Annals of Botany 83 : 235–242, 1999Article No. anbo.1998.0814, available online at http:}}www.idealibrary.com on

Changes in Auxin Patterns in Developing Gynophores of the

Peanut Plant (Arachis hypogaea L.)

EDGAR MOCTEZUMA*

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

431 Koshland Hall, Berkeley, CA 94720, USA

Received: 6 August 1998 Returned for revision: 31 August 1998 Accepted: 9 November 1998

The peanut plant (Arachis hypogaea L.) produces flowers aerially, but buries the recently fertilized ovules in the soilin order for the fruit and seeds to mature underground. The organ that carries the seeds into the soil is called thegynophore. The growth of the peanut gynophore is regulated primarily by indole-3-acetic acid (IAA). A monoclonalantibody raised against IAA was used to successfully detect and localize this growth substance in the tissues ofdeveloping peanut gynophores. Five different stages of development were analysed: (1) before fertilization; (2) afterfertilization; (3) during downwards growth; (4) at soil penetration; and (5) at the early stages of fruit formation.While no auxin signal is visible in the unfertilized ovules and ovary region, an asymmetric signal is observed in thegynophore wall after fertilization. During downwards growth, the auxin signal is located in both the meristematicregion and in the area encircling the seeds, as well as in the cortex and epidermis region of the elongation zone. Uponsoil penetration, the auxin signal in the meristematic region disappears, and most of the signal is detected in thegynophore wall near the tip. At the early stages of peanut fruit development, auxin signal is found at the lowermostarea of the bending fruit, which eventually causes the fruit to be positioned horizontally. The results of this studysuggest that the possible source of auxin within the gynophore may be the area of the gynophore wall close to thetip. # 1999 Annals of Botany Company

Key words : Arachis hypogaea, antibody, auxin, development, groundnut, gynophore, immunolocalization, indole-3-acetic acid (IAA), peanut.

INTRODUCTION

The groundnut or peanut plant (Arachis hypogaea L.) hasan extraordinary growth habit : it is able to ‘sow’ its ownseeds. This phenomenon is called geocarpy, which meansthat the flowers are produced aerially, but the fruit and seeddevelop underground (Smith, 1950). Once the ovules of thepeanut flower are fertilized, a specialized organ called thegynophore begins to form. The gynophore elongates andgrows downwards, exhibiting a positively gravitropic beha-viour (Jacobs, 1951; Shushu and Cutter, 1990; Moctezumaand Feldman, 1998). The gynophore carries the young seedsand buries them into the soil, where fruit and seedmaturation eventually occur (Fig. 1 ; Smith, 1950).

The morphology and anatomy of the growing peanutgynophore and the eventual fruit formation have beenthoroughly described by several workers (Ziv and Zamski,1975; Periasamy and Sampoornam, 1984; Pattee andMohapatra, 1987). It is well established that after fert-ilization of the ovules, an intercalary meristem forms at thebasal region of the gynophore, forming a straight 3–4 mmlong structure. The gynophore then begins to benddownwards towards the ground, and as its tip reaches thesoil the cells of the intercalary meristem elongate and losetheir meristematic activity (Periasamy and Sampoornam,1984). Finally, as the gynophore tip begins to swell

* Fax ­510 642-4995, e-mail edgar!nature.berkeley.edu

underground, the cells of the developing fruit begin toelongate more on the dorsal side (proximal to the basalovule) than at other regions of the fruit—this eventuallyresults in the horizontal orientation of the fruit proper (Fig.1d–f ; Periasamy and Sampoornam, 1984).

The growth and development of the gynophore and thepeanut fruit are controlled by several plant growth sub-stances (Shushu and Cutter, 1990; Shlamovitz, Ziv andZamski, 1995). Shushu and Cutter (1990) studied the rolesof the auxin indole-3-acetic acid (IAA), gibberellic acid(GA) and cytokinin during the growth and development ofthe peanut gynophore: they found that a possible com-bination of IAA and GA may play a crucial role in theelongation of the gynophore. Shlamovitz et al. (1995)measured the levels of ethylene, IAA and abscisic acid(ABA) at three different stages of gynophore and poddevelopment: before soil penetration, after soil penetrationand during pod development. Shlamovitz et al. (1995) foundthat the levels of ethylene increased slightly during soilpenetration, the levels of IAA remained relatively constantat all three stages, and the levels of ABA decreased three- tofive-fold after soil penetration and pod development.

Several studies show that IAA plays a key role in thegrowth of the gynophore and in the development of thepeanut fruit (Ziv and Zamski, 1975; Shushu and Cutter,1990; Moctezuma and Feldman, 1998). IAA has many rolesin plants, including stimulation of cell enlargement anddivision, mediation of tropistic responses, and induction of

0305-7364}99}030235­08 $30.00}0 # 1999 Annals of Botany Company

236 Moctezuma—Auxin Patterns in De�eloping Peanut Gynophores

F. 1. Different stages of peanut gynophore development. The peanutflower before fertilization of the ovules (a) ; peanut gynophore growingdownwards, 4–6 d after fertilization (b) ; 6–10 d after fertilization, withthe gynophore tip penetrating the soil surface (c) ; 3–5 d after soilpenetration, at the early stages of fruit development (d) ; 2–3 weeksafter fertilization, showing the development of the peanut fruit (e andf) ; and a mature peanut fruit, with the seeds exposed (g). Bar¯ 1 cm.

fruit set in many species (Davies, 1995). Since all theseevents take place during the growth of the peanut gynophore,much research has focused on the role of IAA in the growthand development of the peanut gynophore. Previous studies(Jacobs, 1951; Ziv and Zamski, 1975) provide correlativeevidence indicating that IAA regulates the growth of thepeanut gynophore and the development of the peanut fruit.The crucial role that IAA plays in the gravitropic responseof the peanut gynophore has already been described(Moctezuma and Feldman, 1996). Other physiologicalstudies by Shushu and Cutter (1990) and Moctezuma andFeldman (1998) further corroborate the importance of IAA,IAA transport and the seed (tip) region in the growth anddevelopment of the peanut gynophore. Shushu and Cutter(1990) found that both excising the tip or preventing IAAtransport from the tip region to the intercalary meristeminhibited the growth of the gynophore. Conversely, theyalso found that excising the tip region and exogenouslyapplying IAA partially restored growth in the gynophore.

Despite all the previous work on the role of IAA in thegrowth and development of the peanut gynophore, therehas been no study linking anatomical and morphologicaldata to physiological evidence (i.e. the plant growthsubstance interactions within the tissues). Studies havecorroborated the presence and physiological activity of IAAin the growth and development of the peanut gynophore(Shushu and Cutter, 1990; Shlamovitz et al., 1995), butthese physiological events have not been correlated with themorphological and anatomical changes that occur indeveloping gynophores. Therefore, many questions stillremain unanswered: (1) what is the likely source of IAA inthe downward-growing peanut gynophore? Although bothJacobs (1951) and Shushu and Cutter (1990) concluded thatthe probable source of IAA was young seeds, they onlyprovided correlative evidence for this claim (their tip-

excision experiments). The specific source of IAA in thepeanut gynophore is still uncertain: the tip region consistsnot only of the seeds, but also the intercalary meristem andthe gynophore wall. (2) What are the spatial and temporalpatterns of auxin distribution within the tissues of thegynophore at different stages of development (i.e. fromflower to fruit formation)? (3) Where does auxin activitytake place in the tissues of the developing gynophore? (4) Ifthe patterns of auxin localization differ at each develop-mental stage, do these patterns correlate with morpho-logical and cellular changes that occur in the developingpeanut gynophore?

The answers to these and many other questions are nowattainable thanks to the advancement of plant growthsubstance immunoassay techniques (Shi, Miller and Moore,1993; Pence and Caruso, 1998; Caruso, Pence and Leverone,1995). In the present study, a monoclonal antibody raisedagainst IAA was used to successfully detect and localize thisgrowth substance in the tissues of peanut gynophores atdifferent stages of development—from pre-fertilization tothe beginning of fruit formation.

MATERIALS AND METHODS

Peanut plants (Arachis hypogaea ‘Virginia 93B’) were grownas previously described by Moctezuma and Feldman (1998).Gynophores were excised from the plant at different stagesof development. Five to ten sample gynophores of eachdevelopmental stage were used in these experiments, andeach experiment was repeated at least three times. The fivedevelopmental stages studied were: (1) pre-fertilization; (2)10–24 h after fertilization; (3) during downward growth; (4)during soil penetration; and (5) at the early stages of fruitformation.

Excised samples were immediately fixed in freshly pre-pared 3% aqueous 1-ethyl-3-(dimethyl-aminopropyl)-car-bodiimide hydrochloride (EDAC, Sigma, USA) for 1 h at4 °C under vacuum. EDAC crosslinks the carboxyl group ofIAA to structural proteins in the plant tissues, and alsoserves to preserve the antigenicity of IAA to this particularmonoclonal IAA antibody (Shi et al., 1991; Caruso et al.,1995). Subsequently, tissues were postfixed for 2–4 h informalin-acetic acid-alcohol (FAA) at 4 °C under vacuum,dehydrated with a graded ethanol series, embedded inparaffin and sectioned to 10–12 µm. After drying at 42 °C,sections were deparaffinized and hydrated in an ethanol-water graded series. Slides were incubated for 45 min atroom temperature (RT) in a blocking solution [10 m

phosphate solution (pH 7), 0±1% Tween-20, 1±5% glycene,and 5% w}v bovine serum albumin (BSA)]. Sections wererinsed with regular salt rinse (10 m phosphate solution,0±0088% w}v NaCl, 0±1% Tween-20, and 0±8% w}v BSA),and briefly washed with a PBS}BSA solution (10 m

Phosphate solution, 0±8% BSA). A 1:20 dilution of theprimary IAA antibody (1 mg per ml of water) in PBS}BSAsolution was made, and 40 µl were added to each section.The monoclonal IAA antibody, kindly provided by Dr JohnL. Caruso (University of Cincinnati), was raised againstcarboxyl-linked IAA in mice (see Leverone et al., 1991;

Moctezuma—Auxin Patterns in De�eloping Peanut Gynophores 237

Pence and Caruso, 1988; Caruso et al., 1995). Sections wereincubated with this primary IAA antibody in a humidchamber for 4 h at RT in darkness, then washed vigorouslytwice in a high salt rinse solution (10 m phosphate solution,2±9% w}v NaCl, 0±1% Tween-20 and 0±1% w}v BSA), andthen washed again with the regular salt rinse and thePBS}BSA solution. The secondary antibody [Anti-mouseIgG AP conjugate (1 mg ml−"), Promega, USA] was diluted1:20 in PBS}BSA solution, and 50 µl were added to eachsection. Sections were incubated with the secondary anti-body as described above for 4 h, and then rinsed twice inregular salt rinse, and once in distilled water. The slides werethen developed for approx. 1 h by adding 200 µl of WesternBlue stabilized substrate for alkaline phosphatase (Promega,USA). As the blue}purple colour was observed on thesections, they were rinsed with water, dehydrated andmounted with a coverglass for photographing. For eachdevelopmental stage, a representative sample is shown (Figs3–6).

Several controls were included to verify the effectivenessof the immunolocalization technique and the specificity ofthe monoclonal IAA antibody. First, a set of five negativecontrols were performed in cross sections of 30–50 mm-long, downwardly-growing peanut gynophores, at theelongation zone, approx. 2–5 mm from the tip (Moctezumaand Feldman, 1998). In the first three controls, EDAC pre-fixation, the primary monoclonal antibody and the secon-dary antibody were each omitted respectively from theprocedure. In the fourth control, the primary IAA antibodywas substituted with a control antibody (P3X63), whichhas no known antigen (Leverone et al., 1991). Finally, acompetition assay control was also performed as describedby Kerk and Feldman (1995). The primary IAA antibodywas incubated overnight with the antigen (a 1¬10−$

solution of IAA) for a saturation of the binding sites of theprimary IAA antibody. The primary antibody-IAA solutionmixture was applied to the tissue sample. This control wasperformed to determine the specificity of the primaryantibody for IAA (Kerk and Feldman, 1995).

Several positive controls were also included to verify thespecificity of the antibody for IAA. First, 10−% IAA inagar blocks were substituted for the excised tips (approx.0±5 mm) of gynophores for 1 h. Other replicate gynophoreswere given agar blocks without any IAA. After immediatefixation with EDAC, the complete immunolocalizationprocedure was performed on the samples to detect anyauxin signal.

A second positive control performed to verify thespecificity of the monoclonal antibody to IAA was animmunoblot assay. Different concentrations of IAA (from10−# to 10−& ) were blotted (10 µl each) onto a membrane(PVDF, 0±45 µm, Millipore, USA) and dried for 3–5 min.The first column contained BSA, the second aqueous IAA,the third IAA with BSA, and the fourth had IAA, BSA and3% EDAC. After blocking, primary and secondary anti-body incubation, and washing with PBS (as in theimmunolocalization procedure described earlier), themembrane was developed with stabilized substrate foralkaline phosphatase to detect any IAA signal on eachcolumn. Only the column with the IAA, BSA and EDAC

T 1. Radioacti�e "%C-IAA blotting assay to test forEDAC crosslinking of IAA

Treatment: IAA IAA}BSA IAA}BSA­EDAC

Before washing 16130 17500 16860After washing 380 250 2230

All figures are in counts per minute (cpm), and the percent error wastypically 1±5 to 4%.

showed a concentration-dependent, positive signal (datanot shown).

A final control for the IAA immunolocalization techniquewas performed to quantify the percentage of IAA that iscrosslinked with the EDAC. "%C-IAA (0±3 µl ml−" of1–15 mCi per mmol, Sigma, USA) was blotted onto aPVDF membrane as above. Three dots in the membranecontained: (1) a radioactive IAA solution; (2) radioactiveIAA with BSA; and (3) radioactive IAA with BSA and with3% EDAC. After blotting, one replicate membrane wasimmediately cut, placed in a scintillation vial, filled with4 ml of scintillation fluid (ScintiVerse BD, Fisher, USA)and counted on a scintillation counter (Beckman, USA).Another sample underwent the complete blocking and PBSwashing treatment as for the immunoblot assay. Thiscontrol was repeated twice. Table 1 shows the results of thiscontrol.

RESULTS

Controls

The immunolocalization procedure controls corroboratethe specificity of the monoclonal IAA antibody for auxinwithin the tissues. The tissue sections with the completeprocedure (Fig. 2A) show that the auxin signal is localizedin the entire cortex and epidermis of the gynophore; in a‘ring’ that surrounds the vascular bundles. Omitting EDACpre-fixation, primary antibody incubation, and secondaryantibody incubation (Fig. 2B–D, respectively), results insections without any signal staining. Similarly, no auxinsignal was detected in samples in which the primary IAAantibody was substituted with the antibody P3X63 whichhas no known antigen and does not bind specifically to eitherIAA, the secondary antibody or to anything else in thesections. Another control performed for this immuno-localization procedure was a competition assay (Kerk andFeldman, 1995). By incubating the primary IAA monoclonalantibody with the antigen (in this case, IAA), the bindingsites of the antibody became saturated. Thus, the tissuesections of this control showed very little signal staining(Fig. 2F).

Because the antibody used in this work is also reported tobe specific to methyl-IAA (Pence and Caruso, 1988), it wasnecessary to verify that the fixating procedure, including thecrosslinking with EDAC, preserved the antigenicity of IAAfor the antibody. The results of the positive controls verifiedthe specificity of the IAA antibody for IAA in thisimmunolocalization technique. First, adding IAA in agarblocks to the excised tips of gynophores resulted in apositive, blue}purple signal at the zone of IAA application,

238 Moctezuma—Auxin Patterns in De�eloping Peanut Gynophores

F. 2. Controls for the IAA immunolocalization technique. A, Cross section of a 3 cm long gynophore with the complete IAAimmunolocalization procedure, showing the IAA signal in the cortex and epidermis ; B, without EDAC prefixation; C, without the primary IAAantibody; D, without the secondary IAA antibody. E, The primary IAA monoclonal antibody was substituted with the control antibody P3X63,which has no known antigen. F, A competition assay, in which the monoclonal IAA antibody was incubated with a 1¬10−$ IAA to saturate

its epitopes. Bar¯ 0±35 mm.

whereas the blocks without any IAA did not give any signalat the cut surface (data not shown). The immunoblot assayalso showed that the antibody used in this work was able torecognize IAA, since the IAA blotted with BSA and EDACresulted in a positive, concentration-dependent signal in themembrane (data not shown). The results of the final blottingassay, shown in Table 1, demonstrate that EDAC crosslinksto a filter approx. 13% of the radioactive IAA—which isnot removed by either blocking or washing of the membrane.These results also confirm that the immunolocalizationassay used in this work is sensitive enough for detecting IAAwithin the plant tissues. Shlamovitz et al. (1995) reportedthat the amounts of IAA in the developing gynophore varyfrom 0±4 to 0±6 ng mg−". The first immunoblotting assaygives a signal even at the lowest concentration of IAA used(10−& ), which means that the immunolocalization tech-nique can detect at least 1±74¬10−$ ng mg−" of IAA withinthe tissues of the gynophore. Thus, even after losing someendogenous IAA (approx. 87%) by blocking, washing andincubating the tissues, the sensitivity of the immuno-localization technique easily allows the detection of IAAin the amounts naturally found in the gynophore.

IAA patterns at different de�elopmental stages

The patterns of IAA immunolocalization staining variedgreatly in the five developmental stages of peanut gynophorestudied (pre-fertilization, fertilization, downward growth,soil penetration and early fruit formation).

First, a dramatic change in the localization of the auxinsignal was observed as a result of fertilization of the ovules

F. 3. Patterns of IAA immunolocalization during fertilization. A,Longitudinal section of the gynophore before fertilization, showing noIAA signal. B, Recently fertilized gynophore (10–24 h after fert-ilization), showing the IAA signal in the adaxial side of the gynophore.

Bar¯ 0±2 mm.

in the peanut. At the pre-fertilization stage, the tissuesections showed no auxin signal (Fig. 3A). However, oncethe ovules had been fertilized, and the gynophore began togrow, an asymmetric localization of the auxin signalappeared in the post-fertilized gynophore (Fig. 3B). Noticethat only the adaxial side of the gynophore (the left side ofFig. 3B), showed the auxin signal.

Moctezuma—Auxin Patterns in De�eloping Peanut Gynophores 239

F. 4. Patterns of IAA immunolocalization of the peanut gynophore. A, Downward-growing, positively gravitropic gynophore, 3 cm long, withmost of the IAA signal located in the epidermis, cortex and the meristematic region around the seeds. B, A gynophore that has recently penetratedthe soil, with most of the IAA signal located near the tip. C, Early stages of fruit development inside the soil, with the IAA signal located at theventral (lowermost) surface. This ventral surface will grow faster and will eventually lead to the typical horizontal orientation of the peanut fruit.

Bars¯ 0±7 mm.

F. 5. IAA signal in the intercalary meristem. Before soil penetration(A), the IAA signal is localized in the gynophore wall near the tip andin the intercalary meristem (im) region surrounding the seeds (s). Uponsoil penetration (B), the IAA signal at the intercalary meristem regionis no longer present (arrowhead), and most of the signal is localized in

the gynophore wall near the tip. Bar¯ 0±25 mm.

In vertically growing young gynophores (30 to 50 mmlong), staining occurred in the cortex and epidermis (Fig.4A), surrounding the gynophore’s vascular bundles (Fig.2A). Besides the auxin signal detected in the epidermal andcortical region, staining also occurred in the meristematicregion of the gynophore, surrounding the seeds in a ‘halo’-like pattern (Fig. 5A). Once the gynophore tip penetratedthe soil, a strong signal was detected near the tip of thegynophore (Fig. 4B). However, the auxin signal around theseeds in the meristematic region of the gynophore (i.e. the‘halo’-like pattern) vanished during the soil-penetrationstage (Fig. 5B).

F. 6. Cross section of the seed region, approx. 0±7 mm from the tip.No IAA signal was located in the seeds (s) at any stage of gynophore

development. Bar¯ 0±2 mm.

In the early stage of fruit formation (3–5 d after soilpenetration), the auxin signal was detected in the lowermostside of the bending gynophore at the epidermal and corticalcell layers (Fig. 4C). This lower side presumably growsmore in order for the fruit to develop horizontally orparallel to the soil surface (see Fig. 1d–g).

No staining was detected in the peanut seeds at any of thefive stages of gynophore development analysed in this study.In a cross section of the seed region of a downward-growinggynophore, no auxin signal was observed in the seeds(Fig. 6).

DISCUSSION

The different patterns of auxin that occur in the tissues ofthe peanut gynophore at different stages of development(from pre-fertilization to fruit formation) were investigated.Using an immunoassay procedure with a monoclonal IAAantibody, the immunolocalization of endogenous auxin

240 Moctezuma—Auxin Patterns in De�eloping Peanut Gynophores

within the tissues of developing gynophores was successfullyachieved.

The different controls performed for this immuno-localization technique corroborate the specificity of themonoclonal IAA antibody to endogenous auxin in the planttissues (Fig. 2A–F, Table 1). Pence and Caruso (1988)reported the isolation of the monoclonal antibody used inthis work, raised against IAA in mice by a carrier proteincoupled to the carboxyl group of IAA. This monoclonalantibody has a strong crossreactivity to methyl-IAA, andvery weak crossreactivity to free IAA (Pence and Caruso,1988). However, the three positive controls performed inthis work indicate that at least for the immunolocalizationprocedure employed in this work, the use of this monoclonalantibody is effective for the immunodetection of IAA inplant tissues. The controls also highlight the importance ofthe EDAC pre-fixation in the immunolocalization techniqueutilized in this work. More specifically, EDAC may playtwo essential roles in this procedure: first, it fixes andimmobilizes free IAA within the tissues ; second, thiscrosslinking may modify the carboxyl group of IAA enoughto make free IAA detectable with the monoclonal IAAantibody. Previous work reports the modification ofcarboxyl groups of other molecules with EDAC (Mejillanoand Himes, 1991), so it is likely that the carboxyl group ofIAA, during the crosslinking to structural proteins withinthe tissues with EDAC, is modified enough to allowdetection of the crosslinked IAA with the monoclonal IAAantibody used in this work. Thus, although the possibilitythat this IAA antibodymay also detect other IAA conjugatescannot be discarded, all of the positive and negative controls,coupled with the use of EDAC for crosslinking IAA,strongly indicate that the immunolocalization of IAA withinthe tissues of the gynophore was successfully attained in thiswork.

Although many studies have shown a physiologicalimportance for IAA in developing gynophores, these studieshave not been very specific as to which particular tissuescontain IAA. Shlamovitz et al. (1995) measured thequantities of IAA at different stages of gynophore and poddevelopment. They found that the amount of IAA variedbetween 0±4 and 0±6 ng mg−" d. wt by an enzyme linkedimmunosorbent assay (ELISA) method. They did not detectstatistically significant differences in IAA amounts at thethree developmental stages (before soil penetration, aftersoil penetration, and during pod development).One problemwith the data of Shlamovitz et al. (1995) is that they usedwhole tips (0±5 to 1 cm long) of gynophores which includedovaries, gynophore wall, intercalary meristem and adjacentgynophore tissue. In other words, it was impossible todistinguish exactly where the IAA was located within thedifferent tissues at the different stages of development. Animmunolocalization study for auxin at the different phasesof gynophore development was necessary to elucidate thispoint.

A dramatic change in the pattern of IAA localizationoccurs after fertilization of the ovules in the peanut flower.While no signal is visible in the unfertilized ovules and ovaryregion, a very strong signal is detected near the tip and thegynophore wall of the fertilized gynophore (Fig. 3). Thus, it

is possible that the fertilization process itself triggers eitherthe transport or a de no�o synthesis of IAA in the recently-fertilized gynophore. Nevertheless, this increase in auxinsignal is not symmetric : more signal is localized in theadaxial side of the fertilized gynophore (Fig. 3B). Thisasymmetric auxin localization eventually leads to moregrowth on one side of the organ, which subsequently leadsto the downward bending of the gynophore towards thesoil.

When the gynophore is growing vertically downwards,auxin is localized in the epidermal and cortical tissue of theelongation zone, surrounding the vascular bundles (Figs 2Aand 4A). It is well established that auxin stimulates cellelongation by loosening the cell walls of plant organs (Rayleand Cleland, 1992). Thus, it is plausible to find a strongauxin signal exactly in the cortex and epidermis of theelongation zones (Moctezuma and Feldman, 1998) ofgynophores that are growing down towards the soil.

IAA also promotes cell division in plant organs (Davies,1995). In vertically growing gynophores, cell division occursprimarily in the intercalary meristem that is located proximalto the seeds (Shushu and Cutter, 1990). In agreement withthis idea, the auxin signal was located in both themeristematic region and in the area encircling the seeds, ina ‘halo’-like pattern (Fig. 5A). This finding indicates thatthe auxin detected in this zone may be directly involved instimulating and}or maintaining the meristematic activity ofthis region.

A very different pattern of auxin localization was observedin gynophores which had already penetrated the soil : (1) thesignal in the meristematic region disappeared; and (2) mostof the signal was detected in the gynophore wall near thetip (Figs 4B and 5B). The loss of signal in the regionsurrounding the seeds corroborates a previous observationthat the intercalary meristem of the gynophore, soon aftersoil penetration, loses its meristematic activity behind thebasal ovule (Periasamy and Sampoornam, 1984). At thepresent time, it is not known whether the absence of auxin(and therefore auxin signal) causes the loss of meristematicactivity or �ice �ersa, but a strong correlation between thesetwo events was observed in this work.

In addition, the tip of the soil-penetrating gynophorepresents a more rounded shape, as compared to the aerialgynophore. It is possible that this change in shape is causedby the increased concentration of auxin that was foundprecisely in the gynophore wall closest to the tip region.Previous work indicates that the gynophore tip undergoesdramatic changes in cell number, and in the function andvolume of the surface cells once it enters the soil (Periasamyand Sampoornam, 1984; Webb and Hansen, 1989). Thisenhanced accumulation of auxin signal near the tip, and theincreased growth that occurs in this region, may be anecessary step for the gynophore to burrow further into thesoil ; mature peanut fruits can be found 4–8 cm into theground.

At the early stages of peanut fruit development, an auxinsignal can be found along the ventral surface of the bendingfruit (Fig. 4C). This lowermost tissue is presumably thezone that must grow more rapidly in order for horizontalbending of the mature fruit to take place (Fig. 1d–f). These

Moctezuma—Auxin Patterns in De�eloping Peanut Gynophores 241

results agree with previous experiments in which moreradioactively-labelled IAA signal was found in the bottom(lowermost) half of bending gynophores (Shushu andCutter, 1990). In other studies, it has been reported that, atthe early stage of fruit maturation, more rapid cellenlargement occurs at the dorsal side proximal to the basalovule than in other regions of the peanut fruit (Periasamyand Sampoornam, 1984). This unequal cell enlargement,probably attributable to the increased accumulation of IAAin the lowermost, ventral surface, eventually leads to thetypical horizontal orientation in which the fruit proper isusually found underneath the soil (Fig. 1d–f).

Finally, one question still remains unanswered: what isthe source of auxin in the growing peanut gynophore?Previous studies suggest that young seeds are the mainsource of IAA involved in the growth of the peanutgynophore (Jacobs, 1951; Shushu and Cutter, 1990).However, the results of this work indicate that this may notnecessarily be the case : no auxin signal was detected in theseeds of the peanut gynophore at any stage of development(Figs 3–6). One plausible explanation may be that the IAAconcentration in seeds is below the threshold of detectionfor this immunolocalization procedure. Another possibilityis that the seeds produce precursors of IAA, IAA conjugates,etc. which are eventually converted to the IAA detectable inthe wall, cortex and epidermis of the gynophore. Anotherexplanation is that the seeds may not be the source of IAAin the peanut gynophore: IAA may be synthesized de no�oin the gynophore wall near the tip, and then transported tothe elongation zones of the gynophore. In addition, otherplant growth substances in the seed may be present, and,directly or indirectly (through their possible interaction withIAA), may be responsible for the growth and developmentof the peanut gynophore and fruit. Due to the limitations ofthe IAA immunolocalization technique, it is not yet possibleto provide evidence in favour of either of these proposals ;further studies on the synthesis and transport of IAA in thepeanut gynophore are needed.

In conclusion, auxin plays an important role in thedifferent developmental stages of the peanut gynophore:from fertilization to the early stages of fruit maturation.Although other plant growth substances such as gibberellins,cytokinins (Shushu and Cutter, 1990), ABA, ethylene(Shlamovitz et al., 1995) and even brassinosteroids may beinvolved in the growth and development of the peanutgynophore, IAA seems to be of key importance in thisprocess. Similarly, in other developmental processes, suchas the opening of the pea hooks, other plant growthsubstances (like ethylene) are of central importance (Peck,Pawlowski, and Kende, 1998). In the peanut gynophore,ethylene levels increase slightly upon penetration into thesoil (Shlamovitz et al., 1995). The interaction of auxin withother plant growth substances (such as GA or ethylene)during gynophore development, remains a possibility.However, the focus of this study was only on auxin and itslocalization and effects within the tissues of the developinggynophore. In this study, it has been confirmed that auxinis involved in organ extension growth, cell elongation andcell division of the developing peanut gynophore. Theresults obtained in this study correlate the many develop-

mentally-induced cellular and morphological changes indeveloping gynophores with the changes in auxin immuno-localization patterns observed at the different stages ofgrowth. Although further studies are still needed on thespecific sites of auxin production and transport in thedeveloping peanut gynophore, the foundations for thisfuture research have already been laid in this work.

ACKNOWLEDGEMENTS

I thank my thesis advisor, Professor Lewis J. Feldman fromthe University of California at Berkeley, for his invaluablehelp, support, critical reading and scientific advice through-out all stages of this work. I also thank Dr W. Mozingofor supplying peanut seeds, Dr John L. Caruso for providingthe monoclonal IAA antibody, Dr Nancy Kerk for helpfuladvice on the IAA immunolocalization technique, and DrMasao Tasaka for his critical reading of this manuscript.This study was sponsored by a NASA-GSR fellowship. Thiswork represents one chapter of my dissertation submitted inpartial fulfilment of the requirements for the PhD degree atthe University of California at Berkeley.

LITERATURE CITED

Caruso JL, Pence VC, Leverone LA. 1995. Immunoassay methods ofplant hormone analysis. In: Davies PJ, ed. Plant hormones:physiology, biochemistry and molecular biology. Netherlands :Kluwer Academic Publishers, 43–447.

Davies PJ. 1995. The plant hormones: their nature, occurrence andfunction. In: Davies PJ, ed. Plant hormones: physiology, bio-chemistry and molecular biology. Netherlands : Kluwer AcademicPublishers, 1–12.

Jacobs W. 1951. Auxin relationships in an intercalary meristem:further studies on the gynophore of Arachis hypogaea. AmericanJournal of Botany 38 : 307–310.

Kerk NM, Feldman LJ. 1995. A biochemical model for the initiationand maintenance of the quiescent center : implications fororganization of root meristems. De�elopment 121 : 2825–2833.

Leverone LA, Kossenjians W, Jayasimihulu K, Caruso JL. 1991.

Evidence of zein-bound IAA using gas chromatography-selectedion monitoring-mass spectrometry analysis and immunogoldlabeling. Plant Physiology 96 : 1070–1075.

Mejillano MR, Himes RH. 1991. Assembly properties of tubulin aftercarboxyl group modification. The Journal of Biological Chemistry266 : 657–664.

Moctezuma E, Feldman LJ. 1996. IAA redistributes to the upper sideof gravistimulated peanut (Arachis hypogaea) gynophores. PlantPhysiology 111 : S73.

Moctezuma E, Feldman LJ. 1998. Growth rates and auxin effects ingraviresponding gynophores of the peanut, Arachis hypogaea(Fabaceae). American Journal of Botany 85 : 1369–1376.

Pattee HE, Mohapatra SC. 1987. Anatomical changes during ontogenyof the peanut (Arachis hypogaea L.) fruit : mature megaga-metophyte through heart-shaped embryo. Botanical Gazette 148 :156–164.

Peck SC, Pawlowski K, Kende H. 1998. Asymmetric responsiveness toethylene mediates cell elongation in the apical hook of peas. ThePlant Cell 10 : 713–719.

Pence VC, Caruso JL. 1988. Immunoassay methods of plant hormoneanalysis. In: Davies PJ, ed. Plant hormones and their role in plantgrowth and de�elopment. Netherlands : Kluwer Academic Pub-lishers, 240–256.

Periasamy K, Sampoornam C. 1984. The morphology and anatomy ofovule and fruit development in Arachis hypogaea L. Annals ofBotany 53 : 399–411.

Rayle DL, Cleland RE. 1992. The acid growth theory of auxin-inducedcell elongation is alive and well. Plant Physiology 99 : 1271–1274.

242 Moctezuma—Auxin Patterns in De�eloping Peanut Gynophores

Shi L, Miller R, Moore R. 1993. Immunocytochemical localization ofindole-3-acetic acid in primary roots of Zea mays. Plant, Cell andEn�ironment 16 : 967–973.

Shlamovitz N, Ziv M, Zamski E. 1995. Light, dark and growthregulator involvement in groundnut (Arachis hypogaea L.) poddevelopment. Plant Growth Regulation 16 : 37–42.

Shushu DD, Cutter E. 1990. Growth of the gynophore of the peanutArachis hypogaea. 2. Regulation of growth. Canadian Journal ofBotany 68 : 965–978.

Smith BW. 1950. Arachis hypogaea. Aerial flower and subterraneanfruit. American Journal of Botany 37 : 802–815.

Webb AJ, Hansen AP. 1989. Histological changes of the peanut(Arachis hypogaea) gynophore and fruit surface during develop-ment, and their potential significance for nutrient uptake. Annalsof Botany 59 : 351–357.

Ziv M, Zamski E. 1975. Geotropic responses and pod development ingynophore explants of peanut (Arachis hypogaea L.) cultured in�itro. Annals of Botany 39 : 579–583.