JXB Advance Access originally published online on February 8, 2006
Journal of Experimental Botany 2006 57(4):837-847; doi:10.1093/jxb/erj069
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RESEARCH PAPER |
Asymmetric distribution of auxin correlates with gravitropism and phototropism but not with autostraightening (autotropism) in pea epicotyls
Botanical Gardens, Graduate School of Science, Osaka City University, Kisaichi, Katano-shi, Osaka 576-0004, Japan
* To whom correspondence should be addressed. E-mail: k-haga{at}interlink.or.jp
Received 3 September 2005; Accepted 22 November 2005
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Abstract |
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The relationships between the distribution of the native auxin indole-3-acetic acid (IAA) and tropisms in the epicotyl of red light-grown pea (Pisum sativum L.) seedlings have been investigated. The distribution measurement was made in a defined zone of the third internode, using 3H-IAA applied from the plumule as a tracer. The tropisms investigated were gravitropism, pulse-induced phototropism, and time-dependent phototropism. The investigation was extended to the phase of autostraightening (autotropism) that followed gravitropic curvature. It was found that IAA is asymmetrically distributed between the two halves of the zone, with a greater IAA level occurring on the convex side, at early stages of gravitropic and phototropic curvatures. This asymmetry was found in epidermal peels and, except for one case (pulse-induced phototropism), no asymmetry was detected in whole tissues. It was concluded, in support of earlier results, that auxin asymmetry mediates gravitropism and phototropism and that the epidermis or peripheral cell layers play an important role in the establishment of auxin asymmetry in pea epicotyls. During autostraightening, which results from a reversal of growth asymmetry, the extent of IAA asymmetry was reduced, but its direction was not reversed. This result demonstrated that autostraightening is not regulated through auxin distribution. In this study, the growth on either side of the investigated zone was also measured. In some cases, the measured IAA distribution could not adequately explain the local growth rate, necessitating further detailed investigation.
Key words: Autostraightening (autotropism), auxin, epicotyls, epidermis, gravitropism, indole-3-acetic acid, phototropism, Pisum sativum L
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionConcluding remarksReferences |
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The mechanisms of gravitropism and phototropism have long been discussed in terms of the hypothesis known as the Cholodny–Went theory of tropisms (Went and Thimann, 1937
). Results that do not agree with this hypothesis have been reported (Hasegawa et al., 1989; Togo and Hasegawa, 1991), but there have also been results that strongly support the hypothesis (Iino, 2001). A critical condition of the hypothesis is whether the endogenous auxin, indole-3-acetic acid (IAA), is asymmetrically distributed in the stimulated organ. This has been demonstrated in maize coleoptiles for both gravitropism (Mertens and Weiler, 1983; Iino, 1991) and phototropism (Iino, 1991). Another critical issue is the kinetic relationship between auxin asymmetry and growth asymmetry. Work with maize coleoptiles indicated that the former asymmetry adequately precedes the latter asymmetry in both gravitropism and phototropism (Parker and Briggs, 1990; Iino, 1991). More recent evidence for the hypothesis includes that the stem of tobacco plants transformed with the GUS reporter gene fused with the auxin-responsive GH3 promoter gene shows asymmetric distribution of GUS activity in response to gravitropic and phototropic stimulation (Li et al., 1999). Furthermore, the coleoptile of the rice cpt1 mutant was used to demonstrate that asymmetric IAA distribution occurs downstream of the phototropism-limiting CPT1, an orthologue of Arabidopsis NPH3 (Haga et al., 2005).
Gravitropically bent organs, such as coleoptiles and hypocotyls, straighten in their upper parts and finally assume a vertically straight appearance with a curvature retained at the base. This straightening process has long been referred to as autotropism, although it is not a tropism in a strict sense (Stankovi
et al., 1998). Here the straightening process is called autostraightening according to Tarui and Iino (1999). The following results have indicated that autostraightening is not a gravitropic response. First, gravitropically bent oat coleoptiles undertake autostraightening on horizontal clinostats (Dolk, 1936) and even under microgravity (Chapman et al., 1994). Second, autostraightening can be observed in zones of a gravitropically bent organ before these zones reach the vertical position (sunflower hypocotyls and maize coleoptiles; Firn and Digby, 1979) or even before any part of the organ reaches the vertical position (oat and wheat coleoptiles; Tarui and Iino, 1997). Evidence has been provided that autostraightening is based on a unique mechanism that actively counteracts gravitropism (Tarui and Iino, 1997) and is initiated in response to a preceding curvature (Tarui and Iino, 1999). The relationship between auxin and autostraightening has not been investigated. If auxin asymmetry accounts for autostraightening, the asymmetry found during the development of gravitropic curvature is expected to be reversed during the phase of autostraightening.
Pea epicotyls have served as a major material of tropism research. Burg and Burg (1966, 1967) found that radioactivity from applied 14C-IAA is asymmetrically distributed in the epicotyl stimulated for gravitropism (displaced by 90°) in favour of the curvature response. They observed a relatively large gradient between upper and lower quarters of the epicotyl. Iwami and Masuda (1976) could not find a significant gradient when the radioactivity from applied 14C-IAA was compared between the upper and lower halves. However, they found a significant gradient when only epidermal peels were used for the comparison. These results have indicated that auxin is asymmetrically distributed in pea epicotyls, especially in the epidermis or peripheral cell layers, in response to gravitropic stimulation. The results reported for phototropism have been controversial. Kang and Burg (1974) found that radioactivity from applied 14C-IAA is asymmetrically distributed between the irradiated and shaded quarters of the epicotyl. Kuhn and Galston (1992) could not obtain clear evidence that applied 14C-IAA is asymmetrically distributed between the irradiated and shaded halves of the epicotyl. Hasegawa and Yamada (1992) measured the distribution of endogenous IAA in phototropically stimulated epicotyls. They found no asymmetry between the irradiated and shaded sides even though the IAA level in epidermal peels was determined.
In this study, once again pea epicotyls were used to investigate auxin distribution during gravitropism and phototropism. For comparison, the differential growth underlying curvature responses was also investigated. Red light-grown seedlings were used to maximize phototropic responsiveness (Britz and Galston, 1983; Baskin, 1986). Auxin distribution was examined using 3H-IAA as a tracer. The comparison of 3H-IAA levels was made between bisected segments of a defined epicotyl zone and with epidermal peels obtained from these segments. It was also addressed for the first time whether autostraightening is correlated with auxin distribution.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionConcluding remarksReferences |
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Plant material and growth conditions
Seedlings of peas (Pisum sativum L. cv. Alaska; Watanabe Inc., Miyagi, Japan) were raised at 25±0.5 °C under continuous red light (2–3 µmol m–2 s–1) as described in Haga and Iino (1997)
. In brief, pea seeds were surface-sterilized with a sodium hypochlorite solution, rinsed in running tap water, and sown on moist paper towels in trays. After 2 d of incubation under red light, germinated seeds were transplanted into pots filled with 1% agar, placed in red acrylic boxes, and incubated further under red light. About 2.5 d after transplantation, seedlings were selected for the length of the third internodes (11–13 mm) and used for experiments. During the experiment, the selected seedlings were kept in the acrylic boxes, except when they were handled for treatments. Only for the experiments investigating autostraightening were seedlings grown for 3 d after transplantation; the third internodes of selected seedlings were 21–24 mm long.
Marking of third internodes and gravitropic and phototropic stimulation
The third internode of each seedling was marked with Indian ink to delimit 5 mm zones. The marks were placed on the two symmetrical sides of the internode and the top pair of marks was positioned 2–3 mm below the top surface of the hook. Marking was made to delimit one zone (when the third internodes were 11–13 mm; Fig. 1A) or two zones (when the third internodes were 21–24 mm).
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Gravitropic or phototropic stimulation of the epicotyl was initiated 5 h after marking of the third internode. For gravitropic stimulation, the entire seedling was displaced by 90° from the vertical. For phototropic stimulation, the entire epicotyl was irradiated for a defined period with unilateral blue light, which was obtained by passing light from a slide projector (Kodak Ektagraphic III, EXR 300 W lamp) or a light source unit (MHF-D100LR; Moritex, Tokyo, Japan) through a Corning blue glass filter (No. 5-50). The two light sources provided essentially identical photon fluence rate spectra. The direction of either stimulus was perpendicular to the plane of the hook (Fig. 1B). Control seedlings were handled similarly, but without displacement or blue light treatment.
Time-lapse analyses of curvature and differential growth
With the stimulus direction described above, the pea epicotyl developed gravitropic and phototropic curvatures in the direction perpendicular to the hook plane. These curvatures could be observed in either the front or the rear view of the epicotyl (Fig. 1A). However, the epicotyl showed a relatively large nutational movement along the hook plane (Galston et al., 1964; Baskin, 1986). Therefore, the rear and side images of the epicotyl (Fig. 1A) were used to conduct three-dimensional analyses of curvature and differential growth.
The rear and side images of each stimulated epicotyl were recorded simultaneously at 5 min intervals on Kodak technical pan film with two programmable cameras (F-801AF/MF-21; Nikon, Tokyo, Japan). The recorded negative images were expanded by means of a slide projector. The length and angle required for analysis were recorded on paper and measured with a computer-interfaced digitizer.
The length at either side of an epicotyl zone was determined using the rear and side images as described in Tarui and Iino (1999). When the zone had bent exceeding 5° in the rear or side view, it was divided into two or three smaller zones (Yoshihara and Iino, 2005). For determination of epicotyl curvature, the orientation angle 
of the epicotyl tip in the plane parallel to the stimulus direction was calculated using the following equation:
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1 and 2 are the angles made from a reference line by the line tangent to the long axis (elongation axis) at the epicotyl apex in the rear and side views, respectively. For phototropically stimulated epicotyls, the plumb line recorded with the epicotyl was used as the reference line. For gravitropically stimulated epicotyls, a line that was perpendicular to the recorded plumb line served as the reference line. The value of 2 was estimated by drawing a tangent line at the top mark of zone 1 (i.e. just below the hook). The value of represented the curvature of the entire epicotyl. The curvature of a zone was determined by measuring 1 and 2 at both top and base of the zone and subtracting the value of calculated for the base from that calculated for the top.
Application of 3H-IAA and preparation of tissue samples
Tracer experiments with 3H-labelled IAA (3-[5(n)-3H]-IAA, 780–1000 GBq mmol–1, Amersham Pharmacia Biotech, Buckinghamshire, UK) were performed to investigate the distribution of IAA during gravitropism and phototropism. 3H-IAA was mixed with lanolin as described in Haga and Iino (1998) to a concentration of 0.6 µg (2.6–3.4 MBq) g–1 lanolin. The 3H-IAA/lanolin mixture was applied to the upper surface of the plumule (Fig. 1C); a surface area of about 9 mm2 was in contact with the mixture. This application was made 1 h after marking of the third internode (see above) and 4 h before the onset of phototropic or gravitropic stimulation.
Tissues of the investigated zone were harvested at a defined time after the onset of stimulation. The tissues harvested were either the bisected segments of the zone or the epidermal peels obtained separately from the two halves of the zone. The harvested tissues were immediately frozen in liquid N2 and stored at –20 °C. Each tissue sample was obtained from nine seedlings (bisected segments) or 40 seedlings (epidermal peels). Each experiment was repeated to obtain at least three independent sets of tissue samples.
The epidermal peel consisted of either a single cell layer (epidermal cell layer) or two cell layers (epidermal cell layer and an additional cell layer) (Fig. 1D, a). More than 60% of epidermal peels showed two cell layers (Fig. 1D, b). To obtain these data, epidermal peels were fixed in 3.9% formaldehyde for 2 h at room temperature, dehydrated in a graded ethanol series, and embedded in Spurr resin. The embedded peels were cut with glass knives on an ultramicrotome to obtain their cross-sections (0.5 µm). The sections were mounted on glass slides, stained with 0.5% (w/v) toluidine blue O in water, and observed with a light microscope.
Measurement and analysis of 3H-IAA distribution
Extraction of 3H-IAA from the harvested tissues and its purification by TLC were carried out essentially as described in Iino (1991). The extraction and purification procedures consisted of the following steps: (i) incubating the tissues in 2 ml of 80% (v/v) methanol containing 300 µg cold IAA for 12 h (bisected segments) or 1 h (epidermal peels) at 4 °C; (ii) separating the tissues from the aqueous methanol and washing the tissues with a small amount of methanol; (iii) evaporating the aqueous methanol extract to an aqueous phase in a rotary evaporator; (iv) partitioning the aqueous phase (about 1 ml; adjusted to pH 2.5) against ether (0.5 mlx4); (v) partitioning the ether phase against 50 mM K2HPO4 (0.5 mlx4); (vi) partitioning the aqueous phase (pH 2.5) against ether (1 mlx4); (vii) evaporating the ether phase to dryness; (viii) developing the extract on a TLC sheet (0.1 mm, Polyamide-TLC 6UV254; Macherey-Nagel, Düren, Germany) with a solvent system of n-butanol:acetic acid:water (80:5:15, by vol.); (ix) scraping off the IAA zone, identified under UV light, and incubating the scrapings with methanol (1.5 ml) for 1.5 h at 4 °C; and (x) filtering the extracts through a glass filter and evaporating the filtrate to dryness. All organic solvents used were of HPLC or spectrophotometric grades (Nacalai Tesque) and those used before TLC contained butylated hydroxytoluene (Nacalai Tesque) at 0.1 mg ml–1 (Iino et al., 1980). The tissues recovered from the extraction medium were dried at 80 °C for 2 h and weighed.
The sample prepared as described above was dissolved in 1 ml of methanol. A 800 µl aliquot was added to 10 ml of scintillation cocktail (Clearsol 1, Nacalai Tesque) and measured for radioactivity with a liquid-scintillation counter (LS6200; Beckman Coulter, Fullerton, CA, USA). A 100 µl aliquot was diluted to 3 ml with methanol and measured for absorbance at 280 nm with a spectrophotometer. The absorbance value, which almost entirely represented the cold IAA added at the beginning of extraction, was used to estimate the recovery of 3H-IAA. The concentration of 3H-IAA, Bq g–1 dry weight (DW), was calculated on the basis of the measured radioactivity, estimated recovery, and measured DW.
Although the amount of 3H-IAA applied to each seedling could not be controlled exactly, the relative distribution of 3H-IAA between the two sides of an epicotyl zone did not depend on the amount applied, as demonstrated by control measurements. Therefore, the amount of 3H-IAA recovered from each side of the zone of stimulated seedlings was determined as a percentage of the total 3H-IAA recovered from the two sides. The data obtained allowed the relative distribution between the two sides to be examined. The amount of 3H-IAA recovered from each side of the zone of stimulated seedlings was calculated as a percentage of the amount recovered from the corresponding side of the zone in control seedlings. The data obtained in this way, though more variable than those shown above, allowed it to be evaluated whether the absolute level of 3H-IAA was enhanced or reduced.
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionConcluding remarksReferences |
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Differential growth and auxin distribution during gravitropism
Experiments were performed with red light-grown pea epicotyls that were marked to define an apical zone of the third internode (zone 1, Fig. 1A). Gravitropic differential growth that took place in zone 1 of the epicotyl displaced by 90° (Fig. 1B) was investigated by monitoring the lengths along the upper and lower flanks of zone 1. Because the epicotyl showed nutational movement, these lengths were determined three dimensionally using both rear and side images of the epicotyl (see Materials and methods). As shown in Fig. 2A, the differential growth corresponding to negative gravitropism was initiated 20–25 min after displacement. The growth on the upper flank was inhibited and that on the lower flank was stimulated. The overall growth of the zone was not much affected. The data suggested, however, that the growth inhibition on the upper flank occurred more rapidly than the growth stimulation on the lower flank.
The procedures described in the Materials and methods in detail were used to investigate the distribution of applied 3H-IAA between the upper and lower halves of zone 1. 3H-IAA was applied to the plumule (Fig. 1C), and after allowing to stand for 4 h, the seedling was displaced to induce gravitropism. The levels of extractable 3H-IAA in whole tissues and epidermal peels of the halves were determined 30 min after displacement (i.e. at an early stage of curvature development; see the time indicated by an arrow in Fig. 2A, b). Based on these measurements, the relative distributions of 3H-IAA were determined. In whole tissues, no statistically significant difference occurred between the two halves (Fig. 2B, a). In epidermal peels, more 3H-IAA was clearly distributed to the lower half than to the upper half at an approximate ratio of 2:1 (Fig. 2B, b).
To obtain further information, the level of 3H-IAA in either half of the stimulated zone was calculated as a value relative to the level in the corresponding control (see Materials and methods for a methodological limitation). The 3H-IAA level in whole tissues of either half was not significantly different from the control level. The 3H-IAA level in epidermal peels was reduced in the upper half and was enhanced in the lower half. The statistical significance of the difference from the control level was marginal (0.05<P<0.07).
Differential growth and auxin distribution during phototropism
The experimental procedures described above were applied to investigate the differential growth and 3H-IAA distribution during phototropism of pea epicotyls. To induce phototropism, the epicotyl was stimulated with unilateral blue light for 30 s or 20 min at a fixed fluence rate of 0.1 µmol m–2 s–1 (Fig. 1B). The 30 s stimulation provided a total fluence that is optimal for the first pulse-induced positive phototropism (Baskin, 1986; for terminology, see Iino, 1990). Here this phototropism is simply called the pulse-induced phototropism, because it is the only pulse-induced phototropism identified in peas. The 20 min stimulation resulted in a typical time-dependent phototropism (Iino, 1990).
As shown in Fig. 3A, the differential growth for pulse-induced phototropism was initiated about 20 min after stimulation. Comparison with the control measurements indicated that the growth on the irradiated side was inhibited and that on the shaded side was stimulated; the overall growth of the zone was not significantly affected during phototropism. The differential growth for time-dependent phototropism had already been initiated when the 20 min stimulation was terminated (Fig. 4A). The growth on the irradiated side was inhibited, but only slightly. The growth on the shaded side was enhanced substantially. As a result, the overall growth of the zone was enhanced, in contrast to the case for pulse-induced phototropism.
Distribution of applied 3H-IAA between the irradiated and shaded halves of zone 1 was determined 25 min after the onset of blue light stimulation. During pulse-induced phototropism, 3H-IAA was asymmetrically distributed between the two halves, with a greater 3H-IAA level occurring on the shaded half in both whole tissues and epidermal peels (Fig. 3B). During time-dependent phototropism, a clear 3H-IAA asymmetry was detected in epidermal peels with an approximate ratio of 3:5 (Fig. 4B). The extent of asymmetry found in epidermal peels was greater than that found for pulse-induced phototropism. In sharp contrast to the case for pulse-induced phototropism, however, whole-tissue measurements showed no significant difference between the two halves (Fig. 4B).
The level of 3H-IAA in either half was also calculated as a value relative to the control level. As shown in Figs 3C and 4C, the level of 3H-IAA in the shaded half was significantly greater than the control level in all cases where asymmetric distribution of 3H-IAA was detected. On the other hand, in these cases, the level in the irradiated half was not significantly different from the control level. Therefore, during both pulse-induced and time-dependent phototropism, the asymmetric 3H-IAA distribution appeared to occur by an increase of 3H-IAA in the shaded half without any comparable decrease in the irradiated half.
The absolute concentrations of 3H-IAA in the whole tissues and epidermal peels were calculated from the control data obtained for Figs 2–4
(Table 1). The concentration in epidermal peels (16 pmol g–1 DW) was much less than that in whole tissues (2.2 pmol g–1 DW). This is, however, because the concentration was determined on a DW basis (note that the epidermal peel is heavier than other tissues due to the presence of cuticles and thicker cell walls). The mean concentrations calculated on a fresh weight (FW) basis using the measured relationship between DW and FW were 0.62 pmol g–1 (whole tissues) and 0.41 pmol g–1 (epidermal peels). Therefore, on a FW basis, the level of 3H-IAA in epidermal peels was lower by about 30% than the level in whole tissues.
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Differential growth and auxin distribution during autostraightening
The next series of experiments was conducted to examine the differential growth and 3H-IAA distribution during gravitropism, including the phase of autostraightening. For this purpose, longer internodes were used and they were marked to define two apical zones (Fig. 5A). As shown in Fig. 5B (squares), the epicotyl displaced by 90° initially showed a slight downward (positive) curvature for about 20 min and then a large upward (negative) curvature that reached the maximal angle at about 100 min. During the subsequent period of 100 min, the epicotyl maintained an angle of 60–70°; i.e. the epicotyl apex did not reach the vertical position in this period. The curvature of zone 2 (Fig. 5B, circles) was analysed further. After reaching the maximal curvature at 100 min, this zone underwent straightening or autostraightening. This straightening response is clearly not a gravitropic response because it occurred before any part of the epicotyl reached the vertical position.
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Figure 5C shows the growth of zone 2 on the two flanks. The early time-courses, which represent the development of gravitropic curvature, were similar to those shown in Fig. 2A. A difference noted was a somewhat stronger and more rapid growth inhibition on the upper flank. The straightening of the zone after 100 min was clearly caused by the decrease and increase in growth rate that took place on the lower and upper flanks, respectively. In the phase of autostraightening (100–200 min), the growth rate on the upper flank was much greater than that on the lower flank.
Distributions of applied 3H-IAA in zone 2 were investigated. At 30 min after displacement, no asymmetric distribution between the two halves was found when 3H-IAA in whole tissues was determined (Fig. 5D). However, an asymmetric distribution at a ratio of 2:1 was found in epidermal peels (Fig. 5E, a). These results are in accordance with those shown in Fig. 2B. The 3H-IAA level in the upper-half epidermal peels was reduced (by about 30%) and that on the lower-half epidermal peels was enhanced (by about 50%) compared with the control level (data not shown). The increase in the lower half was statistically significant (P=0.0004), whereas the decrease on the upper half showed marginal significance (P=0.051). These results are similar to those shown in Fig. 2C.
As shown in Fig. 5E, the extent of 3H-IAA asymmetry was progressively reduced during autostraightening. However, the direction of asymmetry was not reversed, even when the growth rate on the upper side became substantially greater than that on the lower side (Fig. 5E, c). Therefore, the asymmetry in 3H-IAA level did not agree with the growth asymmetry underlying autostraightening.
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Discussion |
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Tracer investigation of IAA distribution
To use 3H-IAA as a tracer to investigate the distribution of IAA in pea epicotyls, the following technical points were taken into consideration in this study. First, 3H-IAA was applied to the plumule, the expected site of IAA biosynthesis. Second, 4 h was allowed between 3H-IAA application and the onset of phototropic or gravitropic stimulation, so that applied 3H-IAA is well equilibrated with endogenous IAA in the epicotyl at the time of stimulation. Third, because applied 3H-IAA is likely to be metabolized during its transport, the IAA fraction separated from tissue extracts was used for distribution measurement. In addition, corrections were made for losses of 3H-IAA during its extraction and purification.
The distributed 3H-IAA must remain at a negligible level so that it does not alter the distribution pattern of endogenous IAA. The concentration of endogenous IAA in the third internode of red light-grown Alaska pea seedlings was reported to vary from 60 to 325 pmol g–1 FW (Ulvskov et al., 1992). The concentration of 3H-IAA in zone 1 of the third internode was estimated to be about 0.6 pmol g–1 FW (see Results). Therefore, it is expected that applied 3H-IAA enhanced the level of IAA by less than 1%, which can be regarded as negligible.
The concentration of 3H-IAA in epidermal peels, determined on a DW basis, was very low compared with the concentration in whole tissues (Table 1). On a FW basis, however, the concentration in epidermal peels was lower by only about 30% than the concentration in whole tissues (see Results). This distribution pattern is essentially in agreement with the measurements of endogenous IAA in whole tissues and epidermal peels of pea third internodes (Behringer and Davies, 1992; data for a tall cultivar).
Involvement of auxin in gravitropism and phototropism
During both gravitropism and phototropism, the tracer 3H-IAA was found to be asymmetrically distributed in epidermal peels, which is largely composed of the epidermis and an additional cell layer (Fig. 1D). Although asymmetric distribution of 3H-IAA occurred in whole tissues during pulse-induced phototropism, no significant asymmetry could be found during gravitropism and time-dependent phototropism. These results are in agreement with those reported by Iwami and Masuda (1976) for gravitropism and by Burg and his coworkers for gravitropism (Burg and Burg, 1967) and phototropism (Kang and Burg, 1974). The former authors showed that the radioactivity from applied 14C-IAA is asymmetrically distributed in epidermal peels but not in whole tissues of the epicotyl. The latter authors showed that a significant asymmetry could be detected when radioactivity from applied 14C-IAA was compared between the two outer quarters of epicotyl segments. Together, these results strengthen the validity of the Cholodny–Went theory in both gravitropism and phototropism of the pea epicotyl and point to the significance of the epidermis or peripheral cell layers for the establishment of auxin asymmetry in this material. The latter aspect implies that, in pea epicotyls, the distribution of IAA is controlled so that the level of IAA is specifically modified in its target tissue (Kutschera, 1987; Yamagami et al., 2004).
Britz and Galston (1983) could not detect asymmetric distribution of the radioactivity from applied 14C-IAA between irradiated and shaded halves of the epicotyl. The induction of phototropism in the hook plane was considered to be responsible for the failure to detect an asymmetric distribution. Because these authors investigated time-dependent phototropism, it is more likely that the asymmetry was not detected because the comparison was made between two halves. Hasegawa and Yamada (1992) could not detect any asymmetry of endogenous IAA between irradiated and shaded halves, even though they used epidermal peels for IAA determination. The present study can provide no explanation for this controversial result.
Relationship between IAA levels and growth rates
During gravitropism, the concentration of 3H-IAA in epidermal peels was reduced on the upper half and was enhanced in the lower half. Although the statistical significance of the difference from the control level was marginal in many cases, this result was reproduced by two independent series of experiments (Fig. 2; see Results for the other series of experiments). This distribution pattern is in approximate agreement with the growth rate distribution (i.e. growth inhibition on the upper side and stimulation on the lower side; Figs 2A and 5C). It is, however, noted that the growth on the upper flank was inhibited to the extent that the tissues on this flank undergo a slight shrinkage. Apparently the level of IAA was not reduced to that extent.
During time-dependent phototropism, the level of 3H-IAA in epidermal peels was enhanced by about 80% in the shaded half, but no statistically significant change was found in the irradiated half (Fig. 4C). This distribution pattern is in approximate agreement with the growth rate distribution (i.e. a substantial growth stimulation on the shaded flank and a slight growth inhibition on the irradiated flank; Fig. 4A). However, such an agreement was not found for pulse-induced phototropism (i.e. a clear growth inhibition on the irradiated half, but no statistically significant decrease in 3H-IAA level in the irradiated half; Fig. 3A, C).
In conclusion, these results could not resolve the exact relationship between auxin levels and growth rates. Because the epidermal peels were obtained from a half side of the investigated zone, it is not entirely excluded that the 3H-IAA level in a narrow peripheral region more closely correlates with the measured growth rate. Furthermore, the mechanisms of tropisms, though auxin distribution most probably plays a key role, might be more complex than expected from the simple form of the Cholodny–Went hypothesis. For example, tropisms may involve other growth-regulating components that are asymmetrically distributed in parallel to the induction of auxin asymmetry or as a consequence of the induced auxin asymmetry (Iino, 2001).
Relationship between autostraightening and auxin
Autostraightening could be observed in a zone of gravitropically bent pea epicotyls. As found for oat and wheat coleoptiles (Tarui and Iino, 1997), this autostraightening progressed before any part of the epicotyls reached the vertical position (Fig. 5B). Therefore, the pea epicotyl provides another case in which autostraightening occurs entirely against negative gravitropism.
The asymmetry of 3H-IAA established at the beginning of gravitropism was reduced during autostraightening (Fig. 5E). This reduction is explicable because the stimulus strength for negative gravitropism is apparently reduced as the gravitropic curvature progresses. The asymmetry of 3H-IAA was not reversed, however, in the phase of autostraightening. Therefore, the auxin distribution fails to explain the reversed growth asymmetry underlying autostraightening. It is concluded that autostraightening is not achieved by regulation of auxin distribution.
It has been shown that the sensitivity or responsiveness to IAA in the IAA-induced growth response changes in response to a preceding change in the level of IAA with a lag time of a few hours (Haga and Iino, 1998). Autostraightening is the expected outcome if such an adaptive response takes place locally within an organ. In pea epicotyls, an increase in IAA level resulted in a drop in the responsiveness to IAA (Haga and Iino, 1997). However, no clear adaptive response followed a decrease in IAA level (Haga and Iino, 1998). The results cannot entirely explain the changes in growth rate that account for autostraightening.
Further implications of the results for phototropism
The first pulse-induced phototropism of maize coleoptiles is induced by a growth inhibition on the irradiated side and a compensating stimulation on the shaded side (Iino and Briggs, 1984). Such a simple redistribution pattern cannot always be found during phototropism (Iino, 1990). A probable cause of a complex growth distribution pattern is the occurrence of non-phototropic light-growth responses. Such light-growth responses include those that are specifically induced by blue light (Liscum et al., 1992; Folta and Spalding, 2001; Haga et al., 2005).
It is of interest to resolve whether the enhancement of overall growth found during time-dependent phototropism (Fig. 4A) is associated with the mechanism of phototropism or represents a separate light-growth response. Laskowski and Briggs (1989) and Warpeha and Kaufman (1989) found that blue light affects the straight growth of red light-grown pea epicotyls in a complex manner. The reported results, however, indicate that the growth of epicotyls is inhibited by the blue light fluences used in the present study. It is possible that the enhancement of overall growth observed during time-dependent phototropism is associated with the mechanism of phototropism itself.
It is interesting that 3H-IAA was asymmetrically distributed in whole tissues during pulse-induced phototropism (Fig. 3B, a), but not during time-dependent phototropism (Fig. 4B, a). The asymmetry in epidermal peels detected during pulse-induced phototropism (Fig. 3B, b) was not greater than that detected during time-dependent phototropism (Fig. 4B, b). Therefore, the asymmetry detected in whole tissues during pulse-induced phototropism appears to be a property inherent to this phototropism. There has been no direct evidence that the two types of phototropism are fundamentally different and a model has been discussed to relate pulse-induced phototropism to time-dependent phototropism (Iino, 2001). The present finding needs to be considered in further investigation of the relationship between the two types of phototropism.
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TopAbstractIntroductionMaterials and methodsResultsDiscussionConcluding remarksReferences |
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Present tracer experiments have clearly demonstrated that auxin is asymmetrically distributed in the epidermis or peripheral cell layers during gravitropism and phototropism. On the other hand, the results obtained here could not resolve the quantitative relationship between auxin asymmetry and growth asymmetry. Although further investigation is clearly required, it is most likely that the mechanism which allows a specific regulation of the level of IAA in the epidermis or peripheral cell layers is central to the process of tropisms in pea epicotyls. Two models can account for such a mechanism: IAA transport between the peripheral and inner cell layers (Macdonald and Hart, 1987
) and lateral IAA transport along the peripheral cell layers (Iino, 2001).
Autostraightening is a biologically important process that facilitates straightening of organs that have curved by gravitropism and perhaps also by phototropism (Tarui and Iino, 1999). It was investigated for the first time whether autostraightening can be related to auxin asymmetry. It is the most significant finding of this study that autostraightening does not involve auxin asymmetry as a mediating step. How autostraightening is achieved is entirely the subject of future study.
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Acknowledgements |
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We thank Professor Takao Itoh (Research Institute for Sustainable Humanosphere, Kyoto University, Japan) for allowing us to use the Institute's radioisotope research facilities and Dr Satoshi Kimura (Research Institute for Sustainable Humanosphere, Kyoto University, Japan) for his help with the preparation of epidermal peel cross-sections. This work was supported by Grant-in-Aid for Special Research (No. 13139204) from the Ministry of Education, Culture, Sports, and Science.
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Footnotes |
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Abbreviation: IAA, indole-3-acetic acid.
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References |
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