Annals of Botany 84 : 709–714, 1999Article No. anbo.1999.0963, available online at http:}}www.idealibrary.com on

The Role of Amyloplasts during Gravity Perception in Gynophores of the Peanut

Plant (Arachis hypogaea)

EDGAR MOCTEZUMA and LEWIS J. FELDMAN

Department of Plant and Microbial Biology, Uni�ersity of California at Berkeley, 431 Koshland Hall, Berkeley,

CA 94720, USA

Received: 15 March 1999 Returned for revision: 26 May 1999 Accepted: 2 August 1999

Gravitropic perception and response are essential for the completion of the reproductive life cycle of the peanut plant(Arachis hypogaea L.). The developing seeds are buried in the soil by a specialized organ, the gynophore, allowingthe fruit to mature underground. Controversy exists about the site of graviperception in the gynophore: previousworkers suggested that the intercalary meristem was the zone where gravity was perceived. Taking the starch statolithhypothesis for graviperception as a framework, we explored the possibility that the starch-grain filled plastids(amyloplasts) in the starch sheath of the gynophore may be acting as gravisensors. We show that these amyloplastssediment readily with respect to the gravity vector within 30 min of reorientation, and before there is a measurablegravitropic response. Gynophore explants were incubated with gibberellic acid and kinetin, in darkness, to removestarch from the amyloplasts. Destarching the gynophores did not inhibit overall growth of the organ, but reducedthe gravitropic response curvature by 82% compared to water-treated controls. In addition, gynophores placed ona rotating clinostat (without hormone treatment) also showed a reduced gravitropic response. In conclusion, theevidence presented in this work strongly suggests that the amyloplasts of the starch sheath are responsible forgravitropic perception in the peanut gynophore. A model for graviperception in the gynophore is presented.

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Key words : Amyloplast, Arachis hypogaea, gravity perception, groundnut, gynophore, peanut, starch sheath, starchstatolith hypothesis.

INTRODUCTION

Gravity perception and gravitropic response are essentialfor the completion of the reproductive cycle of the peanutplant or groundnut (Arachis hypogaea L.). The peanut plantproduces aerial flowers in the leaf axils of the stem, as inother angiosperm species. What is unusual about the peanutplant is that once the ovules are fertilized, they are carrieddown into the soil where the fruit develop and matureunderground (Smith, 1950). The peanut carries and buriesits developing seeds into the ground bymeans of a specializedorgan, the gynophore (Jacobs, 1947). If the peanutgynophore fails to perceive and respond to gravity, it willnot bury the developing seeds and, as a consequence thefruit will not develop fully.

The gynophore grows downwards by means of anintercalary meristem (Fig. 1A), located proximal to the seedregion (Jacobs, 1947; Shushu and Cutter, 1990), whichincludes an elongation zone found at 2 to 5 mm from the tip(Moctezuma and Feldman, 1998). The downwards gravi-tropic growth of the peanut gynophore consists of threephases : perception of the gravity stimulus, transduction ofthe signal, and the gravitropic growth response (downwardsbending). Thus, to understand the gravitropic response ofthe peanut gynophore, it is first necessary to characterizeand understand its mechanism of gravity perception.

Fax (510) 642–4995, e-mail edgar!nature.berkeley.edu; feldman!nature.berkeley.edu

Although graviperception is of upmost importance in thepeanut life cycle, there have been no studies on how thegynophore perceives the gravity stimulus. Shushu andCutter (1990) briefly mention that the intercalary meristemregion may be responsible for the gynophore’s gravitropicperception. However, they did not provide experimentalevidence to support their statement.

A century ago, the starch statolith hypothesis (SSH) forgravity sensing was established (reviewed by Sack, 1991).The SSH states that specific cells in plant organs containstarch grains (statoliths) that are displaced by the force ofgravity within the cell. The starch grain-filled plastids(amyloplasts) interact with other cell component(s), andprovide the cell with information about its orientation(Hart, 1990; Sack, 1991).

The SSH has been the object of extensive debate becausemost of the evidence to support it is correlative. Althoughopponents of the SSH argue that other cellular componentsmay act as gravity sensors, the amyloplasts remain the mainintracellular elements that readily sediment upon reori-entation (Caspar and Pickard, 1989; Kiss et al., 1989;Sack, 1991).

In plant shoots, the cells of the starch sheath containamyloplasts that readily sediment with the gravity stimulusupon reorientation (Sack, 1991; Fukaki et al., 1998). Thepeanut gynophore has a typical shoot anatomy that includesan amyloplast-filled starch sheath. Despite this typical shootanatomy, the peanut gynophore responds positively togravity. Thus, the following question arises : does the peanut

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710 Moctezuma and Feldman—Gra�ity Perception in the Peanut Gynophore

gynophore perceive the gravity stimulus like a typical shoot,or does it have a different mechanism of graviperception?Sack (1997) points out that gravity perception may haveevolved numerous times in different organisms, so thepeanut gynophore could have an unusual graviperceptionmechanism. The role of the starch sheath and the amylo-plasts of the peanut gynophore in graviperception has notyet been explored; it is the topic of the present work.

Studies that have partially elucidated the role of thestarch sheath and its amyloplasts in shoot graviperceptionhave involved incubating the plants with plant hormones,in darkness, in order to remove all the starch from withinthe amyloplasts (Pickard and Thimann, 1966; Iversen,1969). These destarching experiments have sparked con-troversy about the validity of the SSH, producing evidenceagainst and in favour of the hypothesis due to minormodifications in the destarching protocols.

Using the SSH as a framework, we hypothesize in thiswork that amyloplasts in the cells of the starch sheath areprimarily responsible for gravity perception in the peanutgynophore. Three lines of evidence are presented in thisstudy: (1) an anatomical analysis of amyloplast sedi-mentation correlated with the timing of the gravitropicresponse; (2) experiments in which the starch is removedfrom the gynophores and the subsequent gravitropicresponse measured; and (3) experiments using a rotatingdevice (a clinostat) to verify the importance of thesedimentation of the amyloplasts during graviperception.

MATERIALS AND METHODS

Plant material

Peanut seeds, ‘Virginia 93B’, were kindly provided byDr W. Mozingo (USDA-AREC, Suffolk, Virginia, USA).Plants were grown as described in Moctezuma and Feldman(1998).

Anatomical sections

Peanut gynophores (n¯ 15) were placed either verticallyor horizontally, excised at 6–8 mm from the tip and fixedimmediately in a formalin-acetic acid-ethanol solution (50%ethanol, 10% formalin, 5% acetic acid) overnight (16 h) at4 °C. Of the total samples, 11 were fully developed, fertilized,vertically growing, 20–40 mm long gynophores. The re-maining four sampleswere of gynophores before fertilizationof the ovules (and before any gravitropic response wasobserved). The orientation of the gynophores was main-tained during the fixation process by using metal pins toimmobilize the samples inside the fixation vials. The samples

F. 2. A, Longitudinal section of a vertically oriented, graviresponding gynophore. Notice the sedimenting amyloplasts in the starch sheath cells(arrowhead). This micrograph is also a water-treated control for the destarching experiments. B, Longitudinal section of a gynophore that hasbeen oriented horizontally for 30 min. Notice the sedimentation of the amyloplasts to the new lower surface of the endodermal cells (arrowhead).C, Longitudinal section of a destarched gynophore. Notice the absence of amyloplasts in the cells of the starch sheath (arrowheads). D, Highmagnification view of the intact starch sheath cells showing the sedimenting amyloplasts (arrowhead). E, High magnification view of destarchedstarch sheath cells, showing the absence of starch-filled amyloplasts (arrowhead). c, Cortex; p, pith; vb, vascular bundles ; g and arrow, direction

of the gravity vector. Bars for A–C¯ 80 µm; D–E¯ 20 µm.

were dehydrated, infiltrated with paraffin (Paraplast­,Oxford Labs, USA) and sectioned to 10 µm thickness.Sections were later stained with safranin, tannic acid, ironalum and Orange G, according to the staining protocol ofSharman (1943). Using this technique, amyloplasts of thestarch sheath are stained black.

Destarching experiments

It has been demonstrated previously that gynophoreexplants can be grown in �itro (Ziv and Zamski, 1975).Excised gynophores are able to continue their growth andgravitropic response in the culture media. The ability ofthese gynophore explants to grow in �itro was used to treatthese organs to similar destarching protocols by Pickardand Thimann (1966) and Iversen (1969).

Gynophores 20–40 mm long (n¯ 18) were excised fromthe plant, sterilized, recut to 15 mm in length and placed insterilized Petri plates containing Murashige and Skoogmedia, as in Ziv and Zamski (1975). The vertical orientationof the samples was always maintained during this process.

Two sets of samples were used. The first set was incubatedat 30 °C in a solution of 1¬10−& mol l−" of gibberellic acid(GA

$) and 7¬10−& mol l−" of kinetin for 36 h in the dark-

ness. The second set, a control, was kept under the sameincubation conditions as above, except that distilled waterreplaced GA

$and kinetin. After incubation, samples were

horizontally oriented for 48 h at 22 °C, and the anglesof curvature measured using a protractor as described inMoctezuma and Feldman (1998). In addition, the two sets(hormone-treated and water controls) were further sub-divided and gravistimulated either in complete darkness ordaylight. The final length of all gynophores was recorded inorder to determine the overall rates of growth duringtreatments.

To determine if any starch remained in the hormone-treated samples �s. the water controls, a preliminary test forstarch was performed by adding a few drops of iodine-potassium-iodide to freshly cut samples (Glenn et al., 1992).The hormone treated (destarched) samples showed nostaining, whereas the water controls showed a dark brownstaining, indicating the presence of starch within the tissues.In addition, some of the destarched and control sampleswere fixed, embedded, sectioned and stained with theSharman’s staining technique, as above, in order to detectany starch within the amyloplasts of the starch sheath.

Clinostat experiments

In �itro-grown gynophores were placed on a rotatingclinostat (Model T, 115V, Hurst Corporation, Princeton,Indiana,USA) in order to further investigate the relationshipbetween amyloplast sedimentation and the gravitropic

Moctezuma and Feldman—Gra�ity Perception in the Peanut Gynophore 711

F. 1. Anatomical analysis of amyloplasts in the starch sheath cells ofthe peanut gynophore. A, Longitudinal section of a peanut gynophore.Arrowheads indicate the location of the amyloplasts near the elongationzone, proximal to the intercalary meristem (im) and the seed region (s).Bar¯ 0±5 mm. B, Cross section of a peanut gynophore, showing theamyloplasts of the starch sheath (arrows) surrounding the vascularbundles (vb). C, Cross section of a destarched peanut gynophore.Notice the absence of amyloplasts in the starch sheath (arrows). D,Cross section of a young, unfertilized gynophore. No amyloplasts arevisible in the starch sheath cells (arrows) at this early stage of

development in the peanut gynophore. Bars for B–D¯ 100 µm.

F. 2. For legend see facing page.

response of the gynophore. The constant rotation [1revolution per minute (RPM)] of the clinostat is thought toprevent amyloplasts from sedimenting in a set place withinthe cell. Thus, the gynophore perceives an omnilateralgravity vector, since the rotational movement of the clinostatwill continuously keep changing the direction of the gravityvector.

Gynophores (n¯ 15) were cut to 15 mm in length, leavingthe tip and ovule region intact. Samples were immediatelyexplanted into a glass beaker containing Murashige andSkoog medium, as in Ziv and Zamski (1975). While somesamples (n¯ 9) were horizontally oriented and placed in thecentral axis of rotation of the clinostat, the remainder wereplaced horizontally into a stable (non-rotating) beaker. Theangle of bending after 70 h of gravistimulation was measuredon each of the samples.

RESULTS

The starch sheath of the gynophore consists of the innermostone–three cell layers of the cortex, surrounding the vascularbundles (Fig. 1B). Developmentally, the starch sheath isfirst observed with starch-filled amyloplasts after the ovuleshave been fertilized. Unfertilized gynophores are agravi-tropic (Shushu and Cutter, 1990), and do not exhibit starch-filled amyloplasts within the starch sheath (Fig. 1D). Thestarch sheath of mature (20–40 mm long) gynophores isvisible approx. 2 to 8 mm from the tip, in longitudinalsection. This area includes the main elongation zone of thegynophore, which occurs at 2–5 mm from the tip (Moc-tezuma and Feldman, 1998).

Amyloplasts in the starch sheath of a mature gynophoresediment readily with gravity. In longitudinal sections ofvertically-oriented gynophores, the amyloplasts always

712 Moctezuma and Feldman—Gra�ity Perception in the Peanut Gynophore

T 1. Destarching experiments : mean angles (s.e.) ofgra�itropic response after 48 h of gra�istimulation

Water control Hormone treated

Gravistimulation under continuouswhite light, at 25 °C

45±2 (2±7) 9±2 (0±6)

Gravistimulation under darkness, at 25 °C42±5 (7±5) 7±0 (2±1)

All figures in degrees of curvature, where 0¯horizontal and90¯ vertically downwards.

T 2. Clinostat experiments: mean angles of cur�atureafter 70 h of gra�istimulation

Treatment Mean curvature (s.e.)

Stable controls 58±2 (3±7)Clinostat rotated (1 RPM) 2±3 (0±4)

All figures in degrees of curvature, where 0¯horizontal and90¯ vertically downwards.

sediment to the lowermost surface of the cell, in thedirection of the gravity vector (Fig. 2A and D). However,in gynophores reoriented horizontally for 30 min, theamyloplasts sediment towards the new lower surface of thestarch sheath cells (Fig. 2B). It is important to note that theamyloplasts sediment before a gravitropic response cur-vature occurs, which is 2 h after reorientation (Moctezumaand Feldman, 1998).

In destarched gynophores an 82% decrease in thecurvature angle of the gravitropic response occurs comparedto water-treated controls (Table 1). Amyloplasts are nolonger visible in the destarched gynophores (Figs 1C, 2Cand E) because the plastids have been depleted of starch.Results were not statistically different between gynophoresgravistimulated in light or dark conditions (Table 1).

Both the destarched gynophores and the water-treatedcontrols continued to grow during the treatments (at approx.0±03–0±04 mm h−"), indicating that the destarching hormonetreatment did not affect the growth of the samples.

Results of the clinostat experiments indicate that after70 h of reorientation there was a marked difference in thegravitropic response between the gynophores placed on theclinostat �s. the controls. These experiments corroborate thehypothesis that the bending response in the horizontally-oriented gynophore is a true gravitropic response. Rotationin the clinostat prevented the amyloplasts from sedimentingin a stable place inside the starch sheath cells of thegynophore, thus resulting in a dramatic decrease (96%) inthe mean curvature of the gravitropic response. As in thedestarching experiments, these gynophores also continuedtheir growth in �itro for the duration of the clinostatexperiments.

DISCUSSION

Anatomical sections of reoriented gynophores show rapidsedimentation (within 30 min) of the amyloplasts to the new

lower surfaces of endodermal cells. These amyloplastsappear after fertilization of the ovules, since unfertilizedgynophores do not contain visible amyloplasts (Fig. 1D);nor do they respond to gravity in the same way as fertilizedgynophores. Thus, in the peanut gynophore, there is astrong correlation between amyloplast development in thestarch sheath and the onset of the gravitropic response.

Destarching experiments have produced contradictoryresults in the past. Pickard and Thimann (1966) first used aprotocol in which coleoptiles were incubated in darkness forseveral hours. Although they observed the complete absenceof starch in the samples (under light microscopy), the wheatcoleoptiles were still able to respond to gravity (although ata slower rate than intact coleoptiles). Iversen (1969) alsoperformed similar destarching experiments. By raising thetemperature of the incubation period, Iversen (1969) wasable to completely deplete the amyloplasts of starch and toabolish the gravitropic response of cress roots.

In this paper we used a destarching protocol similar tothat used by Iversen (1969), to fully deplete the amylo-plasts of the peanut gynophore of starch. These destarchedgynophores showed an 82% reduction in the angle ofgravitropic response curvature, compared to water-treatedcontrols. Destarching protocols have been criticized becauseof the possible growth-stunting effects that the hormonetreatments may have on the plant organs as a whole.However, growth measurements in this study showed thatboth destarched and control gynophores continued to growin �itro—only the gravitropic response was inhibited in thedestarched gynophores.

A possible explanation for the results obtained in thedestarching experiments is that the amyloplasts within thegynophore were depleted of starch, leaving only emptyplastids within the endodermal cells. Previous studies(Iversen, 1969; reviewed by Sack, 1991) have shown thatstarch-depleted plastids are less dense than normal, starch-filled plastids, and therefore do not sediment as readily asthe controls. Although the starch-depleted amyloplasts mayhave retained some gravity sensing properties (destarchedgynophores did bend slightly), these empty plastids were notsufficiently dense to produce a full gravitropic response inthe peanut gynophore.

The use of a clinostat for the study of plant gravitropicsensing has been criticized by many (Moore, 1990), mainlybecause of the possible deleterious effects that this devicemay have on the plant. However, for the purposes of thisstudy, the clinostat experiments provided useful data whichcorroborated: (1) that the downwards bending of thegynophore is a true gravitropic response; and (2) bypreventing the amyloplasts from sedimenting during clino-stat rotation, it is possible to reduce gravitropic perceptionand, consequently, gravitropic response. The use of theclinostat provided additional evidence in favour of theamyloplasts in the starch sheath of the gynophore as thelikely gravity perceptors for the organ. Furthermore, theseclinostat experiments also provide an additional control ofan intact graviresponding gynophore grown in �itro, to becompared with hormone-treated and water-treated gyno-phores used in the destarching experiments.

Shushu and Cutter (1990) suggest that the intercalary

Moctezuma and Feldman—Gra�ity Perception in the Peanut Gynophore 713

Vertically oriented Horizontally oriented

A B

g

F. 3. Diagrammatic representation of sedimenting amyloplastsduring gravistimulation. A, Vertically oriented gynophore, withsedimenting amyloplasts (E) located in endodermal cells. B, Hori-zontally oriented gynophore. The strategic location of the starch sheathand the sedimenting amyloplasts produce a structural asymmetrybetween the upper and lower halves of the organ. In the upper starchsheath, the sedimenting amyloplasts are closer to the cells of thevascular bundles (7). The amyloplasts of the lower starch sheath,however, are in direct contact with the cortex cells of the lower surface.This structural asymmetry may be important for the upcomingphysiological events that occur during the gynophore’s gravitropicresponse. g and the arrow indicate the direction of the gravity vector.

meristem (IM) of the peanut gynophore may be the site forgravity perception. This statement is only partially true,since some of the amyloplasts in the starch sheath arelocated near the IM of the gynophore (Fig. 1A). However,the actual meristematic cells of the IM are unlikely to playthe role of gravity perceptors for the gynophore per se.Why? As Bjo$ rkman (1992) calculated in his study, the smallsize of meristematic cells does not physically allow them toperform such a task. In addition, their dense cytoplasmiccontent and their lack of sedimenting amyloplasts or otherorganelles, make the IM a poor candidate for the site ofgravity sensing in the peanut gynophore.

In conclusion, the evidence presented in this study stronglysuggests that the amyloplasts of the starch sheath are primecandidates for the role of gravity perceptors in the peanutgynophore. Recent evidence in other plant systems also pro-vides additional data in support of the starch sheath as thesite of graviperception in plant shoot systems. Kiss et al.(1997) show that hypocotyls of starch-deficient mutants ofArabidopsis exhibit reduced gravitropism. Similarly, Fukakiet al. (1998) provide genetic evidence in favour of theamyloplasts as graviperceptors, with Arabidopsis mutantsthat lack a starch sheath and which fail to perceive andrespond to gravity. In addition, Kuznetsov and Hasenstein(1997) performed magnetophoresis experiments using barleycoleoptiles in which the amyloplasts of the starch sheathwere displaced by a magnetic field, followed by a bendingresponse.

Furthermore, the strategic location of the starch sheath(in a ring surrounding the vascular bundles) may also beimportant in the physiological events that occur during thegravitropic response. The sedimenting amyloplasts create astructural asymmetry between the upper and lower halves ofthe peanut gynophore (Fig. 3). In the upper endodermalcells, the amyloplasts contact the cells of the vascular

bundles, whereas the amyloplasts of the lower starch sheathare closer to the cells of the cortex. This structural asymmetrycreated by the strategic location of the starch sheath cells,may later translate into a physiological asymmetry, whichwould result in an unequal growth response between theupper and lower surfaces—and the eventual downwardbending—of the gynophore. Many workers (Sack, 1991;Bjo$ rkman, 1992) believe that the contact of the amyloplastswith the plasma membrane of the starch sheath cells canmechanically open ion channels in these cells. Calcium andother molecules (such as plant hormones) may be trans-ported from within the tissues upon opening and closing ofthese channels, thus creating physiological differencesbetween the upper and lower halves. As other studies haveshown (Moctezuma and Feldman, 1996, 1999; Moctezuma,1999), the plant hormone indole-3-acetic acid is redistributedasymmetrically to the upper surface of a horizontally-oriented gynophore, thus creating a growth asymmetrybetween the upper and lower surfaces which eventuallyleads to the downwards bending of the organ.

Some final questions still remain open for future study: ifthe positively gravitropic peanut gynophore perceives thegravity stimulus in the same way as a negatively gravitropicshoot, why does the peanut bend down, while a typicalshoot bends up? Which step in the transduction process hasto be ‘switched’ in the gynophore, in order to obtain anopposite gravitropic response? The answers to these andother questions should provide us with important clues toelucidate the signal transduction mechanisms that governgravitropism in higher plants.

ACKNOWLEDGEMENTS

We thank Dr Steven Ruzin and Dr Denise Schichnes fromthe Biological Imaging Facility for their technical help,David Welch for help with the images, Dr W. Mozingofrom USDA for providing the Virginia 93B peanut seeds,and Denise A. Benoit for critically reading this manuscript.This work was supported by a research fellowship from theNational Aeronautics and Space Administration to E.M.

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