Agravitropic Growth and Its Relation to the Formation of the Plumular Hook in Etiolated Shoots of the Pea Mutant, Ageotropum

J. PlantPhysiol. Vol. 138. pp. 216-222 {1991}

Agravitropic Growth and Its Relation to the Formation of the Plumular Hook in Etiolated Shoots of the Pea Mutant, Ageotropum HIDEYUKI TAKAHASHI!'~, HIROSHI SUGE!, 1

2

and MORDECAI J. JAFFE2

Institute of Genetic Ecology, Tohoku University, Katahira, Aoba-ku, Sendai 980, Japan and Department of Biology, Wake Forest University, Winston-Salem, NC 27109, USA

Received February 21, 1990 . Accepted November 21, 1990

Summary Shoots of a pea mutant, ageotropum (Pisum sativum L.) exhibited agravitropic growth and less ability to form a plumular hook in the dark. Decapitated epicotyls of dark-grown ageotropum pea bent away from external IAA when applied to the cut-end asymmetrically. But symmetrical application of IAA to the entire cut-surface of the horizontally placed epicotyls could not induce gravitropic curvature. In horizontally placed epicotyls of dark-grown ageotropum pea, [3H] label applied as [3H]IAA moved basipetally but did not apparently move laterally. Seedlings of ageotropum pea formed the plumular hook to some extent in response to externally applied ethylene. Alaska (normal) epicotyls evolved 2 to 5 times more ethylene than ageotropum epicotyls. Gravistimulation of the shoots increased ethylene evolution in Alaska epicotyls but not in ageotropum epicotyls. These results suggest a positive correlation between auxin asymmetry and ethylene production, which may influence both shoot gravitropism and formation of the plumular hook.

Key words: Pisum sativum L. (pea), ageotropum, Alaska, auxin, ethylene, gravitropism, plumular hook. Introduction Although some earlier works denied the involvement of gravity in the formation of plumular hooks (Darwin, 1880; Rubinstein, 1972), an interaction between gravitropism and hook formation in dark-grown dicotyledonary seedlings has been indicated by other studies (Karve, 1964; MacDonald et al., 1983; Tupper-Carey, 1929). For example, 1) mutant seedlings of A rabidopsis thaliana and Lycopersicon esculentum that have lost orthogravitropism through a single gene mutation are incapable of forming a complete hypocotyl hook (MacDonald et al., 1983; Mirza, 1987; Zobel, 1974), and 2) clinostating of horizontally placed seedlings inhibits the formation of the plumular hook as well as gravitropic bending (MacDonald et al., 1983; Takahashi and Suge, 1988). At this time, however, we do not know the mechanism by which the gravitropic response affects hook formation. Therefore,

* To whom correspondence should be addressed. © 1991 by Gustav Fischer Verlag, Stuttgart

studies with a mutant or clinostat further need to elucidate a mutual factor in gravitropism and hook formation, which is affected by gravity. Both ethylene and auxin have been considered as major factors regulating the formation and opening of the plumular hook (Goeschl et al., 1967; Kang et al., 1967; Kang and Ray, 1969; Klein, 1965; Rubinstein, 1971). These two substances are also involved in shoot gravitropism; an increase of ethylene production as well as auxin asymmetry likely play some role in shoot gravitropism in some plant species (Abeles and Rubinstein, 1964; Bandurski et al., 1984; Clifford et al., 1983; Dolk, 1936; Migliaccio and Galston, 1987; Wheeler et al., 1986). Furthermore, it is known that treatments with an inhibitor of auxin transport and inhibitors of ethylene biosynthesis or action block both shoot gravitropism and hook formation in some plant species (Takahashi and Suge, 1988; Wheeler and Salisbury, 1980, 1981). It was assumed that the increase of ethylene production in the gravistimulated shoots might result from the gravity-induced

Agravitropic growth and hook formation in peas

auxin asymmetry (Abeles and Rubinstein, 1964; Wheeler et a1., 1986). Thus, although as yet there is no direct evidence, auxin asymmetry and an increase of ethylene production, which take place during gravitropic response, could be responsible for the formation of a plumular hook (Abeles and Rubinstein, 1964; Clifford et aI., 1983; Wheeler et aI., 1986). In this study, a pea mutant, ageotropum was used to examine the roles of auxin, ethylene and their relationships in shoot gravitropism and the formation of the plumular hook. Roots of ageotropum pea show agravitropic behavior both in the dark and light (Ekelund and Hemberg, 1966; Olsen and Iversen, 1980 a; Jaffe et aI., 1985), and this was thought to be due to a disruption of the graviperception or signal transduction mechanism in the root tips (Ekelund and Hemberg, 1966; Olsen and Iversen, 1980 b). Ageotropum shoots, on the other hand, were reported to be agravitropic in darkness but gravitropic in light (Blixt et aI., 1958; Scholdeen and Burstrom, 1960). We therefore compared hook formation, auxin asymmetry, and ethylene production of ageotropum seedlings with those of orthogravitropic pea seedlings.

Materials and Methods Plant materials Seeds of a normal pea cuhivar (Pisum sativum 1., cv. Alaska) and those of a pea mutant, ageotropum were planted with radicles pointing downward in plastic boxes filled with presoaked vermiculite. Those boxes were kept in total darkness at 25 °C for 5-6 days. When the epicotyls were about 6 - 8 cm long, the seedlings were used for the experiments, which were done in the dark at 25°C or under a dim green light when necessary. In experiments using lightexposed seedlings, the seedlings were placed vertically under 34-W white fluorescent tubes (F40CW IRS/EW-II, Philips, Bloomfield, NJ, USA) at 3.2 W m - 2 for 10 min.

Gravitropic and auxin· induced curvature of epicotyls The shoot apex above the hook portion and the root were cut off and discarded. The decapitated epicotyls, retaining cotyledons, were placed vertically or horizontally in a plastic box in which humidity was kept high by layers of wet filter paper. An agar block as a control or a block containing 10 - S M buffered IAA with 2 mM MES at pH 6.2 was placed over the entire cut-surface symmetrically or over one edge of the cut-surface asymmetrically. Six hours after the start of the treatment, curvature of epicotyls were measured by protractor. Unless otherwise mentioned, the donor blocks used were 1.5 % agar and were 1.5 x 1.5 x 1.5 mm in dimensions.

Movement ofPH} label applied as PH}lAA Basipetal movement of [3H] label applied as [3H]IAA in the tissues was measured in the horizontally or vertically placed epicotyls. An epicotyl section 5 mm long was obtained from just below the hook portion and placed vertically on a receiver agar block (5 x 5 x 5 mm, 1.5 % in concentration). A donor agar block containing approximately 10,000 dpm eH]IAA (approximately 2.6 x 10- 9 M as IAA concentration; purchased from Amersham Laboratories, Buckinghamshire, England), was applied to the apical cutsurface. Five hours later, radioactivity in the donor blocks, the receiver blocks, and the epicotyl sections were analyzed. In another experiment, the shoot apex and root were removed from an etiolat-

217

ed seedling in a verticalposition, and the donor agar block was symmetrically placed over the entire cut-surface of the decapitated epicotyls. Then, the epicotyl was oriented horizontally. Five hours later, a 42-mm section of the epicotyl including the donor agar block was excised from the distal portion and further divided into three portions: a 2-mm uppermost section with the donor agar block, a 20-mm middle section, and a 20-mm basal section. The amount of [3H] label that moved laterally in the horizontally placed epicotyls was measured using the middle portion of the epicotyl section, which was further divided into upper and the lower halves with a razor blade. Radioactive materials from an excised tissue or an agar block were extracted with 5 mL methanol (99 % v/v) in a 20mL scintillation counting vial. After overnight extraction at 4°C, tissue materials were removed, and a 5 -mL liquid scintillation cocktail, Insta Gel (Packard Instrument Co., Illinois, USA) was added to the extract. The [lH] label of the entire phase of uniformly mixed sample was measured in a Packard TriCARB 300 scintillation counter. Recovery rates of [3H] label and [3H]IAA from the epicotyl tissues 5 h after the administration were approximately 89 % and 60 %, respectively. The data shown were calculated based on dpm's after quenching and background adjustments.

Ethylene treatment Imbibed seeds were planted in a 50-mL beaker containing a soil composite and kept in total darkness at 25°C for 3 days. Then the beaker was enclosed in a 500-mL bell jar, and immediately 2 ILL L -1 of ethylene was injected into the jar through a rubber stopper with a gas-tight syringe. The ethylene treatment was continued for 2 d. Etiolated five-day-old seedlings were also placed horizontally in a 2-L plastic container and treated with 2 ILL L - 1 ethylene for 5 h in the dark.

Measurement of hook angle and ethylene evolution Etiolated seedlings were sampled at different stages for both measurements of hook formation and ethylene evolution. The angle formed by the line parallel to the straight epicotyl, just below the hook portion, and the line parallel to the straight subapical portion was determined by a protractor. In this measurement, growth stages after germination were classified as follows: stage I; the epicotyl retains a strong hook immediately after germination, stage II; the epicotyl hook begins to open to some extent, and stage III; the hook is reformed. To measure ethylene evolution, a 1-cm epicotyl section obtained from just below the hook portion or a 2-cm root tip section of an etiolated seedlings was excised, and 5 such sections were placed in a 5-mL vial. The vial was sealed with a silicone cap. To measure ACCor IAA-induced ethylene production, a 3-cm shoot section with the apex was obtained from the etiolated seedling. The shoot section was vertically placed in a solution of ACC or IAA (10 - 4 M in 2 mM MES buffer at pH 6.2) so that the 1-cm basal part was immersed in the solution in a glass tube for 3 h .Three such sections were then enclosed in a 10-mL vial. One mL of air was withdrawn from the vial after a 2 h incubation at 25°C for ethylene measurement. In order to examine the effect of gravistimulation on ethylene evolution, dark-grown seedlings were placed in a vertical or horizontal position for 3 h .Then, a 3-cm epicotyl section was obtained from below the hook region. Five such sections were, in a vertical position, enclosed into the vial and incubated for an hour. The amount of ethylene was determined by a gas chromatograph (Model GC4CMPF, Shimazu Seisakusho LTD., Kyoto, Japan) equipped with a flame ionization detector and an activated aluminum column.

218

lliDEYUKI

TAKAHASHI, HIROSHI SUGE, and MORDECAI J. JAFFE

Results In Alaska peas, both intact and decapitated epicotyls in a horizontal position showed unequivocal negative gravitropism in the dark (Table 1). Replacing the shoot apex with an IAA-containing (10- 5 M) agar block, by applying the blocks to the entire cut-surface of the decapitated epicotyls, suppressed the gravitropic curvature, but they still showed a considerable negative gravitropism (Table 1). In horizontal epicotyls of Alaska pea, gravitropic curvature was slightly inhibited by IAA applied asymmetrically to the top edge of the decapitated cut-end, whereas the IAA applied to the bottom edge of the decapitated cut-end further stimulated upward curvature (Table 1). On the other hand, both intact and decapitated epicotyls of ageotropum peas in a horizontal position did not bend at all in the dark (Table 1). Exogenous IAA applied to the entire cut-surface of the decapitated epicotyls did not induce gravitropic bending in horizontally placed epicotyls of ageotropum peas (Table 1). However, horizontally placed epicotyls of ageotropum peas responded positively to IAA applied asymmetrically to the top edge or bottom edge of the decapitated cut-end with an induced curvature away from the IAA source; bending downward in the former and upward in the latter (Table 1). In vertically placed epicotyls, IAA applied to one edge of the decapitated cut end induced curvature away from the IAA source in both Alaska and ageotropum peas (Table 1). Curvature induced by asymmetrical IAA application was about 30-35 °C in both vertically and horizontally placed epicotyls of ageotropum pea. As shown in Table2 and Fig. 1, there was not much difference between vertically placed Alaska and ageotropum epicotyls in the amount of [3H] label applied as [3H]IAA, which moved in basipolar fashion. However, the rate of basipetal movement was slightly higher in ageotropum than Alaska when the epicotyls were placed horizontally. Asymmetry of PH] between the upper and the lower halves in horizontally

Table 1: Gravitropic and IAA-induced curvature of Alaska and ageotropum pea epicotyls. Intact or decapitated epicotyls with cotyledons attached were placed vertically or horizontally. Agar blocks containing 10 - 5 M IAA were then placed over the entire cut-surface of the decapitated epicotyls symmetrically or over one end of the cut-surface of the decapitated epicotyls asymmetrically. Experiments were done in the dark. Data show the mean ± SE of 12 plants. Orientation and decapitation

IAA application

Vertical Decapitated NoIAA Decapitated IAA; Asymmetrically Horizontal NoIAA Intact Decapitated NoIAA Decapitated IAA; Symmetrically Decapitated IAA; Upper end IAA; Lower end Decapitated ,. Bending away from the IAA source. ** Bending upward. **" Bending downward.

Curvature after 6 h (degrees) Alaska

Ageotropum

1±1 28±2*

1±1 32±3*

70±4** 63±3** 38±4** 52±3** 81±3 ....

1±1 1±1 1±1 -36±3*** 33±4**

Table 2: Basipetal movement and redistribution of pH], applied as [3H]IAA, in horizontally placed epicotyls of Alaska and ageotropum peas. Experiments were done in the dark. Data in parentheses indicate percent recovery of [3H] label in the upper and the lower halves of the middle section. Shown as the mean ± SE (n= 15). Variety

Radioactivity (%) at different regions of epicotyl 2 mm uppermost section

20 mm middle section

Alaska

61±2

Ageotropum

52 ± 2

3H2 (Upper 41±2) (Lower 59±2) 38±1 (Upper 49±2) (Lower 51 ±2)

ALASKA

13 !1 A

64 ! 3 C

20 mm basal section 7±t

10±l

AGEOTROPUM

13! 1 B

46

!

3

o

Fig. 1: Basipetal movement and lateral asymmetry of [3H] label in the epicotyls of Alaska and ageotropum peas. Closed and open rectangles indicate the IAA-containing agar donor block and receiver agar block, respectively. A (Alaska) and B (ageotropum): epicotyl sections 5 mm long were placed vertically with the apical end upward. Data show the percentage of radioactivity recovered by the receiver agar block, as the mean ± SE (n ~ 11). C (Alaska) and D (ageotropum): epicotyl section 20 mm long were obtained from the subapical portion of the horizontally placed seedlings. Experiments were done in the dark. Data show the percent of radioactivity distributed into the upper and the lower halves of the horizontal sections, as the mean ± SE (n - 11). Arrow (g) indicates the direction of gravitational force.

placed epicotyls of Alaska was obvious, the ratio being approximately 2: 3 (upper half: lower half). On the other hand, no significant asymmetry of [3H] distribution was seen in horizontally placed epicotyls of ageotropum pea. In an observation of the time-course of hook formation in Alaska peas, it was seen that the plumules that had initially arched before imbibition opened slightly after germination (Fig. 2A). Then, they arched back to a greater extent and never opened during the experiment in darkness (Fig. 2A). Ageotropum peas also possessed arched epicotyls initially, which started to open at an early stage soon after the plumules emerged from the cotyledons (Fig. 2 A). The plumular hooks of the ageotropum shoots opened almost straight 4 days after imbibition (stage II), and these epicotyls

Agravitropic growth and hook formation in peas 180 160

tionary rate was unchanged during the time-course study (Fig. 2B). However, both Alaska and ageotropum shoots responded to an externally applied ACC or IAA with an increase in ethylene production (Fig. 3). When the seedlings were reoriented horizontally from a vertical position in the dark, there was an increase in ethylene evolution in Alaska epicotyls but not in ageotropum epicotyls (Table 3). When seedlings were treated with 2 ILL L - I ethylene for 2 days, Alaska shoots showed strongly arched hooks (Table 4). Ageotropum shoots treated with ethylene also showed a hook-like curvature (Table 4). The difference in ethylene effect between treatments with and without ethylene was greater in ageotropum than in Alaska. Both Alaska and ageotropum shoots showed a typical triple response, that is, diagravitropic curvature, thickening, and retardation of the elongation of the epicotyls in response to externally applied ethylene (data not shown). Exogenous ethylene applied to horizontally placed shoots of ageotropum pea induced downward curvature in the dark, whereas the light-exposed shoots bent upward (Table 5).

A ALASKA

(/)

t!:j140 ex: 0120 w

°,100 w

-l 0 80

Z


0 0

J:

40 20 0

'i"

J::

12

o ALASKA

SHOOT • AGEOTROPUM SHOOT o ALASKA ROOT • AGEOTROPUM ROOT

B

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en 10

.>< -l

8

W'

6

::l.-

z

w

-l

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w

4

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Discussion

1

0

80

----"

STAGE I

90

100

110

120

130

219

140

150

STAGE II STAGE III TIME AFTER IMBIBITION, h

It is of interest that some mutant plants with no orthogravitropism show a lesser ability to form plumular hooks

Fig, 2: Time-course studies of hook formation and ethylene evolution in Alaska and ageotropum seedlings. In A (formation and opening of plumular hook): open and closed circles indicate angle of the hook for Alaska and ageotropum peas, respectively. In B (ethylene evolution): open and closed circles indicate ethylene evolution from Alaska and ageotropum epicotyls, respectively, and open and closed squares indicate that of Alaska and ageotropum roots, respectively. Vertical bars indicate standard deviations of 15 samples for each hook angle and of triplicate samples for ethylene evolution. Experiments were done in the dark. See text for classification of the stage.

3

D E2d

ALASKA AGEOTROPUM

Table 3: Ethylene evolution of Alaska and ageotropum shoots in response to gravistimulation. Experiments were done in the dark. Data show the mean ± SE of triplicate samples. Variety Alaska

Ageotropum

Treatment Vertical Horizontal* Vertical Horizontal*

w

Ethylene evolution (JlL kg- I h- I )

z

3.2±O.2 4.l±O.3 1.7±O.2 1.7±O.1

J:

W

-l

>-

~

W

* Seedlings were grown in a vertical position, then placed horizontally for 3 h.

retained the straight form at least during the experiment and did not form complete hooks (Fig. 2 A). In dark-grown seedlings, Alaska epicotyls evolved 2 to 5 times greater amounts of ethylene than did ageotropum epicotyls, with similar results for the roots (Fig. 2 B). Ethylene evolution by Alaska epicotyls significantly increased at stage II, and a somewhat higher ethylene level was maintained thereafter (Fig. 2 B). On the other hand, ageotropum epicotyls evolved much less ethylene, and the lesser evolu-

O~~

__~LL__L-~~L--L__~~

CONTROL

Ace

IAA

Fig. 3: Ace- and IAA-induced ethylene production by Alaska and ageotropum pea shoots. Apical shoots 3 cm long were treated with 10- 4 M ACC or 10- 4 M IAA for 3 h in the dark. Vertical bars indicate standard deviations of triplicate samples.

220

HIDEYUKI TAKAHASHI, HIROSHI SUGE, and MORDECAI J. JAFFE

Table 4: Hook angles of Alaska and ageotropum pea seedlings as affected by an external ethylene application. The 3-day-old seedlings were treated with 2 Jd- L-I ethylene for 2 days in the dark. Curvature data show the mean ± SE (n= 12). Variety Alaska Ageotropum

Treatment Control

Hook angle (degrees) 92 ± 3

C 2H 4

128±2 8±4 106±5

Control C2H 4

Table 5: Effects of light and ethylene on the induction of gravitropic curvature in ageotropum shoots. Intact shoots were treatad with 2 Jd- L -1 ethylene during the horizontal exposure or pretreated with 10 min white light prior to horizontal exposure. Experiments were done in the dark. Curvature data show the mean ± SE (n=5) 5 h after the stan of the treatment. Treatment Dark; Horizontal 10 min light; Horizontal Dark; Horizontal; C2~

Curvature (degrees) Bending direction 1±1 No bending 27±1 Upward -25±2 Downward

{MacDonald et al., 1983; Mirza, 1987; Zobel, 1974}. In this study, the pea mutant ageotropum also showed these characteristics: agravitropic behavior and an opened hook in the dark. This indicates a possible positive correlation between shoot gravitropism and hook formation. The plumular hook is already present at the time of germination in some plant species including peas {Fig. 2}, but it is formed after germination in others {MacDonald et aI., 1983; Takahashi and Suge, 1988}. Even in the former case, however, there are transition phases at which the hook tends to be opened just after germination and to be reformed thereafter in the dark {Fig. 2}. Reformation of the hook which takes place after germination may be easily influenced by gravity as seen in cucumber or sunflower seedlings {MacDonald et aI., 1983; Takahashi and Suge, 1988}. Gravistimulation is known to induce auxin asymmetry in some plant species including peas (Bandurski et aI., 1984; Dolk, 1936; Migliaccio and Galston, 1987; see also reviews by Pickard, 1985; Went and Thimann, 1937) and to increase ethylene production {Abeles and Rubinstein, 1964; Clifford et al., 1983; Harrison and Pickard, 1984; Wheeler et aI., 1986}. Both plant hormones are also important causal factors in formation and opening of the plumular hook {Goeschl et al., 1967; Kang et aI., 1967; Kang and Ray, 1969; Klein, 1965; Rubinstein, 1971}. Therefore, gravity may influence hook formation via either one or both physiological changes. Our current results support this hypothesis as follows. Firstly, in the dark-grown shoots of ageotropum peas the tissues were sensitive enough to IAA to induce curvature as shown previously {Scholdeen and Burstrom, 1960}. However, gravistimulation did not cause lateral IAA movement in the horizontal epicotyls of ageotropum pea despite the normal occurrence of basipetal IAA movement. Secondly, the darkgrown seedlings of ageotropum pea were still sensitive to ethylene and form the plumular hook, but evolved much less ethylene on their own than gravitropically responding epicotyls of Alaska pea. Thirdly, gravistimulation of shoots in-

duced an increase of ethylene evolution in Alaska pea but not in ageotropum pea. Fourthly, ethylene evolution increased significantly at the stage where Alaska pea seedlings started to reform the hook, but no such change was seen in ageotropum pea. It is interesting that gravistimulation caused neither auxin asymmetry nor an increase of ethylene evolution in ageotropum seedlings {Table 2 and 3; Fig. I}. In contrast, in Alaska seedlings, gravistimulation induced both lateral auxin redistribution and an increase of ethylene evolution, although the amount of increase in ethylene evolution was smaller than that reported by others (Abeles and Rubinstein, 1964; Clifford et al., 1983; Wheeler et aI., 1986). The smaller effect of gravistimulation on ethylene evolution may be attributed, in addition to the different methods used, to a shorter period of gravistimulation or vertical placement of the epicotyls for an hour's incubation after gravistimulation. It has been suggested that there is a lag of about 2 h for gravistimulated ethylene evolution (Clifford et al., 1983; Wheeler et aI., 1986). We did not measure the asymmetry of ethylene evolution in pea epicotyls. However, our results are in accordance with the reports that ethylene production caused by gravistimulation seems to be correlated with the increase in auxin concentration on the bottom side of horizontally placed stems (Abeles and Rubinstein, 1964; Clifford et aI., 1983; Wheeler et aI., 1986). A tomato mutant, diageotropica, is known to have both diagravitropic behavior and an opened hook. It is not sensitive to auxin, which induces ethylene production via activation of ACC synthase, but it is sensitive to ACC in the induction of ethylene production (Bradford and Yang, 1980; Zobel, 1973). On the other hand, the ageotropum pea responded to externally applied IAA or ACC accompanying comparable increase of ethylene production to that of Alaska pea (Fig.3). It is possible that the lack of lateral auxin movement resulted in the decreased ethylene production in ageotropum pea. Because auxin appears to move laterally in horizontally oriented epicotyls of Alaska pea but not apparently in those of ageotropum pea, we can assume that agravitropic growth of ageotropum shoots is involved with a factor that blocks auxin redistribution. However, recent studies with some plant species showed that a difference in the tissue sensitivity to auxin between the upper and the lower parts of horizontally oriented sterns may also be important for shoot gravitropism; the lower side becomes more sensitive to auxin than the upper side (Rorabaugh and Salisbury, 1989). We do not know whether the disruption of the differential tissue sensitivity to auxin is involved in the agravitropic behavior of ageotropum epicotyls. But the possibility cannot be ruled out because almost the same degree of curvature was obtained when IAA was applied asymmetrically to the upper or lower end of the horizontally oriented epicotyls, or to the one end of the vertically oriented epicotyls in ageotropum pea (Table 1). The curvature was always away from the asymmetrical IAA source regardless of the epicotyl orientation or the IAA-applied side. In Alaska pea, on the other hand, substantial upward bending still occurred when IAA was continuously applied to the upper end of the horizontally oriented epicotyls; bending toward the IAA source (Table I). The difference in the IAA response between two pea varie-

Agravitropic growth and hook formation in peas

ties may be explainable at least in part, if ageotropum epicotyls were incapable of changing the tissue sensitivity to auxin when oriented horizontally. Though ageotropum pea shoots produce much less ethylene than Alaska pea shoots, exogenous ethylene (2 J.tL L -I) did not induce negative gravitropism of horizontally oriented epicotyls of ageotropum pea (Table 5). Instead, the epicotyls bent downward as occurs in etiolated seedlings of normal pea varieties (Neljubow, 1901). Exogenous ethylene is known to induce upright growth of diagravitropic tomato stems (Zobel, 1973). It is also reported that shoot gravitropism inhibited by an inhibitor of ethylene biosynthesis is recovered following ethylene application (Wheeler et aI., 1986). In ageotropum pea shoots, however, a co-factor to play a role with ethylene, asymmetrical production of ethylene, or an optimal concentration of ethylene may be required for orthogravitropism. On the other hand, the overall increase of ethylene due to gravistimulation may be effective on the formation of the plumular hook by itself since exogenous ethylene induced hook formation to some extent in ageotropum pea (Table 4). Thus, the present study indicates a possible correlation between auxin asymmetry and ethylene production which may be regulated by gravity and may function for both shoot gravitropism and hook formation. The question still remains as to whether either or both factors are essential for gravitropism or hook formation. Since the shoots of ageotropum pea become orthogravitropic under light (Table 5), we are currently studying the light effect on shoot gravitropism and hook formation as well as on auxin asymmetry and ethylene production in ageotropum shoots for further understanding of the above question.

Acknowledgements We thank Dr. H. Morisaki, Tohoku University, for his kind advice on the radioisotope experiments. This work was supported by Grants-in-Aid from the Ministry of Education, Science and Culture of Japan and grants from the Institute of Space and Astronautical Science (Sagamihara, Japan) to H. S. and H. T., and grants from the National Science Foundation (PCM 8206560) and the National Aeronautics and Space Administration (NAGW-96) to M. J. J.

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