Planta (Berl.) 94, 333--354 (1970) 9 by Springer-Verlag 1970

Some Aspects of Geotropism in Coleoptiles BARBARA FILNER a n d R A n ~ HERTEL with participation by CHAI~T~]~SSTE]~LE a n d VICKu FAN MSU/AEC Plant Research Laboratory, Michigan State University, East Lansing Received July 27, 1970

Summary. Auxin transport was studied in coleoptfle sections that were stimulated geotropically. The early time course of auxin-transport asymmetry was measured. An initial phase in which more IAA was delivered into the receptor for the upper half was found after 5 rain of horizontal exposure. After about 15 rain this was followed by the expected known asymmetry in which more auxin flows in the lower side of the coleoptfle. Upon return of the coleoptfle to a vertical position, this asymmetry disappeared within 30 min. Earlier correlations of geosensitivity of the auxin transport system with sedimentation of amyloplasts in comparisons of wild type corn and an amylomaize mutant were confirmed and extended. It was also shown that, in contrast to the geotropic effect, phototropieally induced lateral auxin asymmetry was not significantly different in wild type and amylomaize. Eleven other single-gene endosperm starch mutants of corn were compared to their corresponding normals. I n atl pairs, if a difference in geosensitivity of lateral auxin transport was present, it was correlated with a parallel difference in amyloplast sedimentation: e.g., sugary 1 ("67") had an amyloplast asymmetry index of 0.32 and a 13% gravity effect on auxin transport; the paired wild-type had both a greater amyloplast asymmetry (0.61) and a greater gravity effect on transport (23 % ). Correlations between gravity effects on auxin transport and amyloplasts were also shown in comparisons of apical and basal sections of corn, oat and Sorghum coleoptiles. Further results, confirming the increased effect of centrifugal acceleration grea~er than 1 • g on lateral auxin transport and on curvature, are in agreement with the hypothesis that the pressure exerted by amyloplasts, acting as statoliths, locally stimulates the auxin transport system in the individual cells.

Introduction

W h e n a vertically grown coleoptfle is placed i n a horizontal position the following observations can be m a d e w i t h i n half a n hour. I n m a n y cells there is a m a r k e d displacement of s t a r c h - c o n t a i n i n g a m y l o p l a s t s towards the lower cell surface (N~mec, 1901). There is a n e n h a n c e d lateral a u x i n t r a n s p o r t across the coleoptile towards the physically lower side (Dolk, 1936; Gillespie a n d T h i m a n n , 1963; Goldsmith a n d Wilkins, 1964). Greater cell elongation on the lower side of the coleoptile results i n t h e u p w a r d bending. I t has also been observed t h a t the lower flank of 22 a

Planta (Berl.), Bd. 94

334

B. Filner, R. Hertel, C. Steele and V. Fan:

the horizontally positioned coleoptile becomes electrically more positive (Brauner, 1926). According to the Cholodny-Went theory (see Went and Thimann, 1937) the downward lateral auxin transport is a necessary and sufficient condition for geotropic curvature (see Wflkins, 1966). The geoelectric effect is also considered to be a consequence of the asymmetric auxin flow (Grahm, 1964; Wilkins and Woodcock, 1965). I t was further postulated that the geotropically enhanced downward transport of auxin resulted from a gravity-induced local redistribution of secretion activity in the single cells, and subsequent cell-to-cell auxin movement across the coleoptile (Hertel and Leopold, 1963). Under this working hypothesis the question arises how gravity locally affects the auxin transport system in a single cell. Two different mechanisms have been discussed in relation to this question. One involves the whole cell in the sensing of gravity, via either the weight of the protoplast or the hydrostatic pressure difference across the cell. The other, a statolith mechanism, involves a displacement of and/or pressure from particles or organelles within the cell whose density is not the same as that of the cytoplasm. (For discussions, see Audus, 1962; Brauner, 1962; Piekard and Thimann, 1966; Wilkins, 1966; Ball, 1969). The observation of extensive amyloplast sedimentation prompted the formulation of the amyloplast-statolith hypothesis (Haberlandt, 1900; N~mec, 1900) which ascribes a role in the geosensing mechanism to the dense, starch filled amyloplasts. The list of correlations between movable amyloplasts and geotropic reactivity is impressive, but not without contradictions (e.g., Hawker, 1932; Pickard and Thimann, 1966; Hertel et al., 1969; review, see Brauner, 1962). In the study presented here, the early time course of lateral auxin transport in geotropism was assayed and compared in eoleoptiles which had different levels of amyloplast sedimentation when geotropieally stimulated. The results of these experiments are in agreement with the Cholodny-Went theory, and strongly support the amyloplast-statolith hypothesis. Materials and Methods a) Radioactive Auxins. 3-Indoleacetic aeid-3I-I (IAA; general label, specific activity 1 curie mmole-1) was purchased from International Chemical and Nuclear Corp., Irvine, California. 1-Naphthalene acetic aeid-l-14C (NAA; specific activity 50.6 mcuries mmole-1) was purchased from Nuclear Chicago Corp., Arlington Heights, Illinois. b) Coleoptiles. Corn seeds, Zea mays L., were obtained from two sources. Hybrid WF 9 • Bear 38, "normal" as well as "amylomaize" and "waxy", were purchased from Bear Hybrid Corn Company, Decatur, Illinois. Other corn mutants and the corresponding normals were generously supplied by Drs. E. B. Patterson and R. J. Lambert from the Maize Genetics Group, Department of Botany, University of Illinois, Urbana. Oats, Arena sativa L., cv. Victory, were obtained from H. W. Tomlin, Encino, California. Sorghum vulgare Pers. and whea~, Tritioum

Geotropism in Coleoptiles

335

aestivum L., cv. Henry, were kindly supplied by the Crop and Soil Science Department of Michigan State University. Generally, the seedlings were grown and the coleoptiles harvested as described by Hcrtel and Flory (1968). With several mutants fungal growth developed when the seedlings were grown on paper towels; the seeds of these mutants and of the corresponding normals were grown in about 3-era-deep, moist vermiculite. c) Amyloplast Sedimentation. The lateral distribution of amyloplasts in the coleoptile cells was estimated microscopically according to Hertel et al. (1969) and the amyloplast asymmetry index was calculated as follows: (amyloplasts in the lower cell halves minus amyloplasts in the upper cell halves) divided by (the total number of amyloplasts counted). d) Longitudinal Amyloplast Asymmetry and the E]/ect of Inversion were tested according to Ouitrakul and Hertel (1969). e) Centri]ugal Acceleration was applied as described by Ouitrakul and Hertel (1969). ]) Starch Determination was performed according to Adams (1969). The starch was first separated from soluble sugars and other interfering compounds, and then its glucose content was determined. g) Lateral Auxin Transport was tested either directly by transport into lateral receptors from 5-ram-long, longitudinally split coleoptile sections (Hertel et al., 1969, Method II), or indirectly with 3-ram-long coleoptile cylinders by a modification of the split-receptor technique used by Dolk (1936). This latter method was as follows: Coleoptiles were excised from 4-day-old seedlings and a 3 ram cylinder was cut out 2-5 mm below the apex. A 3 • 3 • 1 m m donor agar block, containing 2.5 • 10-6M 3H-IAA, was put over the apical end, and then the coleoptile section was set vertically on a plain agar block. After 20 rain, the colcoptile section, still retaining the donor, was positioned over the receptor blocks on a glass microscope slide. These were two 5 • 5 • 1 mm plain-agar blocks separated by a 1-~1 disposable capillary micropipette. The vertical coleoptile was positioned so that the two vascular bundles were on a line perpendicular to the capillary. The entire assembly was then rotated 90 ~ so that the long axis of the coleoptile was horizontal. The glass slide was supported in this position by plasticine, and the assembly was placed inside a humid plastic box. The upper receptor block was designated " u p " and the lower block "down". At the stated intervals the assembly was removed from the plasticine supports and returned to the vertical position. The coleoptile section and donor block were transferred to the next pair of receptor blocks, taking care to maintain the original " u p " - " d o w n " orientation. The assembly was placed in the horizontal position and returned to the humid box for the duration of the next interval. The transfer took about 30 sec. The receptor blocks ( " u p " and " d o w n " ) were put in separate vials containing 5 ml each of Brays solution, and radioactivity was determined in a Beckman CPM 100 scintillation counter. The gravity effect, expressed as a percentage, was calculated as (cpm down minus epm up) • (100) divided by (cpm down plus cpm up). This is not a direct assay of lateral transport, as is Method II, but is rather an assay of the effect of gravity (presumably via lateral transport) on the amount of auxin exported basipetally by upper and lower portions of a horizontal coleoptile. Assay of control coleoptile sections which were kept vertical throughout indicated no asymmetries: the average of 4 determinations was 0.5 % for W F 9 • Bear 38 normal corn. Sample data follow for the amylomaize: normal pair 65-610-2/608-2 in the interval 35-50 rain after the start of horizontal exposure. 22b

Planta (Berl.), Bd. 94~

B. Filner, R. Hertel, C. Steele and V. Fan:

336

Normal

Amylomaize

epm down

cpm up

Gravity effect (%)

cpm down

cpm up

Gravity effect (%)

748 799 725 455 599 540

595 373 414 328 313 272

11.4 36.3 27.3 16.2 31.4 33.2

424 388 391 624 433 643

324 308 337 554 350 485

13.4 11.5 7.4 5.9 10.6 14.0

26.0•

10.5 • 1.4

The transport tests were usually carried out in a dark room with a green safe light. Some replicate experiments with the mutants (Results, section 3) were done in daylight, with very similar results. However, all comparisons are based on identical and simultaneous treatment during both growth and assay. h) Statistics. Values are presented as the mean =~standard error of the mean. Student's t test was used to determine whether the means of two samples were significantly different from one another, and whether a mean value was significantly less than zero. The level of significance denotes the probability (0.010 = 1% ) that the difference is not meaningful. Results

1. Time Course o/Gravity E/leer on Lateral Auxin Transport. I f t h e C h o l o d n y - W e n t t h e o r y is correct, a u x i n a s y m m e t r y should precede geot r o p i c bending. Thus, a s t u d y of t h e e a r l y t i m e course of g r a v i t y e n h a n c e d l a t e r a l a u x i n m o v e m e n t seemed w a r r a n t e d . I t h a d been shown t h a t 5 m i n of h o r i z o n t a l e x p o s u r e l e a d s t o a significant l a t e r a l a u x i n a s y m m e t r y in coleoptiles of o a t s (Dolk, 1936) a n d corn (Hager, 1967) This d e m o n s t r a t e d t h a t t h e l a t e r a l d i s p l a c e m e n t of a u x i n is h i g h l y sensitive t o g r a v i t y . I t d i d not, however, allow conclusions on t h e t e m p o r a l sequence of l a t e r a l a u x i n t r a n s p o r t a n d c u r v a t u r e since t h e t r a n s p o r t was a s s a y e d for a t l e a s t one h o u r a f t e r t h e stimulus. I n t h e e x p e r i m e n t shown in Fig. 1, l a t e r a l a u x i n - t r a n s p o r t a s y m m e t r y was a s s a y e d b y t h e m o d i f i e d D o l k m e t h o d for 10-rain i n t e r v a l s d u r i n g t h e i n i t i a l 60 rain of h o r i z o n t a l exposure. E n h a n c e d d o w n w a r d a u x i n t r a n s p o r t can first be d e t e c t e d b e t w e e n 10 a n d 20 rain a f t e r r e o r i e n t a t i o n . This coincides well w i t h t h e s t a r t of u p w a r d c u r v a t u r e (15 rain) as r e p o r t e d ~or o a t s b y B r a u n e r a n d Z i p p e r e r (1961) a n d as c o n f i r m e d b y us for t h i s s t r a i n of corn. I t also coincides w i t h t h e a p p e a r a n c e of t h e geoeleetric effect ( G r a h m a n d H e r t z , 1962; W o o d c o c k a n d Wilkins, 1969). F o r comparison, a m y l o p l a s t d i s p l a c e m e n t was m e a s u r e d in sections f i x e d

Geotropism in Coleoptiles

E E

337

15

2O

I0

10

5 4 3

5

0

0

0

-I0

3'o 4'o

0 102050405060 Minutes Horizontal

Fig. 1

Minutes Horizontal

Fig. 2

Fig. 1. The time course of amyloplast sedimentation and lateral IAA transport asymmetry during the first 60 minutes of horizontal exposure. Assays were performed on apical 2-5 mm corn eoleoptile sections. Transport was assayed by the Dolk method (see Methods). Each point is the average of 15-20 determinations • standard error of the mean Fig. 2. The time course of lateral IAA transport asymmetry in sections of the apical 2-5 mm of corn eoleoptiles, assayed at 5-rain intervals by the Dolk method, during the initial 40 rain of horizontal exposure. Each point is the average of 10-20 determinations 5=standard error of the mean

after similar horizontal exposures. S e d i m e n t a t i o n is m e a s u r a b l e within 1-2 rain, a n d is n e a r m a x i m a l w i t h i n 30 rain. W h e n the lateral t r a n s p o r t a s y m m e t r y was assayed a t 5 - m i n intervals (Fig. 2) a n initial u p w a r d phase became a p p a r e n t . U n d e r these conditions significantly more I A A was detected i n the u p p e r t h a n i n the lower receptor for 15 m i n (levels o~ significance: 5 m i n - ~ 0 . 0 2 5 ; 10 min---0.10; 15 m l n ~ 0.10). This was unexpected, b u t i n retrospect was seen to possibly correspond to the i n i t i a l d o w n w a r d c u r v a t u r e observed b y B r a u n e r a n d Zipperer (1961) i n oat coleoptiles. There may be conditions under which the transient initial "wrong" phase of geotropic response does not occur. This is suggested by the fact that with NAA downward auxin asymmetry can be detected within 10 rain (OuRrakul and Her~el,

338

B. Filner, R. I-Iertel, C. Steele and V. Fan:

25 15 20 '~%

o

lO

~

.'4.--

>,

I0

P ' ~

i \

o " 9,,(.9

5 --5

o

J

-I0

o

i%2'o 3'o4o

o6'o

Minutes Vertical

Fig. 3

o

s'o4'o 5'o

Minutes Horizontal

Fig. 4

Fig. 3. The time course of the decay of lateral IAA transport asymmetry. After a 20-rain horizontal exposure, the 3-ram apical corn coleoptile sections were returned to a vertical position (at time 0), and transport was assayed for 10-rain intervals by the Dolk method. Values are the average of 20 determinations (except the last 2 points which are the average of 6 determinations)4-standard error of the mean Fig. 4. Lateral IAA transport asymmetry of amylomaize (circles) and normal (crosses) W F 9 • Bear 38 corn, assayed by the Dolk method, during the first 50 minutes of horizontal exposure

1969) and by the fact that transient downward curvature does not occur when oat eoleoptiles are geotropieally stimulated trader water (Brauner and Zipperer, 1961). To s t u d y t h e d e c a y of t h e l a t e r a l I A A - t r a n s p o r t a s y m m e t r y , coleoptile sections were r e t u r n e d to t h e v e r t i c a l p o s i t i o n a f t e r 20 mln of h o r i z o n t a l exposure, a n d l a t e r a l t r a n s p o r t was t h e n a s s a y e d for 10-rain i n t e r v a l s . T h e t i m e course of decay, as shown in Fig. 3, was quite similar t o t h e t i m e course of a p p e a r a n c e of t h e t r a n s p o r t a s y m m e t r y (Fig. 1). I n this a c t i v e l y t r a n s p o r t i n g s y s t e m t h e a s y m m e t r y was lost in 20-30 min. Here, too, a t r a n s i e n t p h a s e of e n h a n c e d " u p w a r d " t r a n s p o r t 30-40 rain a f t e r t h e r e t u r n t o t h e v e r t i c a l p o s i t i o n (levels of significance: 30 m i n 0.05; 40 r a i n = 0 . 0 0 5 ) is c o r r e l a t e d w i t h a " d o w n w a r d " b e n d i n g of t h e coleoptile ( P i c k a r d et aL, 1969).

Geotropism in Coleoptiles

339

2. Lateral A u x i n Transport in Amylomaize and Normal Corn. The geotropic response is related to the amount (duration and intensity) of stimulation, e.g., to the amount of centrifugal acceleration over a wide range (see Johnsson, 1965; also below). If geostimulation were effeeted via amyloplasts one would expect a correlation of amyloplast sedimentation and of lateral auxin asymmetry. An amylomaize mutant of corn has already been shown to contain smaller starch plastids which sediment less than those of wild type, and to exhibit less gravity effect on lateral auxin transport as well as less gcotropic curvature (Hertel et ~al., 1969). We sought to extend these observations, and to study further starch endosperm mutants of corn. The time course of appearance of lateral auxin asymmetry was assayed by the modified method of Dolk in eoleoptile sections of normal and amylomaize corn W F 9 • Bear 38 hybrid (used by Hertel et al., 1969). A clear difference between the two types is confirmed by the results shown iu Fig. 4. This simple test was also performed in daylight, with similar results, and as a classroom experiment in which students found a gravity effect of 10 • 2% for wild-type and 4 • 2% for amylomaize during a 50 min horizontal exposure. With W F 9 • Bear 38 hybrid a further suggestive correlation was seen when the sample of amylomaize used in the above experiments (1966 harvest) was compared with another sample (1967 harvest). In coleoptile sections the amyloplast asymmetry index was found to be 0.55 for wild type, 0.20 for "1966", and 0.10 for " 1 9 6 7 " ; the gravity effect on auxin transport was 25 %, 10 % and 2.5 %, respectively. Although in amylomaize eoleoptiles the lateral (Hertel et al., 1969) and the axial (Ouitrakul and Hertel, 1969) transport of auxin are less sensitive to gravity, the basipetal auxin-transport system itself and the "unstimulated" lateral transport in vertical coleoptiles are not significantly different from that in wild-type corn (Hertel et al., 1969). As an additional control that the observed differences between amylomaize and normal are in the geotropie sensing mechanism, photo-induced lateral auxin asymmetry was determined, in a manner similar to that of Piekard and Thimann (1964). Blue light (filtered through three layers of Cinemoid Blue; about 500 ergs em -~ sec-~) was unilaterally applied to 5-ram-long upright eoleoptfle sections between donors and split receptors. The test material had received a saturating dose of red light (Pickard and Thimann, 1964) 2 hrs before the experiment. Geotropically stimulated lateral transport was assayed with a 45 ~ angle of exposure in order to obtain asymmetries comparable to those in phototropism. The results of these tests are presented in Table 1. The auxin asymmetry is very similar in both amylomaize and normal corn when unilaterally illuminated. The differences, if any, in the

340

B. Filner, R. Hertel, C. Steele and V. Fan:

Table 1. Lateral auxin transport asymmetry in phototropically and geotropically stimu-

lated amylomaize and normal corn Sections were cut from the 4th to the 9th mm below the tip. For A, unilateral illumination during hours 1 and 2 was followed by a 1-hr dark period. For B, a 2-hr period ar 45 ~ was followed by 1 hour in the vertical position. Transport was assayed by the Dolk method, and the percentages represent asymmetries in favor of the shaded and lower sides, respectively. Values are the average of 30 determinations :]: standard error of the mean. Hour 1

Hour 2

Hour 3

5.9• 4.6 q- 1.2%

6.3-4-1.2% 6.5 =~ 1.0%

7.0=k0.9% 4.4 • 1.0%

10.7q- 1.6% 4.6 -4-0.9%

13.9q- 1.1% 6.4 =k 1.1%

4.0J= 1.2% 1.7 -4-1.2 %

A. BHect of blue light Normal Amylomaize

B. E]/ect o/gravity Normal Amylomaize

p h o t o s t i m u l a t e d l a t e r a l t r a n s p o r t are m u c h smaller t h a n t h e o b s e r v e d differences in t h e g e o t r o p i c s y s t e m . I t can also be seen f r o m T a b l e 1 ( H o u r 3) t h a t t h e d e c a y in t h e absence of t h e s t i m u l u s is f a s t e r for t h e g e e - i n d u c e d l a t e r a l t r a n s p o r t , in a g r e e m e n t w i t h c u r v a t u r e meas u r e m e n t s ( P i c k a r d et al., 1969). There was an indication in these and other experiments of a slight difference in the phototropically induced lateral transport asymmetry. Interactions between the two tropistic systems are known (Rawitscher, 1932; Dennison, 1964) so that this small difference does not necessarily indicate that the mutation has had a direct effect on the phototropie transport system. A difference in a m y l o p l a s t size a n d s e d i m e n t a t i o n was f o u n d as r e p o r t e d b y H e r t e l et al. (1969). T h e e x p e c t a t i o n f r o m microscopic o b s e r v a t i o n s was c o n f i r m e d b y chemical d e t e r m i n a t i o n of s t a r c h c o n t e n t as ~g glucose e q u i v a l e n t s / g fresh w e i g h t : 0.7 in a m y l o m a i z e a n d 2.4 in wild t y p e . A l l e x p e r i m e n t s d e s c r i b e d a b o v e c o m p a r i n g a m y l o m a i z e a n d wild t y p e were p e r f o r m e d in t h e W F 9 • B e a r 38 strain. To check t h a t t h e o b s e r v e d differences were d u e t o t h e ae m u t a t i o n a n d n o t to t h e genetic backg r o u n d , we t e s t e d a n isogenie a m y l o m a i z e : n o r m a l p a i r in ~ different s t r a i n (Table 2). T h e results w i t h this s t r a i n (65-610-2/608-2) clearly show t h e s a m e correlation as was f o u n d in W F 9 • B e a r 38 (the level of significance of this s t r a i n ' s t r a n s p o r t difference a t 35-50 m i n ----0.005).

3. Lateral A u x i n Transport and Amyloplast Asymmetry in Further Endosperm Mutants of Corn. To a s c e r t a i n t h e g e n e r a l i t y of t h e correlat i o n of a l t e r e d a m y l o p l a s t s e d i m e n t a t i o n a n d of a l t e r e d g r a v i t y effects

Geo~ropism in Coleoptiles

341

Table 2. Amyloplast sedimentation and lateral I A A transloort in amylomaize and normal o] corn strain 6g-610-2/608-2

Dotk assay; test in daylight; values are the average of 6 determinations. Amyloplast % Gravity effect on auxin asymmetry asymmetry 0-20 rain 20-35 rain 35-50 min index Amylomaize Normal

0.25 0.51

2.5 6.1

5.0 9.8

10.3 25.8

on auxin flow, we surveyed other endosperm mutants of corn (see Creech, 1965; Jones, 1967). A number of them (floury 1 and 2, shrunken 1, sugary 2, waxy) did not show a gravity-enhanced lateral transport that was significantly different from the corresponding wild type (Fig. 5). In these eases, no large differences in amyloplast sedimentation were found (Table 3). Chemical determination of starch content, as far as carried out, confirmed the lack of large differences (e.g., ~g glucose equivalents/g fresh weight: waxy eoleoptlles had 2.7, the corresponding normal 2.4; shrunken 1 had 2.4, the isogenie normal 2.1). Etched, opaque 2 and shrunken 2 show a somewhat smaller lateral auxin asymmetry than the paired wild types (Fig. 5); differences in amyloplast sedimentation, though small, point in the same direction (Table 3). Amyloplast sedimentation in brittle was much slower than in the normal counterpart (Table 3 ) ; the geosensitivity of the auxin flow was also significantly less in the mutant (Fig. 5). However, brittle eoleoptfles showed very poor growth and basipetal auxin transport compared to the wild type. I n addition to the isogenic floury 1: normal pair (strain 65-532-2/ 531-7 ; Table 3, Fig. 5), another floury 1 (63-2360 F) was compared with a non-isogenic wild-type (65-M14/531-4). The two strains had a very similar growth rate and basipetal IAA transport. A large difference was seen in lateral transport, floury showing 35 % gravity effect between 20 and 50 rain compared to 14% in the normal (level of significance of the difference=0.025). Amyloplast sedimentation was strikingly correlated (0.6 in floury 1, 0.4 in the non-isogenie normal). The differences observed are probably due to a genetic factor other than floury 1. Sugary 1 and isogenic normals were compared in three different strains. In " 6 7 " (67-923-1/920-1) a difference in amyloplast sedimentation (Table 3), size, and starch content of the coleoptfle (~zg glucose

]3. Fflner, R. Hertel, C. Steele and V. Fan:

342

201

FIouryl

Floury 2

ShrunkenI

Sugary 2

jo 9~ o

50

50

I' Etched 20~

Opaque 2

z ,

~

z o

50

50

Shrunken 2

Waxy

50

Brittle

~x

'~ /~o I0

50

~

-~

2/~

50

50 50 50 Minutes Horizontal :Fig. 5. Lateral IAA transport asymmetry of several endosperm starch mutants (circles) and paired normal (crosses) strains of corn. Transport was assayed in 2-5 mm apical sections by the Dolk method, during the first 50 minutes of horizontal exposure. Values are the average of 8-15 determinations (except brittle which is the average of 4 determinations)

65

25

Sugary I

67

I0

o

5

o .

,o

25 20

,

o

,

,

zb 3b 40 5'o ou,, ,o 20 3o 40 5'0

Young r

~'

Old

~ 5 m Z- 0

,b2b3b&~o

,020304050

MinutesHorizontal Fig. 6. Lateral I A A transport asymmetry of mutant (circles)and normal (crosses) strains of corn. Transport was assayed in 2-5 m m apical sections By the Dolk method, during the :[irst50 minutes of horizontal exposure. Values are the average of 10 determinations (except sugary l" 65" which isthe average o:[2 determinations)

Geotropism in Coleoptiles

343

Table 3. Amyloplast sedimentation in several corn endosperm mutants and the corresponding normals; level o/significance of the lateral transport difference, if any, between mutant and normal (for the time interval 30 or 40-50 min after horizontal exposure) Unless specified, the strain number for normal is the same as that for the corresponding mutant. Strain number

Amyloplast Level of significance, asymmetry lateral transport index difference

Floury I Normal

65-532-2/531-7

0.42 0.38

---

Floury 2 Normal

67-270-1/266-1

0.32 0.39

--

Shrunken 1 Normal

65-716-2/713-1 67-W23/266-4

0.31 0.37

--

Sugary 2 Normal

63-2185-3/2187-5

0.43 0.34

--

Waxy Normal

WF 9 • Bear 38

0.49 0.55

--

Etched Normal

63-2654-6/2651-1

0.39 0.43

0.005

Opaque 2 Normal

65-685-5/683-6

0.36 0.39

0.100

Shrunken 2 Normal

63-2166-5/2168-7

0.27 0.33

0.100

Brittle Normal

65-611-2/614-1

0.23 0.50

0.025

Sugary 1 Normal

6%923-1/920-1

0.32 0.61

0.010

Dull 1 Normal

65-723-7/725-1 : young

0.29 0.36

0.025

Dull 1 Normal

65-723-7/725: old

0.51 0.35

0.050

e q u i v a l e n t s / g fresh weight: 1.6 for sugary 1, 2.2 for normal) was correlated to a clear difference i n g r a v i t y effect on lateral t r a n s p o r t (Fig. 6, " 6 7 "). I n the sugary-1 s t r a i n " 6 5 " (65-567-8/574-1) only two m u t a n t eoleoptiles could be g e r m i n a t e d a n d grown, b u t the following observat i o n was made. The m u t a n t coleoptile c o n t a i n e d larger amyloplasts ( 7 . 4 : ~ 0 . 3 ~ diameter) t h a n the n o r m a l ( 3 . 0 ~ 0 . 1 5 ~ ) . However, the m u t a n t plastids did n o t c o n t a i n m u c h iodine-stainable starch a n d did

344

B. Fflner,

R.

Hertel, C. Steele and V. Fan:

not show any significant settling under the influence of gravity. The smaller wild-type plastids had an asymmetry index of 0.5. The lateral transport level in the mutant tissue was very low (Fig. 6, " 6 5 " ; level of significance for the difference---~ 0.05 for 40-50 rain). Two sections were tested from the only coleopti]e germinated from a third sugary-1 strain (supplied by J.G. Scandalios of this laboratory). This small sample gave the only observation contradicting the correlation between amyloplasts and transport encountered in our experiments: in this case the mutant again had poor geo-enhanced lateral transport, but amyloplast sedimentation was somewhat better than that of the normal. Dull 1 and normal coleoptfles were also compared. In young coleoptfles (about 25 mm total length) the mutant had poorer geoenhanced lateral transport than the wild type (Fig. 6, " y o u n g " ) and amyloplast sedimentation was less in the mutant (Table 3). In older coleoptfles (about 5 cm long) sections from the apical 2-5 mm showed the inverse situation: the mutant had the larger amyloplast asymmetry index (Table 3) and starch content (1.9 ~g/g fresh weight vs. 0.9 for normal). In good correlation with these changed properties of the amyloplasts, the older dull 1 coleoptfles had a sigl~ficantly better lateral transport than the normal (Fig. 6, " o l d " ) . 4. Lateral A u x i n Transport and Amyloplast Sedimentation in Di//erent Zones o/ Coleoptiles. Von Guttenberg (1912) and Dolk (1936) showed that geotropic sensitivity decreased with distance from the coleoptfle tip. The correlation with movable amyloplasts was pointed out b y yon Guttenberg (1912). We found that in Avena the amyloplasta y m m e t r y index decreased in parallel with the increase in presentation time reported by Dolk (1936). We also tested, in the corn coleoptile, lateral auxin-transport asymmetry from an apical section (2-5 mm or 2-7 mm) and from a basal one (just above the mesocotyl, more than 25 mm below the tip). As seen in Fig. 7 and Table 4, the tip region displayed a much stronger georesponsc in the lateral auxin transport than the base (see Hertel and Leopold, 1963, for axial transport). This again correlated with the movable amyloplasts.

Table 4. Lateral transport in corn under gravitational and centri]ugal acceleration Method II assay (2.5 • 10-6~ aH-IAA, 30 rain). Values are the average of 8-15 determinations =Lstandard error of the mean.

Tip Base

1•

10•

20:]:4% 0•

65~:4% 28~10%

Geo~ropism in Coleoptiles

345

25

20

15

l.d

tip

I0

5

se

0

0

0 1020304050 Minul"es Horizontol

Fig. 7. Lateral IAA transporb asymmetry in apical (2-5 mm; circles) and basal (more than 25 mm below the tip; crosses) sections of corn eoleoptiles, assayed by the Dolk method during the first 50 minutes of horizontal exposure. Values are the average of 8 determinations

Table 5. Photo.induced lateral transport asymmetry in corn Dolk Assay. Hour 1 3500 ergs/cm2 sec of unilateral blue light; Hour 2 dark. Values are the average of 15 determinations ! standard error of the mean.

Tip Base

Hour 1

Hour 2

3.4~=0.7% 0.3 ~: 1.2%

5.9-4-0.9% 1.3~- 1.1%

T h e l a t e r a l t r a n s p o r t s y s t e m in t h e b a s a l region was able to develop a significant a s y m m e t r y if s t i m u l a t e d m o r e intensely, e.g. b y 10 • g (Table 4). I t was f u r t h e r assured t h a t t h e n o r m a l b a s i p e t a l m o v e m e n t of I A A was n o t significantly different in t h e sections from t h e t i p region a n d t h e b a s a l region. H o w e v e r , as seen in T a b l e 5, t h e bases were less p h o t o t r o p i e a l l y r e a c t i v e t h a n t h e a p i c a l sections. 5. Georesponses in Di//erent Sections o/Sorghum and Avena. A comparison was m a d e b e t w e e n apical a n d m o r e b a s a l sections of b o t h Arena a n d Sorghum coleoptfles (total l e n g t h was a b o u t 25 r a m ; 5 m m sections were used). Because of t h e small d i a m e t e r of these coleoptfles i t was t e c h n i c a l l y difficult to use t h e u s u a l l a t e r a l - t r a n s p o r t assay. W e therefore

346

B. Filner, R. Herte], C. Steele and V. Fan:

studied the inhibition b y inversion of basipetal N A A transport (Ouitrakul and ttertel, 1969) as a possible assay for amyloplast-auxin transport interaction. Avena had a fairly steep gradient of decreasing amyloplast mobility, and h a d no significant inhibition on inversion in the more basal region. Sorghum, in contrast, h a d considerable amyloplast mobility in the lower region, and was inhibited b y inversion in this region (Table 6). This correlation between amyloplast sedimentation and geosensitivity in different zones of the two grass species agrees fully with findings and conclusions of y o n Guttenberg (1912) (who, however, has been quoted incorrectly on this point b y m a n y reviewers, e.g., Rawitscher, 1932).

Table 6. Inhibition of basipetal N A A transport by inversion Values are the average of 10 determinations 4- standard error of the mean. Amyloplast asymmetry index

Inhibition ( % ) (1 hr)

Sorghum: 2-7 mm 8-13 ram

0.75 0.60

8.9 4-1.5% 6.6 4- 3.9%

A vena : 2.7 mm 8-13 mm

0.25 0.07

11.1 4-3.0% 1.2 4- 3.0%

Table 7. Lateral transport in wheat-gravitational and centri]ugal acceleration Method I I assay (2.5 • 10-6 M ~H-IAA, 60 min). Values are the average of 5 determinations 4- standard error of the mean. Acceleration

Lateral transport

1•

15=]=5%

5• 10 •

374-6% 494-2%

6. E]]ects o/ Gravitational and Centri/ugal Acceleration on Corn and Wheat Coleoptiles. U n d e r the assumption t h a t amyloplasts have a role in the geotropic reaction chain, experiments with accelerations different t h a n 1 • g m a y yield some indirect information on the mode of action of the presumed statoliths. Centrifugal acceleration of 10 • g will enhance

Geotropism in Coleoptiles SET

A

SET B

2 . 5 rn~n :

I0 x g

i m,n: '~ --~

~xQ

2 . 5 rain I 5~ mln

I0 x g

~ ~- I x g

7 . 5 rain :

Repeat 5 times

n

\ "

c.)

I0 x g

mi.:

.,,,~

2.5 rnin : I 5II min :

.

2.~i~

347

.

~m,n

.

.

.

I0 x g .-5 x g

~

.

.

.

:

.

.

.

.

.

,0,0

\ ( 3 0 rain)

( 3 0 m~n)

2

2_

_ 6.2_+1.3o

§ 5.~' -+ I.I ~

Fig. 8. Curvature in corn co]eoptiles subjected to a regime of centri~ugation as indicated. For a fuller discussion see the text: Results, section 6

lateral transport in both coleoptile sections that, under the influence of 1 • g, have strong asymmetric lateral transport and in those that have only little (Hertel et al., 1969). We investigated this phenomenon further. Table 7 presents the effect of centrifugation at 5 • g and 10 • g on the lateral transport in wheat, assayed by the split coleoptile technique. There is a clear increase with the 5 • g force, and an even larger increase with the 10 • g force. Thus, even though the 5 • g force is sufficient to carry the amyloplasts to the side of the cell, the sensitive cell apparatus is not maximally stimulated. The force with which the amyloplasts are pressed down, rather than their mere displacement, m a y again be implicated in the experiment presented in Fig. 8. I n this case excised, but intact corn coleoptfles were subjected to a regime of centrifngation which was designed to distinguish between the effect of the presence of the amyloplasts and their pressure on the cell surface. The sequence was in one case (Set B) to first displace the amyloplasts with a 10 • g force and to follow this with a "pressure" of 1 • g. The amyloplasts were then fully displaced to the opposite side of the cell with another 10 • g force, and again pressed to the side, but with a force of 5 • g. This was repeated 5 times, then the amyloplasts were drawn to the bottom of the cell with a centrifugation at 10 • g, and this was followed with a 30-min development period. The curvature was in the direction expected if the 5 • g force was dominant. This was also the case when the sequence of treatments was changed as shown

348

]3. Filner, R. Hertel, C. Steele and V. Fan:

in Set A. This indicated that the 5 • g force is dominant over the 1 • g force because it is the greater pressure, rather than because it is first or last in the sequence. The above data were presented under the explicit assumption of the validity of the stareh-statolith hypothesis. Stated in a less biased context, the results indicate that the direction in which the product of acceleration and time is greatest determines the outcome of the response.

Discussion The auxin-transport system responds very quickly when the coleoptile section is transferred to the horizontal position. Within 5 rain a significant asymmetry of auxin flow could be observed; surprisingly, more auxin moved on the upper side. After about 15 rain this transient upward transport reverses to the steady downward auxin movement already known (e.g. Do]k, 1936). There is a striking similarity of this time course of transport and that of curvature, which is initially downward and after about 15 rain changes to a steady upward bending (Brauner and Zipperer, 1961). A similar time course has been recorded in several studies of the geoeleetric effect (Grahm and Hertz, 1962; Woodcock and Wi]kins, 1969). The initial, transient transport in the " w r o n g " upward direction may be related to the initial transient inhibition observed after a large step-up in auxin concentration (Rayle et al., 1970). The reported correlation in detail between auxin flow and curvature provides further confirmation for the Cholodny-Went theory. In this context the close coincidence of the appearance of an auxin asymmetry with the onset of asymmetric growth strongly and independently supports the idea that IAA can act on cell elongation without any significant lag period (Nissl and Zenk, 1969; Rayle et al., 1970; see also Pickard et al., 1969). When horizontally exposed tissue is returned to the vertical position, the auxin-transport asymmetry in the short sections used here was found to decay within 30 rain, about 10 rain earlier than the cessation of geotropie curvature in Avena (Pickard e~ at., 1969). The curvature is expressed over a longer section of the coleoptfle, and the asymmetry established at the tip would be expected to travel down to the base after the end of stimulation. Generally, however, the relatively quick loss of asymmetry upon ending horizontal exposure in sections filled with auxin differs strikingly from the prolonged retention of the ability to develop an auxin-transport asymmetry found in auxin-depleted coleoptiles (ttager, 1967).

Geotropism in Coleoptiles

349

Our results also show a correlation between amyloplast sedimentation and gravity-enhanced lateral auxin transport. Some studies correlating amyloplast behavior and geotropic curvature have been criticized because the experimental treatment could also affect the growth or physiology of the test plants. We have attempted to decrease the number of unknown events between the stimulus and the response in two ways. First, We assayed the auxin transport asymmetry directly, so that growth was not necessary to detect a response. We also sought test material that was as similar as possible in all ways but one: amyloplast sedimentation. The closest approximation to this latter criterion was in the comparison of coleoptfles sections from 2-5 mm below the apex in paired normal and single-gene mutant strains of corn. The mutant approach to tropism physiology has been used before: geotropism in lazy corn (Overbeck, 1938), phototropism in albino barley (Asomaning and Galston, 1961) and in Phycomyces mutants (see Bergman et al., 1969). In our case, it was only an approximation of the desired conditions since the mutants that we used were endosperm starch mutants (Jones, 1967) which, in several cases, also had altered amyloplasts in the coleoptile. The physiological state of the pairs may not have been identical since they had differing endosperms. (It was noted that some mutants, e.g. brittle and sugary 1, germinated more slowly than their normal counterparts). I t is likely that the mutations have not only altered the sedimentation properties of the amyloplasts, but the chemical properties as well However, in view of the correlations which we did find, the chemistry does not seem to have a critical role in geosensitivity. For instance, the waxy mutation leads to a chemically altered starch composition (Creech, 1965): nevertheless the geosensitivity of auxin flow (as well as amyloplast sedimentation) appeared unaltered. Pickard and Thimann (1966) have suggested that the weight of the total cell contents might be used by the cells as a means of detecting their orientation with respect to gravity. Pickard (personal communication) has calculated the contribution of the amyloplasts to the total weight of the cell and found it to be minor. The difference in amyloplast weight for the mutant and the normal pairs would be unlikely to account for the observed differences if a " t o t a l weight" mechanism were present. Physiological properties other than those of the amyloplast can be expected to influence geotropic sensitivity. Red light, for example, has a marked effect on geotropism in Arena eoleoptiles (Wilkins, 1965). The genetic and physiological background in which the amyloplasts act may account for the varying sensitivity which is found. (A difference of 0.07 in the amyloplast asymmetry index of floury 2: normal had no 23

I)lanta (Bed.), Bd. 94

350

B. Filner, R. Hertel, C. Steele and V. Fan:

effect, while an even smaller difference of 0.03 for opaque 2: normal did coincide with less asymmetric lateral transport in the mutant.) The decrease from tip to base of the inhibition by inversion of basipetal NAA transport was shown to parallel the extent of the decrease in amyloplast mobility in Sorghum and Arena eoleoptfles. The fact that in the tip region both show a transport inhibition of about 10%, even though their amyloplast asymmetry indices are very different, 0.75 and 0.2~, indicates a difference in geosensitivity between the two species that is due to a factor other than the amyloplasts. The sum of the evidence presented here is clearly in support of the starch statolith hypothesis of Haberlandt (1900) and Ngmee (1900), and our results are in line with the correlations already reported between amyloplast mobility and geotropic sensitivity in higher plants (stems: Hawker, 1932; eoleoptiles: yon Guttenberg, 1912; Hertel e~ al., 1969; roots: Juniper et al., 1966; Konings, 1968; Iversen, 1969). We should point out, however, that in geotropism of lower plants no starch statoliths seem to be involved: no sedimenting particle has been detected in Phycomyces (Bergman et al., 1969), and the statoliths in Chara rhizoids do not contain starch (Buder, 1961; Sievers, 1967). A serious objection to a role of amyloplasts in coleoptile geotropism has been raised by Piekard and Thimann (1966) who were able to obtain significant geotropic curvature in young wheat coleoptiles in which the starch content of the amyloplasts had been depleted by an incubation in gibbere]lin and kinetin. This finding, however, does not seem conclusive evidence against the starch statolith hypothesis for the following reasons: 1. The depleted tissue responds to horizontal exposure after a long lag period (5 hr) which should be compared with a lag of 1.5 hr in the untreated wheat coleoptiles, and with a 15-20 rain lag for Avena coleoptiles (e.g. Brauner and Zipperer, 1961) or for both intact and excised corn eoleoptiles as were used in our work (unpublished observations). [We assayed amyloplast sedimentation ha wheat tissue and found it to be slow, as expected by the longer lag in the georesponse. After 30 rain the amyloplast asymmetry index was 0.10 and after 1 hr it was 0.20, whereas sedimentation reaches an endpoint within 30 rain in corn (asymmetry index=0.55).] I t seems difficult to us to explain the especial sluggishness of the georesponse ha the elongating, starchdepleted eoleoptAles without assuming some decrease in geosensitivity, which would then in turn be correlated to amyloplast sedimentation. 2. I t is possible to lose geosensitivity by depleting wheat coleoptfles of starch under slightly different conditions. Pickard and Thimann (1966) mention such a case ha their Methods section. Using somewhat older coleoptiles, we did not observe any geotropie curvature after 7 hr o~ continuous horizontal stimulation of starch-depleted eoleoptiles (which

Geotropism in Coleoptiles

351

did elongate), while the nondepleted controls began to curve within 1.5 hr. Such a suppression of geotropism by starch depletion is not conclusive evidence by itself for the amyloplast-statolith hypothesis, especially since chemical treatments, e.g. with a-naphthylphthalamic acid, can inhibit tropisms without inhibiting elongation (Steyer, 1969). 3. In the starch-depleted coleoptfles geoeurvature is not only delayed but its final rate is also slower than in the controls. Piekard and Thimann (1966) proposed a correction factor on the basis of the likewise decreased rate of elongation. However, this procedure may not be valid since the rate of geotropic curvature--at least in the one case tested--is not directly related to the elongation rate of the eoleoptile (BlaauwJansen and Blaauw, 1968). The delayed and slow georesponse in starch depleted wheat coleoptiles observed by Pickard and Thlmann (1966) is perhaps the result of a separate, less efficient, gravity-detection mechanism. At the very least, the data are consistent with amyloplast participation in the fast geotropic response of eoleoptfles. Accepting the role of starch statoliths one can then consider the possible mechanism by which they exert their effect on auxin transport. Since sedimentation is correlated with weight, the observed correlation of the geotropie response could also be with weight and pressure of the amyloplasts. An involvement of movable amyloplasts does not necessarfly imply that these particles have to be displaced significantly in order to act. Haberlandt (1902) stresses that if particles act b y mechanical pressure they do not have to move, as long as some are close to the sensitive structure. The effectiveness of short, intermittent stimulation (Giinther-Massias, 1928) and low gravity forces (Jost, 1902) speak against a requirement for a large asymmetry. These observations are consistent with a pressure mechanism, as are the effects, reported here of centrifugal acceleration (even after amyloplast displacement) on lateral auxin transport (el. Ouitrakul and Hertel, 1969) and on curvature. Therefore it is unlikely that the sedimented amyloplasts are involved in the transduction process as a source of some chemical which stimulates transport (as proposed e.g. by Hertel and Leopold, 1963). Localized effects of blue fight on viscosity of the cytoplasm have been reported: after partial illumination, regions of altered and unaltered viscosity were present within the same Elodea cell (Virgin, 1951). Such blue-fight effects in Vallisneria are dependent upon the plane of polarized light (Seitz, 1967). One might conclude that the viscosity is locally controlled by a structured, rigid layer--presumably the plasma membrane. This suggests the possibility of local interactions between the membrane and viscous structures. When amyloplasts are pressed into 23*

352

B. Filuer, R. Hertel, C. Steele a.nd V. Fan:

such viscous structures, the latter m i g h t be changed, and this change could locally affect the plasma m e m b r a n e where auxin transport p r e s u m a b l y occurs. Such a crude model of a sta~olith mechanism does n o t explain the k n o w n stimulus-response kinetics. These functions appear to be simple but are more likely of a compensated complexity. A n y quantitative model of statoliths a n d t r a n s d u c t i o n has to account, 1) for the reciprocity law, 2) for the finding (Pickard, unpublished observations) t h a t geostimulation (as measured b y final curvature) is constant per unit time after a b o u t 3 rain while amyloplast a s y m m e t r y is still increasing, and, 3) if d a t a f r o m oats a n d corn can be compared, for the observation (Pickard, unpublished data) t h a t the rate of curvature is constant v e r y soon after horizontal exposure whereas the lateral auxin-transport a s y m m e t r y increases for a b o u t 40 rain. This work was supported by U.S. Atomic Energy Commission Contract No. AT (11-1)-1338. It is a pleasure to acknowledge the extremely valuable discussions with Dr. Barbara G. Pickard. We also wish to thank Drs. J.D. Bewley and D. P. Weeks for criticism of the manuscript.

References Adams, P. A.: Studies on giberellie acid-induced growth i n / I v e ~ stem segments. Doct. dissert., Univ. of Michigan, Ann Arbor 1969. Asomaning, E. J. A., Ga]sten, A. V~. : Comparative study of phofntropic response and pigment content in oat and barley eoleoptiles. Plant Physiol. 86, 453464 (1961). Audus, L. J.: The mechanism of the perception of gravity by plants. Syrup. Soe. exp. Biol. 16, 197-228 (1962). Ball, N. G. : Tropic, nastic and tactic responses. In: Plant physiology, vol. V A, p. 119-228, F. W. Steward, ed. New York: Aead. Press 1969. Bergman, K., Burke, P.V., Cerda-Olmedo, E., David, C. N., Delbriick, M., Foster, K . W . , Goodell, E.W., Heisenberg, IVL, :~r G., Zalokar, M., Dennison, D. S., Shropshire, W.: Phycomyces. Bact. Rev. 38, 99-157 (1969). Blaauw-Jansen, G., Blauuw, O.H.: Geotropic curvature of Arena coleoptiles in solutions of various osmotic values. Acta bot. n6erl. 17, 273-280 (1968). Brauner, L.: ~ber d~s geo-elektrisehe Phgnomen. Kolloidchem. Beih. 23, 143-152 (1926). - - Prim~reffekte der Schwerkraft bei der geotropischen Reaktion. Encyclopedia Plant Physiol., vol. 17/2, p. 74-102. Berlin, GSttingen, Heidelberg: Springer 1962. - - Zipperer, A. : LVberdie Anfangsphasen der geotropischen Kriimmungsbewegung yon Avena-Koleoptilen. Planta (Berl.) 57, 503-517 (1961). Buder, J.: Der Geotropismus der Charaeeenrhizoide. Ber. dtsch, bot. Ges. 74, 14-23 (1961). Creech, R.G.: Genetic control of carbohydrate synthesis in maize endosperm. Genetics 52, 1175-1185 (1965). Dennison, D. S.: The effect of light on the geotropie responses of Phycomyees sporangiophores. J. gen. Physiol. 47, 651-665 (1964).

Geotropism in Coleoptfles

353

Dolk, H . E . : Geotropism and the growth substance. Rec. Trav. bot. n~erl. 33, 509-585 (1936). Gillespie, B,, Thimann, K. V. : Transport and distribution of auxin during tropistic response. I. The lateral migration of auxin in geotropism. Plant Physiol. 88, 214-225 (1963). Goldsmith, M. H. M., Wilkins, M.B.: Movement of auxin in coleoptfles of Zea mays L. during geotropie stimulation. Plant Physiol. 89, 151-162 (1964). Grahm, L. : Measurement of geoelectric and auxin-induced potentials in coleoptiles with a refined vibrating electrode technique. Physiol. Plantarnm (Cph.) 17, 231-261 (1964). - - Hertz, C. H. : Measurement of geoelectric effect in e01eoptfles by a new technique. Physiol. Plantarnm (Cph.) 15, 96-114 (1962). Giinther-Massias, M. : ~ b e r die Gfiltigkeit des Reizmengengesetzes bei der Summation unterschwelliger Reizung. Z. Bot. 21, 129-172 (1928). Guttenberg, H. yon. : ~ b e r die Verteilung der geotropischen Empfindlichkeit in der Koleoptfle der Gramineen. Jb. wiss. Bot. 50, 289-327 (1912). Haberlandt, G. : ~ b e r die Perception des geotropischen Reizes. Ber. dtsch, bot. Ges. 18, 261-272 (1900). - - ~ b e r die Statolithenfunktion der St/irkekSrner. Ber. dtsch, bot. Ges. 20, 189195 (1902). Hager, A. : Das geotropisehe Ged~chtnis der Pflanzen. Wissenschaftliche Z. Univ. Rostoek, Math.-Naturwiss. R. 16, 549-558 (1967). Hawker, L. E. : A quantitative study of the geotropism of seedlings with special reference to the nature of development of their statolith apparatus. Ann. Bot. 46, 121-157 (1932). Hertel, R., Flory, R.: Auxin movement in corn coleoptiles. Planta (Berl.) 82, 123-144 (1968). - - dela Fuente, R . K . , Leopold, A.C.: Geotropism and the lateral transport of auxin in the corn m u t a n t amylomaize. Planta (Berh) 88, 204-214 (1969). - - Leopold, A. C. : Versuehe zur Analyse des Auxintransports in der Koleoptile yon Zea mays L. Planta (Berl.) 69, 535-561 (1963). Iversen, T.-H. : Elimination of geotropic responsiveness in roots of cress (Lepidium sativum) by removal of statolith starch. Physiol. Plantarum (Cph.) 22, 12511262 (1969). Johnsson, A.: Investigation of the reciprocity rule by means of geotropic measurements. Physiol. Plantarum (Cph.) ]8, 945-967 (1965). Jones, L.M.: The ten chromosomes of maize. Appendix. I n : W. R. Singleton, Elementary genetics, 2nd ed. Princeton: Van l~ostrand 1967. Jost, L. : Die Perzeption des Schwerereizes in der Pflanze. Biol. Zbl. 22, 161-179 (1902). Juniper, B.E., Groves, S., Schachar, B.L., Audus, L . J . : Root cap and the perception of gravity. Nature (Lond.) 209, 93-94 (1966). Konings, H. : The significance of the root cap for geotropism. Acta bot. n6erl. 17, 203-221 (1968). N~mec, B.: ~ b e r die Art der Wahrnehmung des Sehwerkraftreizes bei den Pflanzen. Ber. dtseh, tot. Ges. 18, 241-245 (1900). - - t)~ber die Wahrnehmung des Sehwerkraftreizes bei den Pflanzen. Jb. wiss. Bot. 86, 80--178 (1901). Nissl, P., Zenk, M.H.: Evidence against induction of protein synthesis during auxin-induced initial elongation of Avena coleoptfles. Planta (Berl.) 89, 323-341 (1969). 28 Planta (Berl.), Bd. 94

354

B. Filner et al.: Geotropism in Coleoptiles

Ouitrakul, R., Her~el, R. : Effect of gravity and centrifugal acceleration on auxin transport in corn coleoptiles. Planta (Berl.) 88, 233-243 (1969). Overbeck, J. van: ,,Lazyness" in maize due to abnormal distribution of growth hormone. J. Hered. 29, 339-341 (1938). Pickard, B. G., Dutson, K., tIarrison, V., Donegan, E.: Second positive phototropic response patterns of the oat coleoptile. Planta (Berl.) 88, 1-33 (1969). - - Thimann, K. V. : Transport and distribution of auxin during tropistic response. II. The lateral migration of auxin in phototropism of coleoptfles. Plant Physiol. 39, 341-350 (1964). - - - Geotropic response of wheat coleoptiles in absence of amyloplast starch. J. gen. Physiol. 49, 1065-1086 (1966). Rawitscher, F. : Der Geotropismus der Pflanzen. Jena: Gustav Fischer 1932. Rayle, D. L., Evans, M. L., Hertel, R.: Action of auxin on cell elongation. Proc. nat. Acad. Sci. (Wash.) 65, 184-191 (1970). Seitz, K. : Wirkungsspektren fiir die Starklichtbewegung der Chloroplasten, die Photodinese und die lichtabh~ingige Viskosit~its~nderung bei Vallisneria spirati8 ssp. Z. Pflanzenphysiol. 56, 246-261 (1967). Sievers, A.: Elektronenmikroskopische Untersuchungen zur geotropisehen Reaktion. Z. Pflanzenphysiol. 57, 462,-473 (1967). Steyer, B.: Der Einflull yon Naphthylphthalamids~ure auf die Perception des photo- und geotropen Reizes yon Avena-Coleoptilen. Flora (Jena) 159, 484-493 (1969). Virgin, H.: The effect of light on the protoplasmic viscosity. Physiol. Plantarum (Cph.) 4, 255-357 (1951). Went, 1~. W., Thimann, K. V. : Phyt~hormones. New York: MacMillan 1937. Wilkins, M.B.: Red light and the geotropic response of the Arena coleoptile. Plant Physiol. 4{), 24-33 (1965). - - Geotropism. Ann. Rev. Plant Physiol. 17, 379-408 (1966). - - Woodcock, A. E. R. : The origin of the geoelectric effect in plants. Nature (Lend.) 208, 990-992 (1965). Woodcock, A. E. R., Wilkins, M. B.: The geoelectric effect in plant shoots. I. The characteristics of the effect. J. exp. Bet. 20, 156-169 (1969). Barbara Fflner The Institute for Cancer Research 7701 Fox Chase Philadelphia, Pennsylvania 19111, U.S.A.

Rainer Hertel Institut Biologic I I I der Universit/it Schi~nzlestral3e 11 D-7800 Freiburg i. Br.