Plant Physiol. (1992) 98, 769-7730032-0889/92/98/0769/05/$01 .00/0

Received for publication September 19, 1991Accepted November 4, 1991

Communication

Behavior of Etiolated Peas (Pisum sativum cv Alaska) WhenObstructed by a Mechanical Barrier1David A. Schwarzbach, Ernst J. Woltering, and Mikal E. Saltveit*

Department of Vegetable Crops, University of California, Davis, California 95616 (D.A.S., M.E.S.); andAgrotechnological Research Institute, P.O. Box 17, 6700AA Wageningen, The Netherlands (E.J. W.)

ABSTRACT

Etiolated pea (Pisum sativum cv Alaska) seedlings growingagainst a horizontal barrier in the soil will assume a horizontalorientation and continue to grow for prolonged periods of time.With removal of the barrier or after seedlings grow out fromunderneath the obstruction, seedlings immediately return to nor-mal vertical growth. Ethylene production increased several hoursafter the seedlings began to grow horizontally and not at the firstcontact with a barrier. Increases in ethylene production fromhorizontally growing seedlings were associated with decreasedrates of elongation and increased stem diameter. The data sug-gest that increased ethylene production does not play a mediatingrole in the horizontal growth of pea seedlings when obstructedduring emergence. We conclude that seedlings follow a path ofleast resistance when they grow against a barrier in the soil.

At the turn of the century, Dimitry N. Neljubow (10)observed that exposure of pea seedlings to ethylene inducedthree responses: (a) a reduction in the rate of stem elongation;(b) an increase in stem diameter; and (c) horizontal growth.Collectively, these three responses are known as the tripleresponse. Before the development of the gas chromatograph,the triple response was used as a bioassay for ethylene (1).Other investigators have repeated Neljubow's experiments,and demonstrated a correlation between increasing concen-trations of exogenous ethylene and increases in the deviationof the stem from vertical growth toward horizontal growth(4, 5).

It has been suggested that many of the stress-adapted fea-tures of pea seedlings result from the ability of the seedlingsto produce ethylene (5, 12). The triple response, therefore,may be the mechanism involved in the redirection of growthby pea plants during mechanical stress. Goeschl et al. (6)observed that the rate of ethylene production increased afterseedlings came into contact with a horizontal barrier (a neo-prene stopper in their case) or when growing through glassbeads. In the latter case, they observed a decrease in stemelongation and an increase in stem diameter, which are

I This investigation was supported in part by a President's Under-graduate Fellowship from the University of California, Davis, and anUndergraduate Fellowship from the Department of Vegetable Crops,University of California, Davis.

thought to have resulted from increases in ethylene produc-tion. Similarly, it may be hypothesized that following initialcontact with a barrier in the soil, stress-induced ethyleneproduction could give rise to horizontal growth if endogenousethylene concentrations were to increase sufficiently. To date,however, no one has demonstrated that mechanical stress,under natural conditions, can induce a sufficiently high en-dogenous ethylene concentration to cause diagravitropicgrowth.To test the hypothesis that the triple response accounts for

the capability of plants to grow around impediments in thesoil, we studied the behavior of etiolated pea seedlings underconditions in which vertical growth was restricted by a smoothhorizontal barrier. To determine the ethylene production ofindividual seedlings during mechanical stress, we developed aset of experimental chambers that allowed us to employ laser-driven photoacoustic spectroscopy for our measurements (2,16).

MATERIALS AND METHODS

Plant Material and Growth Conditions

Seeds of pea (Pisum sativum cv Alaska) were allowed toimbibe overnight in aerated, deionized water. Seeds were thenrolled in moist germination paper or were sown directly intomoist vermiculite (W.R. Grace and Company, Cambridge,CT). In all cases, after imbibing, seeds were positioned withthe hyphum in a vertical orientation so that the shoots grewupwards with a minimum of gravistimulation. In the case ofseeds germinated in paper, seedlings of uniform size weretransferred to solution culture after 6 d. All experiments wereperformed in the dark at 20°C. Observations were made usinga safe green light prepared by wrapping a 15 W fluorescentlight (General Electric, model F15T12-SW) with three layersof both green and amber acetate film. Observations weremade of seedlings growing in vermiculite either in air or undera clear plexiglass barrier.

Experimental Chambers for Ethylene Measurement

To measure the evolution of ethylene from single seedlingsundergoing obstruction by a barrier and during the subse-quent horizontal growth, special glass chambers were fabri-cated using 5 mm i.d. glass tubing. These tubes allowedpassage of the seedling with a minimum of stem flexion, a

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Plant Physiol. Vol. 98, 1992

potential inducer of stress ethylene (3). Seedlings first experi-enced physical stress due to contact with a glass surfaceperpendicular to the direction of growth. The shape of thetubes forced seedlings to then grow horizontally. Each cham-ber was equipped with an inlet and outlet port to enable gasexchange and sampling. Chamber bottoms widened to 15 mmi.d. glass tubing to accommodate a rubber stopper.

Seedlings were germinated as described above. Rubber stop-pers were bored at an appropriate diameter to accommodatea single seedling and hold it firmly throughout the experimentwhile avoiding damage to the stem due to compression by therubber. Before placing seedlings in the rubber stopper, eachshoot was wrapped with several layers of teflon tape (Lee-Meyers and Co., San Jose, CA) to maintain epidermal integ-rity and to avoid any contamination from contact with therubber. A radial slit in the stopper facilitated the insertion ofthe seedling. Water-insoluble laboratory grease (Glisseal,Borer Chemie A.G., Solothurn, Switzerland) was then liber-ally applied to the top and bottom of the stopper, ensuringthe separation of the root solution from the shoot chamber.The roots were bathed in a solution ofaerated 0.6 mm CaSO4.Seedlings were observed during the course of the experimentto determine the position of the epicotyl hook.

Laser-Driven Photoacoustic Spectroscopy

Ethylene evolution from the seedlings was measured in aflow-through system. Compressed air was first directedthrough a platinum catalyst (400°C) then into the experimen-tal chambers at a flow rate of 0.5 L/h. After passing throughthe chamber, the air flow was directed through a columnfilled with KOH to remove C02, and then into a flow-throughphotoacoustic cell. A CO2 laser beam, tuned to a frequencyat which ethylene shows high absorption (10.53 mm) andmodulated with a mechanical chopper, was directed into thephotoacoustic cell. Within this system, the ethylene moleculespresent in the sample absorb the incident energy, passing fromthe ground state to a higher rotational energy level. Collisionsbetween the ethylene and gas molecules result in the transferof the absorbed energy from the former to the latter and thereturn of the ethylene molecules to their ground state. Thisproduces an increase in the temperature ofthe gas, leading toa corresponding increase in pressure. By modulating the laserbeam at the resonance frequency of the photoacoustic celland using a microphone, the changes in pressure are detected.This system provides the ability to detect ethylene concentra-tions as low as 0.05 nL/L. For further discussion of thissystem, see Woltering et al. (16) and Harren et al. (7). Duringthe course of the experiments, an empty chamber equippedwith a stopper and sealed with laboratory grease was sampledfor ethylene to control for contamination from the materialsutilized.

RESULTS

Initial experiments were performed by obstructing seedlingsgrown in vermiculite with a horizontal plexiglass barrier.When seedlings encountered the barrier, they continued togrow, adopting a horizontal orientation (Fig. 1A). During

periods of prolonged horizontal growth, we did not observeany visible damage to the seedlings due to contact with theplexiglass. Increased stem diameter (

EMERGENCE BEHAVIOR OF ETIOLATED PEAS

Figure 1. A, Pea seedlings growing in vermiculite and obstructed with flat plexiglass. Seedlings have assumed a horizontal orientation in growth.B, Seedlings after removal of plexiglass obstruction. Seedlings were grown in vermiculite and obstructed with a flat plexiglass barrier. The barrierwas removed after seedlings had grown for approximately 8 cm horizontally. Photograph taken within 1 min of the removal of the barrier.

observed at any time for tissue obstructed with a glass barrier.Biro and Jaffe (3) have found that ethylene production frombean plants increases in response to stem flexion. Because ofthe use of neoprene, a soft, spongy material, in the experi-ments by Goeschl et al. (6), the seedlings were possibly notable to slide over the surface as they encountered the barrier.This may have caused the stems to flex as they grew in length,leading to the production of ethylene due to stress.

In our experiments, we found a pronounced increase inethylene production when seedlings became trapped in the

corner of the experimental chamber instead of turning togrow along the horizontal region. These production rates weresimilar in magnitude to those observed by Goeschl et al. (6).The use of narrow glass tubes for our experimental chambersallowed seedlings to slide into a horizontal orientation withminimal flexing of the stem. Similarly, under natural condi-tions, one would expect that the surrounding soil wouldprevent the stem from flexing. It has been shown that, undercircumstances of elevated exogenous ethylene (10) or aftertreatment with 1-aminocyclopropane- 1 -carboxylic acid (our

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Table 1. Stem Diameter of Pea Seedlings Growing Horizontally andVertically at 1, 3, and 5 cm from the Top of the Epicotyl Hook

Seedlings were grown in vermiculite and either obstructed with abarrier or allowed to grow into the air. Seedlings obstructed with abarrier grew into a horizontal orientation. Measurements were takenafter seedlings had grown approximately 7 cm horizontally orvertically.

Stem Diameter

Orientation Distance from hook (cm)

1 3 5

mm

Horizontal 2.07 2.06 2.16Vertical 2.01 1.91 2.10LSD (0.05) 0.030 0.030 0.029

unpublished results), true diagravitropic growth may be ob-served. The concentration of ethylene required to initiatediagravitropic growth, however, is likely in excess of theendogenous levels present for the ethylene production rateswe have measured.Growth rates of vertically growing seedlings exceeded those

of horizontally growing seedlings (Fig. 2). Additionally, thestem diameters of seedlings growing horizontally were greaterat distances of 1, 3, and 5 cm from the top of the epicotylhook than those of seedlings growing vertically (Table I).When considered with the measurements of ethylene produc-tion, these results may indicate that elevated levels ofethyleneinduce the first two c-omponents of the triple response (i.e.reduced elongation and increased stem diameter) for seedlingsgrowing horizontally due to obstruction by a barrier (10).With the removal of the flat barrier, seedlings growing in a

horizontal orientation were found to spring upward in asimilar manner to that observed for cocklebur, tomato, andcastor bean by Wheeler and Salisbury (14). The tension in

60

50 -

Vertical40-

E

E

30

*g+Horizontal

10

0 20 40Time (h)

Figure 2. Measurement of cumulative growth of seedlings growingin vermiculite and obstructed by a flat plexiglass barrier (@) and ofseedlings growing vertically (0). Measurements began after the seed-lings had grown for 1 cm. Data presented as mean ± SE.

-C

0C.)

0._Qa1)irC-

C0

02

ii)C

C)

0.14-

0.12

0.10

0.08

0.06

0.04

0.02

0

-0.02

4 8 12 16 20Time (h)

Figure 3. Typical measurement of ethylene production rates byseedlings growing vertically (0) or horizontally beneath a barrier (@).Measurements were made on single seedlings using laser-drivenphotoacoustic spectroscopy and specially fabricated experimentalchambers.

the stem indicates the presence of the normal gravitropicresponse and the absence ofdiagravitropic growth. Ifa realign-ment in the orientation of growth with respect to gravity hadtaken place, one would expect a lag period between removalof the barrier and the commencement of vertical growth.When the barrier was removed and the seedlings sprang up,the stem bent, with increasing curvature, from the point ofemergence from the soil to the epicotyl hook. This differentialin curvature may be accounted for by elongation. Schierle etal. (13) have shown that differentials in stem growth occurapproximately 1 cm beneath the epicotyl hook. As expected,this is the region of greatest curvature (Fig. 1B). With elon-gation ofthe cells, the ratio of cell length between the top andbottom decreases, resulting in a reduction of curvature overthe length of the stem.Many plant parts produce increased levels ofethylene when

placed horizontally (1, 8, 15). Despite this, the role ofethylenein the gravitropic response remains poorly understood (17).Gravitropic stimulation may account for our observations ofincreased ethylene production during horizontal growth ofetiolated pea seedlings. In addition, the continuous upwardbending ofthe hook region due to gravistimulation may havealso caused a thigmotropic-like stress that may have resultedin increased production of ethylene (3, 9).

In conclusion, our data show that true diagravitropic growthis not required for etiolated pea seedlings to grow aroundbarriers to emergence in the soil. Seedlings most likely followa path ofleast resistance and slide into a horizontal orientationwhen encountering a horizontal obstruction. The poor cor-relation between the rise in ethylene production and the startof horizontal growth indicates that ethylene does not play asignificant role in the growth of peas around impediments.The horizontal orientation ofthe stem is the most likely causeof the observed increase in ethylene production, although theexact nature of the stimulus remains unknown.

.. Regression line* . ..****--

------ --.

j Horizontal

.. , ~~~~~~~Vertical

n A o ~~ ~ ~ ~~ ~~~4el4 ell%

772 SCHWARZBACH ET AL.

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EMERGENCE BEHAVIOR OF ETIOLATED PEAS

ACKNOWLEDGMENTS

We would like to thank Jian Guo Dong, Juan Carlos Fernandez,and Aron Silverstone for technical advice, and Dr. Frans Harren forhis cooperation with laser-driven photoacoustic spectroscopy.

LITERATURE CITED

1. Abeles FB (1973) Ethylene in Plant Biology. Academic Press,New York

2. Bicanic D, Harren JR, Woltering E, Snel J, Voesenek LACJ,Zuidberg B, Jalink H, Bijnen F, Blom CWPM, Sauren H,Kooijman M, van Hove L, Tonk W (1989) Trace detection inagriculture and biology. In P Hess, ed, Topics in CurrentPhysics: Photoacoustic, Photothermal and PhotochemicalProcesses in Gasses, Vol 46. Springer-Verlag, Berlin, pp213-245

3. Biro RL, Jaffe MJ (1984) Thigmomorphogenisis: ethylene evo-lution and its role in the changes observed in mechanicallyperturbed bean plants. Physiol Plant 62: 289-296

4. Goeschl JD, Kays SJ (1975) Concentration dependencies ofsome effects of ethylene on etiolated pea, peanut, bean, andcotton seedlings. Plant Physiol 55: 670-677

5. Goeschl JD, Pratt HK (1968) Regulatory roles of ethylene in theetiolated growth habit of Pisum sativum. In F Wightman, GSetterfield, eds, Biochemistry and Physiology of Plant GrowthSubstances. Runge Ltd., Ottawa, Canada, pp 1229-1243

6. Goeschl JD, Rappaport L, Pratt HK (1966) Ethylene as a factorin regulating the growth of pea epicotyls subjected to physicalstress. Plant Physiol 41: 877-884

7. Harren FJM, Reuss J, Woltering EJ, Bicanic DD (1990) Pho-toacoustic measurement of agriculturally interesting gases and

detection of C2H4 below the PPB level. Appl Spectrosc 44:1360-1368

8. Harrison M, Pickard BG (1986) Evaluation of ethylene as amediator of gravitropism by tomato hypocotyls. Plant Physiol80: 592-595

9. Jaffe MJ (1976) Thigmomorphogenisis: a detailed characteriza-tion of the response of beans (Phaseolus vulgaris L.) to me-chanical stimulation. Z Pflanzenphysiol 77: 437-453

10. Neljubow DN (1901) Uber die horizontale Nutation der Stengelvon Pisum sativum und einiger anderer Pflanzen. Beitr BotZentralbl 10: 128-138

11. Salisbury FB, Ross CW (1986) Plant Physiology, Ed 3. Wads-worth Publishing Company, Belmont, CA, pp 362-370

12. Saltveit ME, Dilley DR (1978) Rapidly induced wound ethylenefrom excised segments of etiolated Pisum sativum L., cvAlaska. Plant Physiol 61: 447-450

13. Schierle J, Rohwer F, Bopp M (1989) Distribution of ethylenesynthesis along the etiolated pea shoot and its regulation byethylene. J Plant Physiol 134: 331-337

14. Wheeler RM, Salisbury FB (1981) Gravitropism in higher plantshoots. I. A role for ethylene. Plant Physiol 67: 686-690

15. Wheeler RM, White RG, Salisbury FB (1986) Gravitropism inhigher plant shoots. IV. Further studies on participation ofethylene. Plant Physiol 82: 534-542

16. Woltering EJ, Harren F, Boerrigter HAM (1988) Use of a laser-driven photoacoustic detection system for measurement ofethylene production in Cymbidium flowers. Plant Physiol 88:506-510

17. Woltering EJ (1991) Regulation of ethylene biosynthesis ingravistimulated Kniphofia (hybrid) flower stalks. J Plant Phys-iol 138: 443-449

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