the lateral transport of iaa in intact coleoptiles of avena sativa l. and zea mays l. during...

Planta (Berl.) 115, 97--111 (1973) �9 by Springer-Verlag 1973

The Lateral Transport of IAA in Intact Coleoptiles of Avena sativa L.

and Zea mays L. during Geotropic Stimulation

Stanley Shaw *, Gary Gardner **, and Malcolm B. Wilkins

Department of Botany, The University, Glasgow G12 8 Q Q, U.K.

Received September 24, 1973

Summary. Movement of IAA was studied in excised coleoptile apices and whole seedlings of Zea mays L. and Avena sativa L. during geotropic stimulation. A micropipette technique permitted the application of [5-aH]IAA at predetermined points on the coleoptiles with minimal tissue damage.

When [5-aH]IAA was applied to the upper side of a horizontal excised Zea coleoptile, about 60% of the recoverable radioactivity had moved into the lower half after 2 h. In contrast, when application was made to the lower side of a horizontal excised coleoptile, only 4% of the radioactivity migrated to the upper half. There was, thus, a net downward movement of 56%. Similar patterns of distribution were found for radioactivity in both the tissue and the basal receiver blocks. In horizontal shoot tissues of intact Zea seedlings a net downward movement of about 30% of the recoverable radioactivity occurred after 1 h of geotropic stimulation. Comparable experiments with Avena indicated a net downward movement of 6-12 % in excised apices of coleoptiles and in the intact shoot. In both Zea and Avena chromatographic analyses of tissue and receiver blocks indicated that the movement of radioactivity reflected that of IAA.

In Zea coleoptiles, the lateral migration of radioactivity after 2 h was 3 to 4 times greater in the apical tissues than in the basal tissues. A significant net downward movement of radioactivity was detected after 10 rain of geotropic stimulation in the extreme apex of Zea coleoptiles but not in the more basal regions.

These experiments show that downward lateral transport of IAA occurs in intact shoots of Zea and Avena seedlings upon geotropic stimulation. Lateral transport of IAA had previously been demonstrated only in sub-apical segments of Zea coleoptiles.

In t roduct ion

Although there is evidence t ha t a lateral redis t r ibut ion of endogenous growth regulators takes place in geotropically s t imula ted coleoptile apices (Dolk, 1936; Brauner and Appel, 1960; Gillespie and Briggs, 1961), a metabol ical ly-dependent , polarised downward lateral movemen t of radioactive indolyl-3-acetic acid (IAA) has only been demonst ra ted in horizon-

* Present address : Dept. of Chemistry and Biology, John Dalton Faculty of Tech- nology, Manchester Polytechnic, Manchester M15 6BX, U.K. ** Present address: MSU/AEC Plant Research Laboratory, Michigan State University, East Lansing, Michigan 48824, USA.

7 Plan ta (Berl.), Yol. 115

98 S. Shaw et al.

ta l sub-apical segments of Zea coleoptiles (Goldsmith and Wilkins, 1964;

dela Fuen te and Leopold, 1968; Will, ins and Whyte , 1968; Her te l et al., 1969; Cane and Wilkins, 1969).

This paper reports the results of exper iments in which glass micro-

p ipet tes were used to apply high specific ac t iv i ty t r i t i a ted I A A to intact ,

geotropical ly s t imula ted coleoptiles of A r e n a and Zea. This technique

enabled the t ranspor t of I A A to be inves t iga ted in re la t ive ly undamaged

coleoptiles which had an endogenous supply of growth regulators and

which had, therefore, the capac i ty to undergo normal geotropic cur-

va ture .

Materials and Methods Plant Material. Coleoptile apices, 15 mm in length, were excised from 41/2 to 51/2

days-old seedlings of Zea mays L. var. Giant White Horsetooth (Martin's Seeds Ltd., Norwich, England), the wild type and the amylomaize strains of the Hybrid WF9 • Bear 38 (Bear Hybrid Corn Company, Decatur, Illinois) and Arena sativa L. var. SvalSf Victory I (Allm~nna Svenska UtsS~des A.-B., Sval5f, Sweden). The seedlings were grown in moist vermiculite at 25 ~ C in complete darkness.

Some experiments were carried out on 3 to 31/2 days-old intact seedlings. Fruits of Zea may8 L. var. Snowcross (W. Atlee Burpce Co., Philadelphia, Pa., USA) were soaked for 14 h in flowing tap water and then planted out, embryo uppermost, on moist paper towels. About 32 h later when the roots had attained a length of about 5 ram, the seedlings were transferred individually to vials which had been filled previously with 1% (w/v) agar ("Purified", Oxoid Ltd., London). The seedlings were used 90 h after soaking began, having been exposed to red light for mes0cotyl suppression from the 53rd to 55th h. Dehusked fruits of Avena sativa were soaked for 2 h in deionized water, planted out on wet filter paper, and, beginning 8 h later, were exposed to red light for 4 h. After a further 12 h, the seedlings were transferred to vials containing 1% agar; the experiments were carried out about 72 h after soaking began. Except as specified above, the seedlings were grown at 25~ in darkness; the soaking and planting procedures were carried out under dim green light.

Indole-3-Acetic Acid. The specific activity of the [5-aH]IAA was 29 Ci. mM -1 (C.E.A., Gif-sur-Yvette, France). Thin layer chromatographic analyses revealed less than 10% impurities.

Application o/the I A A to the Coleoptiles. IAA was applied to the coleoptiles as an aqueous solution at 10 -a ~ in glass micropipettes similar to those used previously by Shaw and Wilkins (1973). The pipettes were made from Pyrex electrode glass tubing (external diameter 2.5 ram) on a Narishige PE-2 vertical glass-microelectrode puller (Narashige Scientific Instrument Laboratory, Tokyo). The tips of the pipettes were broken to a diameter of 100 to 200 ~xm. Pipettes with wedge-shaped tips were filled by capillarity with about 10 [xl of the IAA solution and then supported in a hand-held Narishige H-3 perspex electrode holder.

Experimental Procedure. The primary leaves were removed from five excised coleoptile apices which were then mounted on either a No. 1 glass coverslip for Zea, or stainless steel razor blade for Arena. The coverslip or razor blade penetrated the base of the coleoptiles to a distance of 5 mm and a 1.5% (w/v) agar block (3 • 3 • 1 ram) was placed in contact with the cut surfaces at the base of each half of the coleoptile (Fig. 1A). The assemblies were held in a plastic frame and placed in a humid atmosphere.

Lateral Transpor t of IAA during Geotropic Stimulat ion 99

B

C Fig. 1. A Method of mount ing the excised coleoptiles on the eoverslip or razor blade (black). The agar blocks (cross hatched) are in contact with the bases of the two halves of the coleoptile. The ar rogs show the points of application of the [5-3H]IAA 1 mm (a) or 5 m m (b) from the apex. B Diagrammatic cross section of coleoptile indicating position of donat ion of [5-zH]IAA 5 m m below the apex. Donations were made at point (a) before a 2-h t ranspor t period and at point (b) before a 10-min t ranspor t period. The direction of gravi ty is perpendicular ~ the plane of bisection

of the eoleoptile (broken line)

The pipette containing the radioactive IAA was placed against the surface of the coleoptile so that the sharp tip just penetrated the tissues for a few seconds before being withdrawn. In this way IAA was donated to one side of the co]eoptile at a predetermined point ei ther 1 mm or 5 m m behind the apex (Fig. I A and B). Depending on the length of t ime the pipet te was allowed to remain in contact with the tissue a dose of between 2000 and 200000 dpm of radioact ivi ty was donated to a single coleoptile. This represented a volume of between 0.0003 and 0.03 lxl. The vials containing the intact seedlings were supported in wooden holders, and applications of [5-sH]IAA were made in a similar fashion.

After application of the IAA, the eoleoptiles or seedlings were orientated either vertically, or horizontally with the point of donat ion on the upper or the lower side. Unless otherwise specified the experiments were set up under normal laboratory lighting and temperature,l and t ranspor t was allowed to take place in darkness at 25 ~ C. At the end of the t ranspor t period the tissue was bisected longitudinally and then divided transversely into portions for radioassay. Since the absolute amount of IAA supplied to each eoleoptile varied, the results were calculated as percentage distr ibutions of radioactivi ty within a given coleoptile,

The data presented in the figures are ei ther from an individual experiment or the mean result of a number of experiments. All experiments were carried out at least twice, and generally on more t han four separate occasions. The level of significance, t ha t is, the probabil i ty p t ha t the difference between two mean values was not meaningful, was assessed by means of Student ' s t test. A difference was considered significant if p < 0.05.

Radioassay. The tissue and receiver blocks were placed in scintillation vials and extracted for a t least 24 h in 1.4 ml of absolute ethanol a t room temperature. Ten ml of scintillation fluid (0.4% w/v, 2,4-diphenyloxazole in toluene) were then added, and the samples were counted by liquid scintillation spectrometry.

Analysis o/Radioactivity. The radioact ivi ty in the coleoptile tissues and receiver blocks was analysed by paper and silica-gel thin-layer chromatography. After

7*

100 S. Shaw st al.

30 min or 60 min of transport, tissue portions from at least 20 coleoptiles were extracted at room temperature in 1 to 2 ml of absolute methanol for 48 h in darkness. After this period about 10% of the total recoverable radioactivity remained asso- ciated with the tissue or receiver blocks. An aliquot of each extract containing about 5 • 10 a cpm was chromatographed on paper (Whatman No 1) with either n butanol: glacial acetic acid: water (5:1 : 2.2) or isopropanol: ammonia: water (8:1 : 1), and on silica-gel thin layer plates with chloroform :glacial acetic acid (95:5). The distribution of radioactivity on the chromatograms was assessed by liquid scintillation spectrometry. Unlabelled IAA (Sigma Chemical Company) was used as a marker in each chromatographic system and located by Ehrlich's reagent or U.V. fluorescence.

Results

1. E//ect o / the Applicat ion Technique on Geotropic Curvature

Wate r or IAA (10 -a M) was applied asymmetr icMly to the tips of excised, 15-mm coleoptile apices of Avena and Zea. The coleoptiles were t hen or ienta ted vert ical ly or hor izontal ly so t ha t the point of dona t ion was on the upper or the lower side. The curva ture developed by these coleoptiles after a period of 4 hours was compared with that. developed by horizontal un t r ea t ed coleoptiles (Table 1). The applications had no effect on the geotropic curva ture of Zea coleoptiles, nei ther did they induce any curva ture in vertical coleoptiles. Vertical Avena coleoptiles which had been supplied with either water or IAA remained straight bu t the curva ture of the horizontal t rea ted coleoptiles appeared to be less t h a n tha t of the coleoptiles which had received no application. I n fact, a horizontal Avena coleoptile supplied with IAA on its upper side (Table 1, column d) ben t to an angle which was less t h a n half t ha t a t ta ined by an un t rea ted coleoptile (Table 1). All the horizontal coleoptiles used in the t ranspor t experiments had developed visible geotropic curvatures after 2 h.

Table 1. Curvature (degrees • S.E. of mean) of excised horizontal and vertical coleoptiles of Zea mays and Avena sativa 4 h after they had been supplied with either water (a, b, c) or IAA (10 -4 M) (d, e, f) from a micropipette, and of horizontal coleoptiles which received no treatment. The treated coleoptiles were orientated horizontally with the point of application on the upper (a and d) or lower (b and e)

sides, or vertically (c and f)

:No treatment H20 IAA

Horizontal Horizontal Vertical Horizontal Vertical

Upper Lower Upper Lower donation donation donation donation (a) (b) (c) (d) (e) (f)

Zea 36.44-5.0 33.1=~4.0 35.84-4.7 0.2• 34.1• 39.4• --0.4• Avena 45.1• 37.4i2 .7 28.2i3 .5 --0.8• 19.2• 32.6• --0.14-0.6

Lateral Transport of IAA during Geotropic Stimulation 101

Z E A D E T A C H E D AP ICES 2 h o u r s

(eo.71 ~ ~ - 7 ~ ; .... ~ .....

19 1,ei 9.1

( 9 5 ' 7 ) L ~ 20"41-i0-612-'4~8/~.. N t

Fig. 2. Percent distribution of radioactivity after 2 h in horizontal and vertical 15 mm-long excised coleoptile apices of Zea mays L. var. Giant White Horsetooth. The tissue was bisected longitudinally and then transversely into three 5-ram long portions (dotted lines). The solid line bisecting the agar receiver blocks and the base of the coleoptiles represents the supporting coverslip. Figures in brackets show the total percent radioactivity in the tissue and receiver blocks in each half of the coleoptiles. Arrows indicate the position of o~pplication of the [5-SH]IAA. The direction of gravity is shown by the arrow g. The data presented are representative of

experiments carried out on five separate occasions

Z E A SEEDLINGS 1 h o u r

t i t , - - - ~ . . . .

(66"1) 1 12"1 17"7 9 3 1 2 7 2 ' 5 : 1 8 0 i i i d 0.2 i- . . . . . . . . . . r . . . . . . . } _ _ _ i . . . . i I I 4 .9 ~14-51 6"91 7-4/] 3 .31224 I I , , / .__~ . . . . .

(33"7)

1 I g 2"3110"6

I I ', I I ' (2"2) 0"5 i 0-7~ 0-4 ~ 0-6~ . . . . _l . . . . i i i i ~

0'1 ~- . . . . . . * - - - 4 - - - J - - - 0 ~ 7 0 1 J I 1 (97"9) 10-4 ~20 -9d6"5~

I I I I ~ ~ (10"5) ( 8 9 6 )

Fig. 3. Percent distribution of radioactivity after 1 h in horizontal and vertical shoots of intact seedlings of Zea mays L. var. Snowcross. The shoot tissue was bisected longitudinally and then divided transversely into three 5-ram portions and one 10-ram portion (dotted lines). The figure at the base of the shoots shows the total radioactivity in the rest of the shoot above the seed. The figures in brackets are the total radioactivity in each half of the shoots. The arrows indicate the position of application of the [53ttJIAA. The direction of gravity is shown by the arrow g.

The data presented were obtained from one of two separate experiments

2. Movement o/ Radioactivity in Zea Coleoptiles

T h e d i s t r i b u t i o n of r a d i o a c t i v i t y in h o r i z o n t a l a n d v e r t i c a l exc i sed co leopt i les of Zea mays L. var . G i a n t W h i t e H o r s e t o o t h 2 h a f t e r a sym-

m e t r i c a p p l i c a t i o n of [ 5 - a H ] I A A is s h o w n in Fig . 2. I n h o r i z o n t a l coleop-

102 S. Shaw et al.

Table 2. Percent radioactivity (4- S.E.) in the tissue and receiver block on the side opposite to the point of application of [5-3H]IAA for excised apices of three varieties of Zea mays L. after 2h. The IAA was applied to a point l m m (a, b, c) or 5 mm (d, e, f) below the apex of the coleoptiles which were then orientated horizontally with the point of donation on ~he upper (a and d) or lower (b and e) side, or vertically (f and g). The experiments were carried out entirely under dim

green light

1 mm from apex 5 mm from apex

Horizontal Vertical Horizontal Vertical

Upper Lower Upper Lower donation donation donation donation (a) (b) (c) (d) (e) (f)

Giant White 54.95• 3.73• 11.64=~2.08 16.684-2.44 4.444-t-0.75 6.41• Horsetooth

WF9 x Bear 38 51.80• 8.92• 16.69• 17.114-2.13 6.844-1.44 7.15~1.35 "Wild type"

"Amylomaize" 44.53~-3.17 8.904-1.65 20.904-2.44 10.894-2.32 5.35• 7.19•

t i les the p ropor t ion of r a d i o a c t i v i t y recovered f rom both the eoleopti le t issue and receiver b lock on the side opposi te the po in t of dona t ion was 60.7% when the d o n a t e d side was uppe rmos t and on ly 4.4% when the dona t ed side was lowermost . The difference be tween these two values is 56.3% and represents the net downward movement of r a d i o a c t i v i t y in the hor izon ta l eoleoptile. Some 16 % of the appl ied r a d ioa c t i v i t y moved l a t e ra l ly in the ver t ica l eoleoptiles. S imi lar d a t a were ob ta ined from expe r imen t s wi th excised eoleopti les of Zea m a y s L. var . Snoweross.

The resul t of an ident ica l expe r imen t carr ied out on in tac t seedlings of Zea m a y s L. va t . Snoweross is shown in Fig. 3. The d i s t r ibu t ion of r a d i o a c t i v i t y in the apical 25 m m of the shoot was de te rmined . I n horizon ta l seedlings the downward m o v e m e n t of r a d i o a c t i v i t y exceeded the upward m o v e m e n t b y 31.5% af ter 1 h. A b o u t 10% of the r a d ioa c t i v i t y app l ied to ver t ica l seedlings was recovered f rom the side oppos i te the po in t of donat ion .

The nex t exper iments were designed to de te rmine the ex t en t of l a te ra l m o v e m e n t of r a d i o a c t i v i t y af ter a symmet r i c app l ica t ion of [5-aH]IAA to a po in t e i ther 1 m m or 5 m m behind the apical e x t r e m i t y of an i n t ac t eoleopti le (Fig. 1). The appl ica t ions were made to coleoptilcs of th ree var ie t ies of Zea may8 and t r a n s p o r t was al lowed to t ake place for 2 h (Table 2). The exper iments were carr ied ou t en t i re ly under d im green l ight . The u p w a r d mig ra t ion of r a d i o a c t i v i t y f rom a dona t ion to the lower side of the hor izonta l eoleopti le was no t inf luenced b y the d is tance


_ 4 o A

~z 3O o 2 o ' ~ O 30 60 90 120

Ld ~12 ; . . . . . a '-

10 z

>- 8

6 b

4 0

O I I

0 I'0 20 ' 30

TIME (minutes)

lfig. 4. A Lateral movement of radioactivity as a function of time. Total radioactivity (percent) in the tissue and receiver blocks on the side opposite to the point of application of [5-3tI]IAA are shown for excised apices of Zea mays L. vat. Giant White I-Iorsetooth. The inset diagrams show the point of ~pplication of IAA (arrow) on the upper (a) or lower (e) side of a horizontal coleoptile, or the side (b) of a vertical coleoptile. The shaded portions in the inset diagrams show the portions of the tissue and receiver blocks for which radioassay data are presented. B As for A, except that the data refer to a time course of 30 rain in a separate experiment

of the point of appl icat ion from the apex. On the other hand, when IAA was supplied to the upper side of a horizontal eoleoptile 1 mm from the apex, between 3 and 4 t imes more rad ioac t iv i ty moved la teral ly t h a n when appl icat ion was made to the more basal regions. Nevertheless, signifiean~ ne t lateral movemen t did occur after basal applicat ion to all three varieties. I n vert ical coleoptile apices there was more lateral move- m e n t of radioact iv i ty after apical t h a n after basal appl icat ion of the IAA. After both apical and basal applicat ions of I A A there appeared to be less downward lateral movemen t of rad ioac t iv i ty in the " a m y l o m a i z e " W F 9 • Bear 38 t h a n in the "wi ld t y p e " WF9 • Bear 38. These differences between the two strains yielded a p value which was jus t greater t h a n 0.05.

I n excised coleoptile apices of the corn var ie ty Giant Whi te t torse- tooth, the a m o u n t of radioact iv i ty recovered from the side opposite the point of dona t ion was determined as a func t ion of time. The data are i l lus t ra ted in Figs. 4 A and B. After 15 rain significantly more radioact iv i ty could be recovered from the lower side of a horizontal coleoptile after

104 S. Shaw et al.

Table 3. Percent radioactivity (--S.E.) in the tissue on the side opposite to the point of application of [5-*HJlAA for excised apices of Zea mays L. var. Giant While ttorsetooth and WF9 • Bear 38 "Wild type" after 10 rain of geotropic stimulation. The mean values are from 3 to 4 separate experiments, all carried out entirely

under dim green light

1 mm from apex 5 mm from apex

Upper Lower Upper Lower donation donation donation donation (a) (b) (c) (d)

Giant White ttorsetooth 3.98 • 0.53 1.71 • 0.30 4.63 • 1.06 5.68 -c 1.19 Number of coleoptiles 26 34 37 40

(p<0,001) (p~0.5)

WF9 • Bear 38 "Wild type" 3.05 • 0.45 1.59 :i: 0.31 5.32 -~ 0.94 3.92 • 0.68 Number of coleoptiles 33 35 60 59

(p<0.01) (p~0.2)

upper donat ion (Fig. 4 A and B, curves a) than from the upper side of a coleoptile after lower donat ion (Fig. 4A and B, curves c). This represents a net downward movemen t of 4.1% (p <0 .02) in Fig. 4A and 3.0% (p <0 .02) in Fig. 4B.

This finding differs f rom earlier reports on the pa t te rn of I A A transpor t during the initial phases of the geotropic response of corn coleoptiles (Filner et al., 1970 ; t I i ld and Hertel , 1972).

The next experiments were, therefore, carried out to examine in detail the distr ibution of radioact ivi ty in horizontal coleoptiles of two varieties of corn 10 rain after horizontal orientation. Asymmetr ic applications of IAA, in the manner il lustrated in Fig. 1 A and by the arrow b in Fig. 1B, were made under dim green light to a point either 1 m m or 5 m m below the apex of the coleoptiles. I n the tip region of both varieties of corn significantly more radioact ivi ty moved laterally downwards than up- wards, but in the basal region no significant net downward lateral movemen t could be detected after 10 rain (Table 3).

3. Movement of Radioactivity in Avena Coleoptiles

I n both horizontal, excised coleoptile apices (Fig. 5 A and B) and horizontal in tact seedlings (Fig. 6) the downward lateral movement of radioact iv i ty is significantly greater than the upward movement after asymmetr ic donation. After 1 h, the magni tude of the net lateral movement in excised Avena coleoptiles (Fig. 5 B) is about 12 % compared with t h a t of about 45% in Zea coleoptiles (Fig. 4A, curves a and c).


AVENA 30 rain

AVENA 60 min

Fig. 5. A Percent distribution of radioactivity after 30 min in 15 mm-long excised apices of Avena sativa L. The dotted line represents the plane of longitudinal bisection of the coleoptile. The solid line separating the agar receiver blocks and the bases of the coleoptiles represents the supporting coverslip. Arrows indicate the position of application of the [5-3ItJIAA. The direction of gravity is shown by the arrow g. Data are the mean values for four separate experiments and net downward transport was 8.6% (p < 0.001). B As for A, except that the data refer to percent distribution of radioactivity after 60 min. Data are the mean values for two separate

experiments and net downward transport was 12.3% (p < 0.01)

4, Analysis o/Radioactivity

After 60 rain in Zea coleopti les and 30 rain in Avena coleoptiles the r a d i o a c t i v i t y in the donor micropipe t te , co]eoptile t issue and agar receiver blocks was ana lysed b y means of pape r and th in - l aye r ch romatography . Fig. 7 A and B show the d i s t r ibu t ion of r a d i o a c t i v i t y ex t r ac t ed f rom tissue on silica-gel th in - l aye r eh romatograms deve loped in the chloro- f o r m : a c e t i c acid so lvent sys tem. I n bo th the Zea and Avena t issue ex t rac t s a p ropor t ion of the r a d i o a c t i v i t y d id no t co -chromatograph with [AA. The p ropor t ion of recoverable r a d i o a c t i v i t y which was indis t inguish- able f rom I A A could thus be assessed. The d i s t r ibu t ion of ex t r ac t ab le [5 -a t I ] IAA in coleopti le t issue was de t e rmined and compared with the d i s t r ibu t ion of r ad ioac t iv i ty . The d a t a are shown in Fig. 8 A and B. I n bo th Arena and Zea the d i s t r ibu t ion p a t t e r n of I A A is closely s imi lar to t h a t of the r ad ioac t iv i ty . However , in Zea, the m o v e m e n t of I A A appea red to be m a s k e d to a small ex ten t b y metabo l i t e s of IAA. The analysis revea led abou t 65% ne t downward m o v e m e n t of I A A whereas the ne t l a te ra l m o v e m e n t of r ad ioac t i v i t y was only 53%. Chromatograph ic analys is of the rad ioae t i , - i ty in the I A A solut ion in the mie rop ipe t t e and of the smal l a m o u n t of r a d i o a c t i v i t y which reached the agar receiver

106 S. Shaw et al.

AVENA SEEDLINGS

o ~ i ~o.~ __~ . . . . . . . . . . . . . . . - /

/ i 16.9 ._~

r ....... ......... \

30 rain

i

i i

17"318 2 . ,

I ,, i i i i

___4 . . . . .

0 . 4

Fig. 6. Percent distribution of radioactivity after 30 min in horizontal and vertical shoots of intact seedlings of Avena sativa L. The apical 25 mm of the shoot were bisected for radioassay (dotted lines). The figure at the base of the shoot shows the total radioactivity in the rest of the shoot above the seed. The arrows indicate the position of application of the [5-SH]IAA. The direction of gravity is shown by the arrow g. Mean values for data pooled from four separate experiments are shown.

Net downward transport was 12.7 % (p < 0.001)

2 0 0 0

1600

1200 -/ 8 0 0 u

v

4OO

0

>..

~- 5 0 0 >

t- 4 0 0 0

g 300 < rr 200

100

IAA u

A ZEA ~ (60 rain)

IAA B ~

; ' g2 & '&'ols' ~Jo Rf

:Fig. 7A and B. Chromatographic analysis of radioactivity in the tissues of excised apices of coleoptiles of Zea may8 L. vat. Giant White Horsetooth (A) and Avena sativa L. (B) after transport periods of 60 rain and 30 rain respectively. The analyses were carried out on silica-gel thindayers developed in chloroform : acetic acid (95 : 5). The shaded part of the inset diagram shows the 5-ram portion of tissue (for both Zea and Avena) to which these particular analyses refer. The shaded bar represents

the position of IAA


ZEA 60 min RADIOACTIVITY IAA

A

t 9.6 i34./ (53-3)

t o9

AVENA RADIOACTIVITY

B

Kwi:;;/ (6"8)

s.o !sl.X, ~

, 2 9

32., 138.s/ I

(68.5)

l-s 11~ . . . . . . . . . . J . . . . .

57"6 i40-Q~,,

30 rrlJn IAA

95 j72 3 5

Fig. 8. A Percent distribution of [5-aH]IAA compared with that of total radioactivity after 60 min in horizontal excised apices of Zea mays L. var. Giant White tIorsetooth. The distribution of [5-3H]IAA was determined from the kind of chromatographic data illustrated in Fig. 7. The eoleoptile tissue was bisected longitudinally and then divided transversely into a 5-ram and a 10-mm portion. Correspond- ing portions from 25 eoleoptiles were combined for extraction, chromatographic analysis, and radioassay. Figures in brackets between the eoleoptiles represent the net downward movement within the tissues. The arrows indicate the position of application of the [5-~II]IAA. The direction of gravity is shown by the arrow g. B As for A, except that the data refer to horizontal excised Avena apices after a

30-min transport period

blocks revealed only one d i s t inc t zone of a c t i v i t y which was indis t inguish- able f rom IAA. Similar resul ts were ob ta ined from analyses carr ied out on pape r wi th o ther solvent systems.

Discussion

The mie roapp l i ea t ion techniques r epor t ed in this paper had no effect on the no rma l nega t ive geot ropic response of excised Zea coleoptiles. Hor izon ta l Avena coleopti les which h a d been suppl ied with I A A developed somewha t smal ler curva tures t h a n those which had received no appl ica- t ion. Never theless , all the hor izon ta l coleopti les h a d ben t subs t an t i a l l y a f te r 4 h. W e conclude, therefore, t h a t the mie roapp l ica t ion technique caused ve ry l i t t le t issue damage and h a d a m i n i m u m physiological effect.

The d a t a p resen ted here ex tend to in t ac t coleopti les and seedlings of Zea mays the f inding in sub-apica l coleopti le segments t h a t I A A under-

108 S. Shaw et al.

goes a polarised lateral redistribution within the tissues upon geotropie stimulation (Goldsmith and Wilkins, 1964; dela Fuente and Leopold, 1968; Wilkins and Whyte, 1968; Cane and Wilkins, 1969; Hertel et al., 1969). The magnitude of the gravity-induced net lateral movement of radioactivity applied within 1 m m of the apex of an intact Zea coleoptile was, in fact, more than twice tha t reported by Goldsmith and Wilkins (1964) for sub-apieal eoleoptile sections.

There is also a significant lateral t ransport of IAA within the tissues of horizontal intact Arena eoleoptiles. The magnitude of the net downward movement of radioactivity in the horizontal oat eoleoptiles is, however, about 6% to 12% eompared with that of about 45% in Zea

coleoptiles. Gillespie and Thimann (1961) did not observe any difference between the amounts of radioactivity in the upper and lower sides of horizontal sub-apical Arena coleoptile segments following symmetrical donation of IAA from an apical agar block. This finding was at tr ibuted to immobilisation of radioactivity in the eoleoptile tissues obscuring the redistribution of the mobile auxin (Gillespie and Thimann, 1961; Gold- smith and Thimann, 1962 ; Gillespie and Thimann, 1963). However, with our techniques the distribution of radioactivity which co-chromatographs with IAA closely parallels tha t of the total radioactivity (Figs. 7 and 8). The relatively small lateral movement of radioactivity in Arena as compared to Zea does not result, therefore, from a masking of the transport of IAA by immobile radioactive metabolites, but rather from a smaller lateral movement of the IAA molecule itself. I t is possible that some IAA is bound to a cellular site and is released during our relatively mild extraction procedures. The failure of Gillespie and Thimann (1961) to observe a significant lateral t ransport of radioactivity in Arena

coleoptile segments may have been due to the absence of the extreme apex of the coleoptile or to other experimental procedures different from those used in the present study.

In horizontal eoleoptiles from a number of varieties of Zea mays the lateral t ransport of radioactivity was found to be 3 to 4 times greater after application 1 mm from the apex than after application 5 mm from the apex. This result is consistent with the suggestion of Goldsmith and Wilkins (1964), and the findings of dela Fuente and Leopold (1968) tha t lateral redistribution could occur along the whole length of a 15mm-long sub-apical segment. Hertel et al. (1969) and Filner et al. (1970) have also reported tha t lateral t ransport of radioactivity from IAA was more efficient in sections excised from the tip region of the eoleoptile than in sections excised from the base of the coleoptile. I-Iertel et al. (1969) showed tha t in horizontal coleoptile segments excised 3 mm behind the apex, downward transport in the wild type and the amylomaize strains of the corn WF9 • Bear 38 occurred in the proportion 1.4:1. Our data


(see Table 2) indieate tha t lateral movement in the wild type and the amylomaize strains is in the proportion 1.2:1 in the apex and 1.6:1 in the basal region. In both the tip and basal zones of the coleoptiles, however, the differences between the amylomaize and wild type just failed to achieve the 0.05 level of probability. The trends are, nevertheless, consistent with the earlier findings of Hertel eta/. (1969) and Filner et al. (1970) tha t the geotropieally-induced lateral auxin transport capacity of amylomaize eoleoptiles is less than that of the wild type coleoptiles.

When IAA is applied 1 mm behind the apex of coleoptiles of Zea mav.s a significant net downward movement of radioactivity eould be detected after 10 min of horizontal Stimulation. This finding indicates tha t lateral movement of auxin occurs at t ime intervals which appear to preeede the onset of other gravity-induced phenomena sueh as the geoelectrie effeet (Grahm and Hertz, 1962; Woodcock and Wilkins, 1969, 1970, 1971)and the negative geotropie eurvature (Brauner and Zipperer, 1961; l~ilner et ctl., 1970; Hild and Hertel, 1972). These data do not agree with the findings of Filner et al. (1970) and Hild and Hertel (1972) suggesting tha t an upward displacement of applied IAA is correlated with the positive geotropie curvature observed in Avenct (Branner and Zipperer, 1961) and Zea (Hild and Hertel, 1972) after about 10 min. Filner et al. (1970) and Hild and Hertel (1972) carried out experiments on segments excised 2-5 mm behind the apex of the eoleoptile, and the inconsistency between their findings and ours might be at tr ibuted to the initial upward IAA movement occurring in the more basal regions of the eoleoptile but not in the extreme tip. In the present study, however, after 10 rain no significant net lateral movement of radioactivity in any direction was observed after application of IAA g m m behind the apex of an intaet eoleoptile.

The differences between our data and those of Filner et ctl. (1969) and Hild and Hertel (1972) could also result from differences in the methods used to assay the lateral movement of auxin in the eoleoptiles. Firstly, our experiments were carried out on intact coleoptiles and lateral transport was thus assayed in the presence of endogenous growth regulators which may modify the pat tern of t ransport of the applied auxin during the early phases of the geotropic response. Secondly, Filner et ctl. (1970) and Hild and Hertel (1972) assayed only agar receiver blocks which had been in contact with the cut surfaees of the coleoptiles. The methods of determination of lateral t ransport by assaying only agar receiver blocks have been critieised on the grounds tha t gravi ty could effect the export of radioactivity into the blocks independently of movement within the tissues (Wilkins, 1966 and 1971). Until, therefore, an initial upward movement of auxin has been shown in the plant tissues as well as in reeeiver blocks, this point will remain unresolved.

110 S. Shaw et al.

We wish to thank Professor R. Hertel for the gifts of the amylomaize mutant and wild type strains of the corn variety Zea mays L. var. W:F9 • Bear 38 and for his helpful comments. This work was conducted while S. Shaw was in receipt of an I.C.I. Post-Doctoral l~eseareh Fellowship and G. Gardner was in receipt of a NATO Post-Doctoral l~esearch l%llowship.

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the lateral transport of iaa in intact coleoptiles of avena sativa l. and zea mays l. during...

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