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(American Journal of Botany. 1998;85:1369-1376.)
© 1998 Botanical Society of America, Inc.

Growth rates and auxin effects in graviresponding gynophores of the peanut, Arachis hypogaea(Fabaceae)1

Edgar Moctezuma2,a and LewisJ. Feldmana

a Universityof California at Berkeley, Department of Plant and Microbial Biology,431 Koshland Hall, Berkeley, California 94720


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The gynophore of the peanut plant (Arachis hypogaea) is aspecialized organ that carries and buries the fertilized ovules into thesoil in order for seed and fruit development to occur underground. Therates of growth of vertically and horizontally oriented gynophores weremeasured using a time-lapse video imaging system. We found that theregion of maximum extension growth due to elongation (termed the CentralElongation Zone) is located on average at 2-5 mm from the tip. In thefirst 0-4 h after horizontal reorientation (gravistimulation), new zonesof growth emerge on the upper surface, while the elongation zone of thelower side decreases in size and magnitude. Four to six hours afterreorientation the zones of maximum growth are almost equal in size andlocation on the upper and lower sides. The growth rate and thegravitropic response decreased dramatically upon the excision of theovule region (terminal 1.5 mm), but a gravitropic growth response couldbe restored by applying the auxin indole-3-acetic acid exogenously tothe excised tip. The addition of napthylphthalamic acid (an auxintransport inhibitor) at the ovule region allowed some growth to occur,but the gynophores do not respond normally to gravity upon horizontalreorientation. We discuss the role of auxin in the gravitropic responseof the gynophore.

Key Words: Arachishypogaea • auxin • Fabaceae • gravitropism • growthrates • gynophore • indole-3-aceticacid • napthylphthalamicacid • peanut


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Gravity perception and response play a crucial role in the successfulcompletion of the peanut plant (Arachis hypogaea L.) lifecycle: fruit and seed development do not occur unless the fertilizedovules are buried underground. This positively gravitropic response isfirst evident just after fertilization of the ovules (Fig. 1). Following fertilization,embryo development is arrested, and the fertilized ovules are carrieddown and buried inside the soil, where they may mature and develop intothe peanut fruit and seed. This "self-sowing" event (knownas geocarpy) is accomplished by the downward growth of a specializedorgan called the gynophore. [Note: According to Smith (1950), this organ is not really a"gynophore" (i.e., a gynoecium-bearer); rather it is anelongated ovary. Although almost all peanut researchers prefer to usethe colloquial name "peg" for this structure, in this paperwe will use the commonly accepted term gynophore to refer to suchorgan].



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  		Fig. 1. Three stages of peanut gynophore development. (a) Before fertilization (the flower parts have been removed). (b) Five days after fertilization and responding positively to gravity. (c) Two weeks after fertilization, with developing immature fruit buried underground. Bar = 1.5 mm.

 
The peanut gynophore is a unique organ. Although it is astem-derived structure and has anatomical features common to dicotshoots, the gynophore responds positively to gravity, similar to atypical primary root (Jacobs, 1947). The peanut gynophore has a gravitropic set-point angle (GSA) of~0° (i.e., it grows vertically downwards), whereas most mainshoots have a GSA of ~180° (vertically upwards) (see Digby and Firn, 1995). Thus, several questionsarise: How does a stem-like organ such as the peanut gynophore respondpositively to gravity? What is the nature of this gravitropicresponse?

Although the peanut gynophore exhibits one of the most extreme anddramatic developmentally regulated changes in gravitropic response inthe plant kingdom (Hart, 1990), verylittle is known about this interesting gravitropic phenomenon.Ziv and Zamski investigated the role of light, touch, and gravity duringthe reproductive growth of the peanut (Ziv andZamski, 1975; Zamski and Ziv,1976). Their studies pointed to the role of auxins andcytokinin in the development of the peanut fruit, as well as theimportance of a touch stimulus and darkness in this process. Pattee and Mohapatra (1987) performed a seriesof elegant studies in which they describe the anatomical changes duringthe growth and development of the peanut fruit. Shushu and Cutter (1990a, b), through a number of ovule excision andexogenous plant hormone application experiments, concluded that acombination of indole-3-acetic acid (IAA) and gibberellic acid (GA),produced at the ovule region near the tip, were responsible for thegrowth promotion of the vertically growing gynophore. Shushu and Cutterfocused their research on the normal, vertically oriented gynophore, butdid not explore the gravitropic response of this organ. A recent studyby Shlamovitz, Ziv, and Zamski(1995) also investigated the role of light, darkness, andgrowth regulators (e.g., IAA, ethylene and abscisic acid) in gynophoreelongation and pod formation in the peanut plant. IAA is believed to beinvolved in the growth and development of vertically growing gynophores(Shushu and Cutter, 1990b), as well asin the gravitropic response of roots and shoots (Hart, 1990). Jacobs found that IAA is located inthe distal 10 mm of vertically growing gynophores, and it is believed tomove basipetally along the length of this organ (Jacobs, 1951).

In this paper, we describe the spatial and temporal growth patternsof intact and graviresponding peanut gynophores. We then investigatethe role of the ovule region and IAA in this gravitropic response. Thespecific objectives of this investigation were: (1) to define moreprecisely and characterize the existing data on the growth patterns ofvertically growing peanut gynophores; (2) to analyze the patterns ofgrowth of gravistimulated (horizontally oriented) gynophores atdifferent time points after reorientation; (3) to investigate the roleof the ovule region in the normal growth and development of thegynophore; and (4) to study the role that IAA may play in thegynophore's development and gravitropic response. Byknowing the kinematics of growth of the gynophore, we may begin tounderstand the cellular and physiological basis by which this organresponds positively to the gravity stimulus.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plant material
Plants of Arachis hypogaea, cultivar Virginia 93B, weregrown in a greenhouse from seeds kindly provided by Dr. W. Mozingo fromthe Tidewater Agricultural Research and Extension Center in Suffolk,Virginia, USA. Virginia 93B was chosen because it is an erect,two-seeded cultivar that rapidly produces large numbers of gynophores. Plants were grown in cylindrical pots (21 cm diameter x 17 cmheight) with a combination of peat moss, redwood soil conditioner,vermiculite, and coarse sand (2:2:3:5) at 27°C (±2°C)under a 16-h/d natural daylight regime supplemented with halogen lamps. Plants were watered daily and provided with a one-half strengthHoagland nutrient solution every day. Plants began flowering 4–6wk after planting.

Growth ratemeasurements
Plants were transferred to a room for videotaping while beingmaintained under similar temperature and irradiance conditions of thegreenhouse. Young gynophores (15–30 mm long) were used for the measurements because at this stage of gynophore development the overallgrowth rates are stable and reproducible, and the gravitropic responseis stronger than at other developmental stages. Vertical growthmeasurements were repeated using 15 gynophores, whereas 5–10gynophores were measured for each of the gravistimulation experiments. With the use of a special marking device (described in Kim and Mulkey [1993]), we carefullymarked the surfaces of either the vertical, intact, or horizontal,gravistimulated gynophores with waterproof black India ink (Koh-I-Noor,Bloomsbury, New Jersey, USA). The ink marks were ~0.4–0.6 mmapart. The gynophores were videotaped using a time-lapse videocassetterecorder (JVC, Yokohama, Japan) and a video camera (Javelin, Torrance,California, USA) with a Macro-zoom close-up lens (Nikon, Tokyo, Japan)over 0–6 h of growth. The recorded images were displayed andmeasured on a computer screen, using the NIH Image Analyzer (NationalInstitute of Health, Bethesda, Maryland, USA) program. The images wereenlarged in the computer screen for measurements with a resolution of upto 0.02 mm (i.e., 1 pixel = 0.02 mm). Measurements of thedisplacement of the marks with respect to the tip along the gynophorewere made every 0.5 h on each sample. Rate of displacement from the tip({Delta}x/{Delta}t) was calculated for each of the marks alongthe length of the gynophore according to Silk(1984). A cubic spline program was used to interpolate at0.5-mm segments the rate of displacement raw data calculated from thevideo images (Bret-Harte and Silk,1994). The relative elemental growth rates (REGR) werecalculated for each point along the gynophore using Erickson's5-point formula for numerical differentiation (Erickson, 1976):

where -2 to +2 areconsecutive numbers from the rate of displacement from the tip data, andh is the distance between the marks, 0.5 mm). The growth ratedata were analyzed using the Sigma Plot (Jandel, San Rafael, Calfornia,USA) program and averaged, smoothed, and plotted using the Kaleidagraph(Apple Computer Inc., Cupertino, California, USA) graphing program. Thecurvature measurements were taken from printed images of gravirespondinggynophores by measuring the angle with respect to the horizontal(0°) every hour for a total of 12 h (Harrison and Pickard,1989).

Microscopy andcell length measurement
Vertically growing gynophores were excised from the plant and fixedimmediately in 50% formalin-acetic acid-alcohol (FAA). Afterfixation, the tissues were dehydrated in increasing concentrations ofethanol:water, infiltrated with Hemo-De (Fisher, Pittsburgh,Pennsylvania, USA), embedded in paraffin, and sectioned at 10 µm on arotary microtome. Once mounted on slides, the sections were stainedusing a 1%, aqueous (pH 7.5) toluidine blue solution, asdescribed in Shushu and Cutter (1990). The slides were observed under a microscope and the images were recordedwith a video camera. The images were transferred to a computer screenand the lengths of ~20–40 pith cells per every 0.5-mm segmentwere measured using the NIH Image Analyzer program, previouslycalibrated to measure distances in micrometers on the computer screen. Cell length measurements were repeated for five differentgynophores.

Excisionexperiments
On vertically and horizontally oriented gynophores, the distal 1.5 mmof the tip were excised with a sharp razor blade, making sure only theovule region was removed. The intercalary meristem and the elongationzone were left intact on the gynophore. The tips of 200-µL plasticpolymerase chain reaction tubes (Phenix Research Products, San Leandro,Calfornia, USA) were used to apply the IAA in agar in place of theexcised portion of the gynophore tip. The tubes contained 40 µL of1% low-gelling agar (SeaPlaque Agarose, FMC Bioproducts,Rockland, Maryland, USA) and 1 x 10 mol/L IAAin water. In other treatments, we filled the tubes with 5 x10 mol/L napthylphthalamic acid (NPA), which is anIAA transport inhibitor (Hertel, Lomax, andBriggs, 1983). It is important to note that the ovules werenot excised in the NPA-application experiments, because they are thepresumed sources of IAA. Only a small portion of the tip (<0.3 mm)was excised in order to facilitate the uptake of the NPA inside thetissues of the gynophore. Excising <0.3 mm from the tip does notsignificantly affect the gravitropic response or curvature of thegynophore. In presenting the data the growth rate (percentage growthper hour) graph, we compensated the x-axis for the excisedsegment by shifting the curve to the right by an amount corresponding tothe length of the excised tissue.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Vertically growing gynophores
The growth rate patterns of intact, vertically growing gynophoreswere characterized using the tip of the gynophore as a reference pointfor calculating how fast each individual mark was displaced away fromthe tip. This procedure provided an accurate measurement of the overallrates of growth of the organ, and of which segment of the organundergoes the most extension growth (Silk,1984). The mean rate of displacement of the ink marks fromthe tip (i.e., the overall growth rate of the gynophore) varied fromsample to sample, but it is, on the average, ~0.31 mm/h (SD =±0.07 mm/h) (Fig. 2). The region beyond 10 mm from the tip did not contribute significantly tothe overall growth of the gynophore (as indicated by the plateau of thegraph at 10–12 mm from the tip).



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  		Fig. 2. Mean rate of displacement of the marks from the tip of the gynophore vs. distance from the tip of the vertically growing gynophore. Graph is a composite of 15 gynophore measurements, averaged, and with seventh-degree polynomial smoothing. Error bars are ±1 SD.

 
The growth data must also be expressed relative to the region oftissue producing the growth (Selker and Sievers,1987). The REGR indicates the percentage of growth per hourcontributed by each individual segment of the growing organ. Forexample, for a plant organ that is 10 mm long, to increase its overalllength by 2 mm, each of the ten 1-mm segments would need to increasetheir length by 20%. Similarly, the same organ could have a 2-mmsegment increasing its length by 100%. Although both growthpatterns result in an overall increase of 2 mm in length, the relativegrowth rate and the physiological processes involved in such growth aremuch faster in the latter case (the 2-mm segment increasing by100%) than in the first case. In Figs. 3 and 6–9, the REGR isexpressed as the growth rate (percentage growth per hour) in they-axis, since the units for the REGR are millimetres per hourper millimetre.



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  		Fig. 3. Relative Elemental Growth Rate (expressed as the Growth Rate) as a function of distance from the tip of vertically growing, intact gynophores. DEZ, Distal Elongation Zone; CEZ, Central Elongation Zone; PEZ, Proximal Elongation Zone. Graph is a composite of 15 gynophore measurements, averaged, and with seventh-degree polynomial smoothing. Standard errors are typically 0.2–0.5 %/h.

 
Figure 3 shows the meangrowth rate of 15 vertically growing gynophores. One segment along thelength of the gynophore contributes the most to the overall growth ofthe organ. This segment not only varies in size from sample to sample(between 2 and 4 mm long), but it also fluctuates in its location withinthe length of the growing gynophore: it can be located between 1.5 and8.0 mm from the tip. On average, this segment is found between 2.0 and5.0 mm from the tip. We designate this region of maximum growth as theCentral Elongation Zone (CEZ). The CEZ was arbitrarily determined bytaking the average growth rate values >0.85 of the peak rate value onthe plot. The CEZ segment contributed the highest percentage of growthto the elongating gynophore. The CEZ is only 2-4 mm long, but it canaccount for >50% of the total extension growth in the distal10 mm of a vertically growing gynophore. The distal region (with growthrate values <0.85 of the peak rate) was designated the DistalElongation Zone (DEZ). The ovule region and the meristem are located inthe DEZ region (the distal 2 mm of the gynophore). Similarly, theProximal Elongation Zone (PEZ) is characterized as that proximal growthregion with a value <0.85 of the peak growth rate, and is located, onaverage, between 5 and 10 mm from the tip.

Noticeable changes in cell length take place in the 3-mm segment ofthe gynophore designated as the CEZ. For example, Fig. 4 shows that the greatestdifference in the lengths of pith cells occurs in a 3-mm regionlocated, on average, between 2 and 5 mm from the tip. The cells inthese segments are undergoing the greatest increase in cell size, andthey correspond to the average location of the CEZ. Therefore, growthin the CEZ is due to cell elongation (Fig. 4). Figure 5 illustrates diagramaticallythe relative size of these zones of growth, together with the mitoticindex data that constitute the "H-shaped" intercalarymeristem that is characteristic of the peanut gynophore (Jacobs, 1951; Brennan,1969; Shushu and Cutter,1990a). Some overlap of cell division and cell elongationoccurs between 2.0 and 3.0 mm from the tip, especially in the epidermaland cortical tissues (Fig.5).



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  		Fig. 4. Average pith cell length vs. distance from the tip in the distal 8 mm region of the vertically growing, intact gynophore. Data are means of 20–40 different cells in each 0.5-mm segment from the tip of the gynophore, ±1 SD (N = 5).

 


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  		Fig. 5. Growth zones of the peanut gynophore. Notice the overlap of the Central Elongation Zone (zone of maximum growth due to cell elongation) and the Zone of Cell Division between 2 and 3 mm from the tip.

 
Horizontallyoriented, gravistimulated gynophores
When gynophores are oriented horizontally, a downwards bendingresponse occurs within the next 6 h of horizontal reorientation. Theoverall growth rates from the upper and lower sides of a horizontallyoriented gynophore were measured during the first 6 h ofgravistimulation for comparison with the growth rates of verticallygrowing gynophores, before gravistimulation (Fig. 6).



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  		Fig. 6. Growth rates vs. distance from the tip of horizontally oriented gynophores during the first 6 h of gravistimulation. The graphs represent averages of growth rate measurements taken during vertical growth (before gravistimulation) (A, B), during the first 2 h after gravistimulation (C, D), at 2–4 h after gravistimulation (E, F), and at 4–6 h after gravistimulation (G, H). (C), (E), and (G) represent the growth rate of the upper side, whereas (D), (F), and (H) represent the lower side of the horizontally oriented gynophore. The diagrams between the graphs depict the bending gynophores at the respective times, where the peaks of high growth in the upper and lower surfaces of the gravistimulated gynophore are represented by the black bars. In (C-H), the dashed line represents the growth rate of the intact, vertically oriented gynophore; the solid line represent the growth rate at the particular time and side. Graphs are composite measurements of nine gynophores, averaged by computer, with ninth-degree polynomial smoothing in the y -axis. Standard errors were typically 0.2–0.5 %/h.

 
The most dramatic changes in growth rate occur in the first 2 h ofgravistimulation (Fig. 6C, D). The growth rate on the upper side of the horizontally oriented gynophoreincreased by ~40% with respect to the vertical rate ofgrowth, whereas the rate on the lower side decreased by ~50%(Fig. 6A, B). In the upperside, at 0–2 h after horizontal reorientation (Fig. 6C) a new region of growthappeared near the ovule region (at 1–2.5 mm from the tip, alongwith another greater peak (6–7 mm from the tip), which divided intwo the CEZ of the vertical control. The CEZ in the lower side wasdivided into two peaks of growth at 0.5–1.5 mm and 4.5–6 mmfrom the tip (Fig. 6D).

In the next stage of gravitropic response, 2–4 h afterreorientation, the rates of growth of both the upper and lower sidedecreased as compared to the vertical control (Fig. 6E, F). The upper side, however,still showed a rate growth twice as high as the overall rate in thelower side (0.11 mm/h). Thus, the gravistimulated gynophore continuedbending downwards at 2–4 h after horizontal reorientation. Thegrowth rate data of the upper side (Fig.6E) show that the small peak near the tip in the upper sidethat had been present during the first 2 h of gravistimulation haddisappeared after 4 h. As a result, most of the growth in the upperside at 2–4 h now occurs in the region of the vertical CEZ(~2.5–5 mm from the tip; Fig.6E). The location of this peak matched the location of thepeak of maximum growth in the vertical control. However, another newand more proximal peak (beyond 10 mm from the tip) along the uppersurface accounted for the increased overall growth in the upper side,and thus the continued gravitropic bending of the organ. In the lowerside at 2–4 h after horizontal reorientation (Fig. 6F), the growth rate was stillmuch lower than the upper side, with a small peak of growth locatedcloser to the tip (at 1–2 mm from the tip) than in the previous 2h.

In the final gravitropic response stage measured, at 4–6 hafter gravistimulation, the gynophore was approaching a verticalorientation, and the growth rates on both the upper and lower sides werealmost equal, but still lower than the rate in the vertical control(Fig. 6G, H). The growth ratedata of this time point (4–6 h) indicate that the peaks in boththe upper and lower sides are located approximately at the same locationas the CEZ of the vertical control. The growth rate peak in the lowerside (Fig. 6H) is slightlyhigher than the peak of the upper side (Fig. 6G), indicating a small reversalof growth once a vertical orientation has almost been reached after 6 hof gravistimulation. The gynophore starts a relatively verticaldownward growth after this time, but we did not observe any"overshooting."

Therole of IAA in the gynophore's gravitropic response
Three different procedures were performed on the gynophores: (1)excision of the ovule region; (2) addition of IAA to the excised ovuleregion; and (3) addition of an IAA transport inhibitor (NPA) to the tipsof vertically oriented and gravistimulated gynophores.

The overall growth rate is greatly reduced in vertical andgravistimulated gynophores when the ovule region is excised (Fig. 7). During the first 2 h ofgrowth, the rate of mark displacement from the tip of the vertical,excised gynophore is reduced by ~60% as compared to thevertical control. Although both the upper and lower side reduced theirgrowth rate by ~80% in the horizontally oriented, excisedgynophore, the upper side maintains a slightly higher rate of growththan the lower side at 0–2 h of gravistimulation. A small growthrate peak in the upper side of the excised, gravistimulated gynophoreshowed that some growth occurred in exactly the same CEZ as in thevertical control. We also observed (data not shown) that if afterexcising the tips we wait 24 h in the vertical orientation, even lessvertical growth is observed and no downwards bending occurs when theexcised gynophores are oriented horizontally. These results could beexplained by the presence of small residual amounts of growth substances(such as IAA and GA) that may still be active during gravistimulation atthe time the ovules were recently excised, but that were depleted (ortransported away) after 24 h of vertical growth.



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  		Fig. 7. Ovule excised. Comparison of the growth rates of the upper (open circles) and lower (filled circles) surfaces of a horizontally oriented, representative gynophore in which the ovule region has been excised. The growth rates of the same gynophore intact vertical control before gravistimulation (dashed line) and vertically growing with ovule region removed (solid line), are presented for comparison. Sample measurements were done in the first 2 h of vertical growth or gravistimulation after excision. Graph is a composite of measurements of six ovule-excised gynophores, averaged by computer and with a seventh-degree polynomial smoothing. Standard errors were typically 0.2–0.4%/h.

 
By adding IAA in place of the excised ovule region, a growth ratepattern for vertical and graviresponding gynophores was restored(Fig. 8). The growth rate plot(Fig. 8) during the first 2 hof gravistimulation revealed that this increase in growth in the upperside occurred due to a very high growth peak zone near the tip. Thelower side also decreased its growth.



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  		Fig. 8. IAA added. Comparison of the growth rate of the upper (open circles) and lower (filled circles) surfaces of a horizontally oriented gynophore from which the ovule region has been removed, and which has been provided with 10mol/L indole-3-acetic acid (IAA) in place of the excised tissue during the first 2 h of horizontal reorientation. The curve with the dashed line represents the measurements of the intact, vertically growing gynophore before excision of the ovules and before gravistimulation. The solid line represents the same gynophore growing vertically (before horizontal reorientation), but with the tip excised and with added IAA. Graph is a composite of four IAA-treated gynophores, averaged by computer and with a seventh-degree polynomial smoothing. Standard errors were typically 0.3–0.7%/h.

 
The effects of an IAA transport inhibitor on the growth of verticallyoriented and gravistimulated gynophores are illustrated in Fig. 9. Vertically oriented gynophorestreated with NPA reduced their overall rate of mark displacement by anaverage of 50%. Horizontally oriented gynophores also treatedwith the IAA transport inhibitors did not bend down during the first 2 hof gravistimulation, but they continued to elongate horizontally,although at a much reduced rate (Fig.9).



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  		Fig. 9. NPA added. Comparison of the growth rates of the upper (open circles) and lower (filled circles) surfaces of a horizontally oriented, representative gynophore in which 5 x 10 mol/L N-1-napthylphthalamic acid (NPA) has been added to the tip during the first 2 h of gravistimulation. The ovules remain in the gynophore, and only 0.3 mm from the tip has been removed in order to ease the uptake of the NPA. The curve with the dashed line represents the growth rate measurements of the same gynophore, but intact and vertically growing. The solid line represents the same sample, growing vertically after NPA was added to the tip. The graph is a composite of three gynophores, averaged, and with ninth-degree polynomial smoothing. Standard errors were typically 0.3–0.7%/h.

 
Curvature patterns of the horizontally oriented gynophores can alsobe mimicked and restored under the different treatments. Figure 10 exhibits the curvatures indegrees of the different treatments during 12 h of gravistimulation.After 8 h, the curvature pattern of the IAA-treated sample(-70°) resembled much more the curvature pattern of theintact, horizontally oriented gynophore (-88°) than thepattern of a gravistimulated sample in which the ovule region had beenexcised (-8°) or in which an IAA transport inhibitor had beenadded (-6°) (Fig.10).



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  		Fig. 10. Curvature measurements vs. time during the first 10 h of gravistimulation of horizontally oriented gynophores under different treatments. The different treatments are: intact, no treatment (open circles); ovule region removed (open triangles); ovule region removed with addition of 10 mol/L IAA (filled circles); and with the addition of 5 x 10 mol/L NPA, ovules still present (filled triangles). SD were typically 1–8° (N = 5).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Measurements using improved techniques
The data presented in this paper were obtained using improved, moreaccurate techniques for growth rate measurements, over shorter timeintervals than previous studies (hours instead of days). In addition,these results were analyzed employing better mathematical formulas forgrowth analysis than those used in previous works (Jacobs, 1947; Shushu andCutter, 1990a). Shushu and Cutter(1990a), for example, showed that extension growth occurred inthe first 6-mm region from the tip in the gynophores of cultivar BukeneNo. 2, and that the region between 2 and 3 mm from the tip wasundergoing the greatest growth (Shushu andCutter, 1990a). Our results corroborate these conclusions,but we also found that the region of greatest extension growth can bemuch larger and can also vary in its location along the length of thegynophore. Shushu and Cutter incompletely determined the zone ofmaximum growth because they spaced their marks in the gynophore too farapart (1 mm instead of <=0.5 mm) and because they waited too long fordoing the measurements (several days instead of hours). We observedthat the gynophore can undergo extension growth throughout the distal 10mm from the tip, with most of the growth taking place at the CEZ, whichis located on average at 2–5 mm from the tip. The improved andmore precise techniques we employed in this work and further examinationof the data with Erickson and Silk's 5-point formula for plantgrowth analysis resulted in a better understanding of the complex growthkinematics of the peanut gynophore.

Analysis of the growth rate results
The growth rates of the upper and lower side of gravistimulatedgynophores show how new peaks of growth (and growth asymmetries) seem toemerge at the tip and travel basipetally along the length of theelongation zones of the gynophore, in a "wave"-like motionpattern (Fig. 6). The upperside particularly shows the formation of new zones of growth (i.e., newpeaks of growth in the graphs). These new basipetally moving"waves" of growth, observed in the upper side during thefirst 2 h after gravistimulation, may be caused by a growth signal(possibly IAA) that is sent from the ovule region into the area near theCEZ.

By 6 h of horizontal reorientation—when the gravistimulatedgynophore has almost reached a vertical orientation again— peaksof growth of both sides shift back to their original location as in thevertical, intact controls (Fig. 6G,H). "Overshooting", or bending beyond90 from the horizontal plane, is commonly observed ingraviresponding roots and shoots (Cosgrove,1990; Ishikawa, Hasenstein, andEvans, 1991), but did not occur in the graviresponding peanutgynophore.

The growth rate patterns of the gynophore are complicated duringnormal gravistimulation, but they become even more complex under thedifferent treatments performed in this study. Jacobs had already shownthat IAA is produced at the tip of vertically growing peanut gynophores,and he demonstrated that this IAA is transported basipetally in astrictly polar manner (Jacobs, 1947,1951). Our ovule-excision resultsconfirm that IAA produced at the ovule region may play an important rolein the normal growth and gravitropic response of the gynophore, but theyalso suggest that other factors may also be involved in such response(Fig. 7). In addition,gravistimulation caused a dramatic decrease of growth in the upper sideof horizontally oriented, ovule-excised gynophores (~80%),but only a lesser decrease in growth in the ovule-excised, verticallygrowing gynophores (~60%).

The data showing that the growth rate can be partially restored bythe addition of IAA to the excised ovule region suggest that the ovuleregion of the gynophore may be the major producer and distributor of IAAupon gravistimulation. It is important to point out, however, thatalthough the final bending curvature of the horizontal control is almostthe same as the IAA-treated sample, the growth rate results indicatethat the samples reach such a bending curvature with different growthrate patterns. For example, the peak of growth rate of the upper sideof the IAA-treated sample is much higher than the vertical andhorizontal controls. Another important difference is that the growthrate in the lower side of the IAA-treated gynophore does not decrease asmuch in magnitude in relation to the vertical or horizontal controlgrowth rates (Fig. 8, filledcircles) as in the intact, horizontally oriented gynophore (Fig. 6D). This difference could be dueto the fact that other growth-promoting substances, such as GA, maystill be involved in the normal vertical growth of the gynophore. Furthermore, both the upper and lower sides may be receiving the sameamount of IAA, and although the upper side seems to be more responsiveto this hormone, the artificial amounts of IAA that are beingexogenously applied to the tip may produce a slightly higher rate ofgrowth on the lower side.

One important result we observed in the ovule-excised, IAA-treatedsamples is that the peak growth rates or zones of maximum growth of theupper side were shifted slightly away from the tip compared with thepeak in the vertical, intact control (Fig. 8). The cells near the meristem(1.5–2.5 mm from the tip) and the CEZ may now be responding to theexogenously applied IAA, and may now be more directly involved in thegravitropic response of the gynophore. Nevertheless, although wesuccessfully restored a gravitropic response to the ovule-excisedgynophores by adding IAA, it is important to notice that the growth ratecurves demonstrate that such a response was accomplished in a differentmanner than in the intact, gravistimulated gynophore. The differentgrowth rate patterns found in Fig.8, as compared to the control growth rate pattern in Fig. 6C, indicate that IAA may not bethe only factor involved in the normal gravitropic response of thegynophore. Other factor(s) such as growth promoting substances, hormonecatabolists, growth inhibitors, etc., which may have been modified bythe removal of the ovary region, may also play a role in thegynophore's graviresponse.

Adding an IAA transport inhibitor (NPA) to the tip of a verticallygrowing gynophore reduced the overall growth rate by an average of50% as compared to the intact control. However, the verticalgrowth rate was not completely inhibited (treated gynophores maintaineda relatively constant rate of growth) (Fig. 9), and the gynophore did notrespond to gravity when IAA transport was inhibited (Fig. 10). These results indicate, asShushu and Cutter (1990b) suggest intheir work, that other growth hormones such as GA may also be involvedin the normal growth and development of the gynophore. Our data suggestthat transport of IAA from the tip to the CEZ may be very important forthe gravitropic response and bending of gravistimulatedgynophores.

Physiologicalimplications of the gynophore's growth rate patterns
The Cholodny-Went Hypothesis (CWH), proposed in 1926, is the leadingmodel to explain the gravitropic response of plant roots and shoots interms of differential growth rates and hormone redistributions withinthe tissues (reviewed by Hart, 1990). Part of the CWH states that the gravity stimulus triggers aredistribution of IAA to the lower half of a horizontally oriented stem,which would lead to an overall increase in the growth rate in the lowersurface, coupled by an equal magnitude decrease in the growth rate ofthe upper surface. When interpreted in this broad sense, the CWH is inagreement with our results only for the first 2 h of gravistimulation: we observe an overall increase in the growth rate on the upper surfacethat is coupled by a decrease of growth in the lower surface (oppositeof what occurs in typical shoots). Nevertheless, we must admit that theCWH is too simplistic to explain the series of complex, new peaks ofgrowth that emerge and disappear in the following 4 h ofgravistimulation. The CWH fails to take into consideration the temporaland spatial variations in the growth rate patterns of thegraviresponding gynophores, i.e., how specific regions of maximum growthare fluctuating along the length of the organ through time.

Nevertheless, the IAA redistribution patterns that the CWH proposeshas sparked the interest of many workers and much research in an attemptto explain the mechanisms that lead to the growth rate patterns observedin gravistimulated stems. An asymmetric redistribution of IAA towardsthe lower side in the elongating region of stems during gravitropism hasbeen established in many plant model systems (reviewed by Hart, 1990; Li, Hagen, andGuilfoyle, 1991). Downward bending, however, can occur inupwards-growing tomato hypocotyls when extremely high amounts of IAA (1mol/L) are externally applied to horizontally oriented samples (Harrison and Pickard, 1989). Harrison andPickard's work suggests that unusually high amounts of IAA may becapable of changing the gravitropic response of a shoot, from upwards todownwards curving. In a different case, IAA redistribution to the upperside occurs in the gravistimulated lazy-2 tomato mutant, which,like the gynophore, is a stem that responds positively to gravity(Kim, Rayle, and Lomax, 1994). In thegynophore, since presumably high amounts of IAA are produced at theovule region (Jacobs, 1951), it may bepossible that this IAA supply may be partially responsible for thegynophore's positive gravitropism.

Thus, we hypothesize that when a gynophore is placed horizontally,important changes in IAA may be occurring within the tissues. Althoughother factors may also be involved in the gynophore'sgraviresponse, IAA seems to play a very important role in thisgravitropic growth response. There is the possibility that IAAsensitivity may be higher in the lower side, where high amounts of IAAtransported there would cause growth inhibition. However, previousresults obtained in another study suggest that more IAA is localized inthe upper side than the lower side in gravistimulated gynophores(Moctezuma and Feldman, 1996).

Several possible mechanisms may be responsible for suchgravity-induced IAA redistribution. Possibilities include: an increasedtransport of the IAA to the tissues of the upper side; a decreased IAAsignal being sent to the lower side; and a breakdown of IAA, a decreasein IAA sensitivity, or the presence of growth inhibitors also in thelower side. But regardless of the mechanism or combination of mechanismsinvolved in the gynophore's putative upwards redistribution of IAA,it is apparent that some crucial step(s) in the signal transductionpathway of normal gravitropic stems have been reversed in the gynophore. Further physiological studies are still necessary in order to determinewhat mechanisms are involved in this presumed gravitropically inducedIAA redistribution, as well as in the complex, succeeding growth rateasymmetries it generates and the other factors possibly involved in thisresponse. The nature and the causes of this atypical IAA redistributionin the peanut gynophore are currently beinginvestigated.


   FOOTNOTES
 
1 The authors thank Dr. Wendy K. Silk for her invaluable advice at the beginning stages of this work; Dr. Syndonia Bret-Harte for allowing us to use her cubic spline computer program; Dr. Ian Sussex, Dr. Frederick D. Hempel, and Stacy Steinberg for their critical reading of this manuscript; and Dr. Steven E. Ruzin for his expert technical assistance. This work was supported by a research fellowship from the National Aeronautics and Space Administration to E. M. This study represents a chapter of a dissertation to be submitted by the first author in partial fulfillment of the requirements for the Ph.D. degree at the University of California at Berkeley. Back

2 Author for correspondence. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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