanical stresses ofleaf flopping, or it could be caused by gravity compensation ratherthan or in addition to mechanical stresses.

Perhaps the best way to duplicate the mechanical stresses of leafflopping experienced by plants on a clinostat is one in which sixplants were attached to the turntables of the large clinostat butleft in the vertical position over 90%o of the time. Once each 4 min(horizontal clinostat rotation time), the clinostat was tipped sothat the plants were horizontal and turned on with the speed setso that one rotation was complete in 10 s (6 rpm). This was slowenough so that centripetal forces were negligible (about 0.002 to0.007g, 6 to 16 cm from the axis of rotation) but fast enough thatthe gravity perception mechanism had little time to respond.Leaves flopped in a manner closely similar to their action on aslowly rotating, horizontal clinostat. Plants were returned to thevertical, the entire operation requiring about 20 s. Leaf epinastieswere compared with those of a plant on the single-turntableclinostat and with six stationary controls. Results are shown inFigure 6. Epinasty developed on the horizontal clinostat as usual,but leaf curvatures of plants receiving the intermittent clinostatingwere virtually identical to those of stationary control plants. (Noteagain the sleep movements, in this case mostly a leaf straightening

after dark, with little epinasty just before dark.) Tomatoes andcastor beans respond similarly to intermittent clinostating.

Reducing Mechanical Stresses by Inverting Plants. If epinastyis caused by upsetting gravity perception during clinostating (assuggested by failure to produce epinasty by gentle mechanicalstresses), then plants that are inverted at intervals should also havetheir gravity perception mechanism upset and thus develop epi-nasty, even though leaf displacements are greatly minimized.Figure 7 shows the results of an experiment in which plants weremanipulated in 4 ways, half the plants receiving no AVG (top offigure) and half being treated with AVG (bottom of figure). Thefour manipulations were as follows: first, plants inverted at 20-minintervals (left upside down half the time); second, plants invertedat 20-min intervals but immediately returned to the upright posi-tion (to produce leaf displacements but without significantly per-turbing gravity perception); third, plants on a horizontal clinostat;and fourth, vertical control plants that were left upright all thetime but moved at 20-min intervals so all were exposed to thesame possible environmental gradients from floor to ceiling on theracks in the greenhouse.As to plants not treated with AVG: epinasty developed only on

the clinostated and inverted plants but not on the inverted-and-returned or the upright plants. Strong sleep movements(straightening after dark) occurred on clinostated and invertedplants as well as on controls, but there was no epinasty beforedark (compare Fig. 4). AVG not only greatly delayed the devel-opment of epinasty but also eliminated the sleep movements. Theexperiment (without AVG) has been repeated several times withcockleburs and once with tomatoes, castor beans, and peppers.Intervals of 10 to 30 min between inversions have been used withcockleburs. There is no doubt that reducing leaf displacementsdoes not reduce epinasty significantly if gravity perception canstill be upset. And gravity perception may also involve productionof ethylene in gravitropic bending (44).We have immobilized leaves by packing plants in vermiculite

and then clinostating or inverting them. Virtually no epinastydevelops in response to such treatment. This surprised us becausebending energy and perceived stimulus can both be stored ingravitropic bending of immobilized plants laid on their sides, aswe describe in the companion paper (44). Apparently, stimulusfor epinasty cannot be stored; it must be expressed or it is lost. Itseems possible that CO2 builds up in the vermiculite, antagonizingethylene action.

CONCLUSIONS

All the evidence so far clearly indicates that ethylene plays arole in the epinastic leaf bending of plants on a clinostat. Previousresults showed that ethylene was evolved (24, 32) and that l1o0CO2 prevented clinostat epinasty measured on petiole explantsafter 24 h (24). Our results show that Ag+, AVG, and CO2 delaydevelopment of epinasty on a clinostat, thus confirming andextending the earlier results. Further data are required to assessaccurately the kinetic and quantitative aspects of ethylene evolu-tion of clinostated plants and to study the roles ofother hormones.It has been suggested that ethylene influences auxin transportduring clinostating, accounting for the observed growth responses(22). Interactions of auxins and ethylene in leaf epinasties havealso been discussed by Hayes (12), although her hypotheses pro-vide no obvious help in interpreting our results.

All of our attempts to induce epinasties by mechanical stresses(horizontal and vertical shaking, twisting, intermittent horizontalrotating, and hand shaking) have failed to produce epinasties thateven approach those observed on the slow clinostat; reducing leafdisplacements by inverting plants (upsetting graviperception) does




Plant Physiol. Vol. 67, 1981

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FIG. 5. Epinasty as a function of time for cocklebur plants on a horizontal clinostat, a vertical or horizontal shaker, and a twister. The data are thecombined results of two experiments, performed 1 day apart in the greenhouse (begun Feb. 19 and 20, 1980). On the first day, there were three plantseach on the horizontal clinostat, as stationary vertical controls, and on the horizontal shaker (slow speed). On the second day, the clinostat was convertedto a twister (holding three plants), and one plant was used as a clinostat control (single-turntable clinostat). There were two plants on the vertical shaker.

Differences between leaf curvatures of plants on the twister and vertical shaker are probably not significant from those of stationary controls. The 3- and6-h plants on the horizontal shaker may have had significantly straighter leaves than the others at those times, but this is doubtful. Light conditions are

indicated at the top of the figure. The slight hump in all but the clinostat curves at 12 h (21:00 h) expresses the slight epinasty that is sometimes part of

the daily sleep movement. Tomatoes, tested simultaneously, produced similar results with slightly more epinasty produced by shaking and twisting (datanot shown).

FIG. 6. Epinasty of intermittent horizontally rotated cocklebur plants, slow clinostated plants, and stationary controls. Each point represents theaverage for two leaves (half and three-quarters expanded), six plants for intermittent clinostating and stationary controls, but only one on the horizontalclinostat. The experiment was carried out in the greenhouse (light conditions at top). Twelve h after starting the experiment, intermittent clinostatingwas stopped (arrow). Data for stationary controls and intermittent-horizontal-clinostat plants are indistinguishable from each other. Experiment of Jan.25, 1980.

FIG. 7. Upper, epinasties of cocklebur plants on a horizontal clinostat, inverted at 20-min intervals and left upside down half the time, inverted at

20-min intervals and immediately returned to the upright position, and left upright. Lower, identical treatments of plants treated with AVG (0.1 mM).Each point represents averages for two young leaves (one-half and three-quarters expanded) and four plants (except AVG on clinostat: only three plantsper point). Light conditions are indicated at the top. Inverted plants develop epinasty as fast and nearly as much as those on the clinostat (upper). In thisexperiment, the epinasty that sometimes develops as part of the normal sleep movements just before dark (compare Figs. 3-5) does not appear, but thesleep movement is expressed by a strong straightening of the leaves, beginning about when the lights go out, not only for controls and plants invertedand returned, but also for clinostated and inverted plants (top part of Figure). AVG (bottom part of Figure) not only delays the epinasty that developson clinostated and inverted plants (by about 20 h), but also virtually eliminates the sleep movements of all treated plants. (Dashed lines suggest that no

measurements were taken from 02:00 until 08:00 h, but plant inversions were continued.) Experiment of Jan. 30 and 31, 1980.

not significantly reduce epinasty. We conclude that the importanceof mechanical stresses in causing clinostat symptoms (especiallyepinasty) is considerably minimized.

Since leaf flopping alone is not sufficient to account for theobserved epinasty, other features of clinostating, particularly grav-

ity compensation, may well be of paramount importance in caus-

ing the epinasty (presumably via ethylene). Our results stronglysupport the idea that clinostating is, to an approximation at least,

a simulation of weightlessness. Thus, we would expect epinasty to

develop on plants in a satellite (as in Biosatellite II, see ref. 19).We acknowledge the statistically different behavior between cli-nostated pepper plants (ground controls) and those on BiosatelliteII, as reported by Brown et al. (4), but we doubt that thesedifferences were caused by leaf flopping on the clinostat.

Acknowledgments-We acknowledge the technical assistance of Mary Jo Hansen

683

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0 6 12 Is 24TIME (h)



SALISBURY AND WHEELER Plant Physiol. Vol. 67, 1981

Table I. Growth and Epinasty of Plants Subjected to Clinostating or to Various Kinds ofMechanical Stress, withand without Silver Treatment

The experiment was conducted in the greenhouse, with 18-h days at 24 C and lasted for 1 week. Plants weresprayed each day either with 2.0 mM AgNO3 in 8.0 mM Na2S203 or with 8.0 mM Na2S203 about I h prior totreatments.

Internode Internode Diame- Epinasty:Average Growth ter Growth Average

Treatment Shoot ChangeGrowth First Second First Second after 7

Days'I/R x 103mm mm mm mm-

Horizontal clinostat 26.3 0.0 10.7 0.4 0.7 8.5Clinostat + Ag+ 21.0 1.7 4.5 0.4 1.0 9.7Nonstressed controls 40.7 3.4 11.0 0.2 0.6 0.0Continuous + Ag+ 37.0 1.8 10.4 0.35 0.55 3.1Shaker, 8 h/day 44.5 2.4 11.8 0.55 0.8 1.0Shaker, 8 h + Ag+ 39.2 2.0 9.5 0.65 0.75 3.0Shaker, 120 s/day 45.3 6.4 13.2 0.4 0.75 2.1Shaker, 120 s + Ag+ 30.5 1.1 5.7 0.4 0.5 3.5Hand shake, 120 s/day 24.0 1.1 5.7 0.2 0.5 1.2Hand shake, 120 s/day + Ag+ 27.0 0.0 5.0 0.5 0.55 4.4Controlsb 40.0 1.5 10.8 0.6 0.6 0.0Controlsb + Ag+ 32.0 3.0 10.0 0.35 0.60 0.9

a Average change in 1/R x I 03 caused by silver, 4.12; by Na2S203 applied to controls, 2.04.b Measured only at the beginning and the end of the experiment.

(who, with Jill Richards, also typed the manuscript). Julianne Sliwinski and WesleyJ. Mueller also participated in many experiments and many discussions.

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Interpreting Plant Responses to Clinostating · influence plant growth, it seems certain that mechanical stresses mustinfluence plant responses to clinostating. It seemsalmostas certain