Planta (Berl.) 105, 139--154 (1972) 9 by Springer-Verlag 1972

Transport of Indoleacctic Acid in Intact Roots of Phaseolus coccineus P. J. Davies a n d E. K. Mitchell Section of Genetics, Development and Physiology, Division of Biological Sciences, Cornell University, Ithaca, N.Y. Received September 27, 1971 / February 2, 1972

Summary. Indoleacetic acid (IAA)-5-aH (2 • 10-9) was applied to intact roots of Phaseolus coccineus seedlings at the apex or 2 cm above the apex, and the movement of IAA-aH and its metabolites traced by sectioning and chromatography. Basipetal movement of label occurred for 2 em or less, declining exponentially, and the amount increased with time. Acropetal transport from above the apex showed quantitatively less movement of radioactivity. After a 6 h treatment period a decline of label occurred in the first 0.5 cm, below which there was a long distance movement of small amounts of label, mainly in IAA, towards the apex where the label concentrated by a factor of approximately 2. Short-distance basipetal movement consisted of about equal amounts of IAA and metabolites, and only metabolites were found in areas more basipetal than 2 cm. Label from solutions of sucrose-laC and sHoO followed the same general pattern of movement as label from IAA-SH, except that aeropetal movement of water showed a steady decrease in the amount of label as the distance from the area of application increased. The short distance basipetal transport of label with the breakdown of IAA-aH indicates that the extent of basipetal movement was limited by catabolic processes. The acropetal pattern of IAA-aH movement with the concentration of the transported material close to the apex, is possibly the result of transport in the phloem. Introduction T h e u n d e r l y i n g question in the m o v e m e n t of a u x i n i n roots is its role i n geotropism. I t has been shown t h a t the root cap cells c o n t a i n starch statoliths which fall u n d e r g r a v i t y (Audus, 1962, 1964; O u i t r a k u l a n d Hertel, 1969) a n d t h a t the r e m o v a l of the root cap eliminates the response to g r a v i t y ( J u n i p e r et al., 1966; Gibbons a n d Wilkins, 1970). F r o m analogies to work with coleoptile tips (Gillespie a n d T h i m a n n , 1963) we have a s s u m e d t h a t a u x i n r e d i s t r i b u t i o n gives a differential growth response on the u p p e r a n d lower sides of the growing zone of the root, a n d a c o n c e n t r a t i o n of indole-3-acetic acid (IAA) i n the lower p a r t of a h o r i z o n t a l root has in fact been d e m o n s t r a t e d (Konings, 1967)

140

P.J. Davies and E. K. Mitchell:

The role of auxin is therefore hypothesised to be t h a t of a messenger formed in the root-tip and transported back to the growing zone. Most work with 14C-IAA in root sections has, however, shown acropetal transport towards the root-tip (Wilkins and Scott, 1968; Scott and Wflldns, 1968 ; Cane and Wilkins, 1970 ; Kirkland and Jacobs, 1968), which thus carries the message in the " w r o n g " direction, though some work has demonstrated basipetal polarity (Leopold and Guernsey, 1953) or no definite polarity (Ycomans and Audus, 1969). These differences m a y be due to the use of different species (Wilkins and Scott, 1968; Hertel and Leopold, 1963). There is also uncertainty about the effect of technique on the results obtained (Iversen and Aasheim, 1970; Mann and Jaworski, 1970). One of the causes of uncertainty about the usefulness of some techniques for an understanding of natural conditions in plants comes from the continued use of relatively short sections, rather than intact plants, and the collection of the auxin in agar b]ocks, originally used for auxin bioassays. The advent of radiotracers (particularly IAA-SH) enables measurement of auxin with an equivalent sensitivity to bioassays. Results obtained with whole plants can be interpreted less ambiguously, as there is no need to damage the experimental organism. Such injury has been demonstrated to affect the results obtained because of the stimulation of I A A oxidases on sectioning (Iversen and Aasheim, 1970). The agar block technique has, however, been retained for work with IAA-14C which, due to its low specific activity compared to IAA-3H, still requires a method for collection and concentration of the radioactivity. A related disadvantage of IAA-14C is that high IAA concentrations, 10-7 to 10 -5 M (Scott and Wflkins, 1968; Hil]man and Phillips, 1970), must be used for treatment if appreciable counts are to be obtained. These concentrations are sufficiently high to be inhibitory to root growth (Fiedler, 1936; Thimann, 1937; Pilet et a/.,1960; Diez et al., 1971). IAA-aH is of sufficiently high specific activity t h a t it can be used to 10-9 M, which is much nearer the concentration which promotes root growth. Work done with intact plants indicates that IAA transport is from shoot tip to root tip. Application of IAA-14C to the stems of plants resulted in the transport of label to the roots, both in the form of IAA-~4C and as metabolites (Morris et a/.,1969; Haissig, 1970), and endogenous IAA also appears to move in a similar direction (Phillips, 1964). These results support the theory of acropetal transport of IAA in roots. The present experiments were undertaken with a dual purpose in mind. We wished to determine directions and characteristics of IAA transport in intact roots and to establish whether auxin could fulfill the role of messenger in the geotrophic response. By use of intact plants

Auxin Transport in I n t a c t Roots

141

a n d I A A - ~ H w e h o p e t o a v o i d c e r t a i n l i m i t a t i o n s of t h e m o s t c o m m o n l y used techniques and to approximate the natural conditions in the plant m o r e closely.

Materials and Methods Plant Material. Seeds of Phaseolus eoccineus L. ev. Emperor Scarlet R u n n e r (Kellogg Seed Co., Ventura, California) were washed with distilled water and soaked with one or more changes of distilled water with vigorous aeration for 24 h. They were planted in vermiculite, kept moist with distilled water, and grown in the dark at 25~ until the primary roots were 5-7 cm long ( 3 4 days). The seeds were oriented vertically a t planting so t h a t the roots grew straight downwards. Damaged, bent, or abnormal roots were discarded as were seedlings t h a t h a d developed lateral roots. The shoots of the seedlings used in all the experiments, except those involving shoot application of IAA, h a d not yet emerged from the seed. Treatment Solutions. IAA-5-aH, specific activity 17e/mmole, was obtained in benzene/ethanol solution (Sehwarz BioResearch, Orangeburg, N.Y.) with a stated radiochemical purity of 98.5%. Chromatography of the solutions on silicic-acidimpregnated glass fiber (Gelman ITLC-SA) with the sample overlayed on aseorbic acid as an anti-oxidant (Mann and Jaworski, 1970), showed t h a t the [AA-5-aH was 85-90% pure. Chromatograms were developed with methyl acetate:isopropanol : 25 % ammonia (9 : 7: 4) ; chloroform: 96 % acetic acid (95: 5) ; n-butanol: ammonia: water (4:1 : 1) ; a n d isopropanol : ammonia: water (8:1 : 1). Label was traced by cutting the ehromatograms into 0.5- or 1-cm strips (an average of 24 strips per ehromatogram) a n d counting the strips using 2,5-diphenyloxazole (PPO) a n d 1,4-bis-[2-(-4-methyl-5-phenyloxazolyl)] benzene (dimethyl POPOP) in toluene, in polyethylene vials, in a Packard model 3375 scintillation counter. A parallel IAA s t a n d a r d was detected with an ultraviolet lamp. Impm, ities were extracted with ether from an alkaline solution of IAA-3I-I. Chromatograms of these impurities showed less t h a n 10% IAA and were tested for t r a n s p o r t characteristics and physiological effects on roots. The IAA-aH was not further purified because the impurities were less readily transported t h a n IAA and were physiologically inactive, even at concentrations of l0 -s M. The IAA-aH was applied to the roots in aqueous solution at 2.0 • 10 -9 M. The t r e a t m e n t solutions were prepared in glass double-distilled water, kept a t 2~ in the dark and used within 6 h of preparation. Solutions were prepared with selLmeasuring pyrex mieropipettes (Will Scientific) after early work showed t h a t neither Coming disposable soda-glass micro-pipettes nor Hamilton or Terumo micro-syringes gave reproducible measurements of label when IAA in organic solvents was measured, probably due to adsorption of IAA-aH onto the glass. This effect became very noticeable a t the low concentration of IAA-aH used in our experiments. Sucrose (420 mc/mmole) was applied as a 2 • 10.7 IV[ aqueous solution and aH20 as water with a radioactive concentration of 0-1 ixe/ml. Treatmen~ Methods. All soIutions were applied in 0.3 ml quantities a t one of two positions: 1) at the apex, in a polyethylene "Caplug" (6 mm diameter, 11 m m high) (Protective Closures Inc., Buffalo, N.Y.) with the terminal 1 m m of the root immersed in the solution; 2) 2 em up the root in silicone rubber cups molded from G.E. Silicone Sealant (Fig. 1). The silicone rubber cups h a d small holes in the b o t t o m through which the roots could be pushed without damage. To ensure t h a t the recorded movement of radioactivity was not caused b y the water supply of the IAA solution, glass-distilled water was also supplied a t the position, either at the tip or in the rubber cup 2 cm up the root, at which IAA was not being supplied. 10 Pl~nta 03erl.), Bd. 105

142

P . J . Davies and E. K. Mitchell:

movable

~

f balsa wood support

nylon s c r e w _ , . ~ ~ fixed

molded silicone

w

~-,/rubber

cup

plexigtas support ~-...~ T

~

~1"I~"----p~

t hylene

"Caplug"

Fig. 1. The method used to t r e a t portions of bean roots with IAA-3H. To account for root growth during the t r e a t m e n t period, the bean seedling was pinned in a vertical position to a plexiglas support which could moved in a vertical direction. For treatment, the IAA solution was placed in a small polyethylene container (0.3 ml volume) a t the root tip or, for t r e a t m e n t further up the root, in a moulded silicone r u b b e r cup which made a watertight seal against the root without damage to the tissues

Handling was always done in a water-saturated atmosphere under dim green light. During the t r e a t m e n t periods the plants were kept in a vertical position in the dark in water-saturated air at 25 ~: 2 ~ C. The t r e a t m e n t period was 6 h unless otherwise noted. To accommodate growth during the experiment, the seeds were mounted on a n adjustable back-support (Fig. 1) which was raised every 30 min so t h a t the tip of the root was just in the t r e a t m e n t solution supplied at the apex. The area of elongation was determined b y marking the roots at 2-ram intervals with a n organic-based ink. After a period of growth the distance between the marks was determined. All experiments were performed at least twice. Analysis o/ Radio-Label Distribution. The roots were sectioned into 1.17-mm sections (approximated to full millimeters in the figures) with a seetioner constructed of stainless-steel razor blades. I n order to avoid any slight rise of the t r e a t m e n t solution on the surface of the root, sectioning was begun 2-3 m m from the treated area of the root. Basipetal movement was generally followed b y sectioning upwards from the point of treatment. I n order to show the effect of growth on the measurement of transported radioactivity from t r e a t m e n t at the apex sectioning in one experiment (Fig. 4) was begun from a fixed point originally 2 cm above the apex and continued to the apex of the root with the least growth. I n order to obtain synehrony of sections when measuring acropetal movement from 2 cm above the apex, 9 sections were cut from the apex upwards a n d 9 from the zone of t r e a t m e n t downwards (in the figures n u m b e r e d 1-9 and 10-18, respectively). The variable central zone (2-8 ram) between these two sectioned areas (labeled as "region of elongation" in the figures) contained a level of radioactivity similar to the level in adjacent portions of the root. Similar sections from 6-12 separate roots were pooled in polyethylene scintillation vials, and were treated with t w e n t y times their volume of Soluene, a corn-

Auxin Transport in Intact Roots

143

inertial tissue solubilizer (Packard Instrument Co., Downers Grove, Illinois) after which the vials were capped and heated at 55 ~ C for 12 h to complete digestion. The vials were then filled with 10 ml of scintillation fluid (POPOP-PPO in toluene), shaken for an hour and counted in a Packard model 3375 with approximately 30 % effeciency as measured by automatic-external-standard channels ratio. Counting times were 10, 20, or 50 min. Background radiation was 8-10 cpm. In all figures radioactivity is expressed as dpm per single section (i.e., corrected dpm/vial+ no. of sections/vial). Analysis o] Transported Label. For the identification of transported label 2 • 10-SM IAA-aH was used in order to increase radioactivity. Extraction of transported label was done with ethanol, acidified chloroform or peroxide-free ether, with essentially similar results, with e-n-tocopherol-acid succinate (10-a M) added to act as an antioxidant (similar to the method of Mann and Jaworski, 1970). In this experiment 35-80 roots were sectioned below and above the area of application which was originalIy 2 cm up the root, but after 6 h growth closer to 3 cm. The root acropetal to the area of application was extracted whole while the basipetal portion was sectioned into two parts, under and over 2 cm above the source of IAA. In the latter part, all of the root up to the cotyledons was included. Similar sections were homogenized with 20 ml of solvent for 2 rain in a Sorvall omnimixer at 5000 rpm at 2~ C. The homogenate was centrifuged, and the supernatant evaporated nearly to dryness under a stream of nitrogen, and chromatographed immediately in the manner previously described. For measurement of IAA-SH movement from shoot to root tip, small amounts of 10-s M IAA-SH in lanolin/ethanol (10:1) were applied to the shoot tips of 5-dayold plants growing in the dark in vermiculite. After 24 h the lower quarter of the primary roots of the treated plants was extracted according to the procedure outlined above. In this portion of the root there was no lateral root formation.

Results I t was f o u n d t h a t short (less t h a n 2.8 cm) roots co n t ai n ed slightly m o r e label a t a distance f r o m t h e p o i n t of ap p l i cat i o n t h a n did long (longer t h a n 7.8 am) roots, b u t this could be caused b y t h e thickness of t h e short roots r e l a t i v e to t h e long ones. Th er e was also some variability b e t w e e n similar t r e a t m e n t s f r o m different batches of seed in t h e absolute a m o u n t of label which m o v e d , b u t t h e p a t t e r n of t r a n s p o r t was closely replicated. To ensure c o m p a r a b l e results each e x p e r i m e n t was r u n w i t h seedlings from a single b a t c h a n d with roots in t h e range of 5-7 c m (measured f r o m th e a p e x to t h e c o t y l e d o n stalk). A p p l i c a t i o n of I A A - 3 H was carried out in w a t e r or as a lanolin emulsion. Th e p a t t e r n s of t r a n s p o r t were similar in b o t h cases. W a t e r was used as t h e m e d i u m of a p p l i c a t i o n in all e x p e r i m e n t s r e p o r t e d below. Th e zone of m a x i m u m elongation was f o u n d to be localized in an area t h a t e x t e n d e d no f u r t h e r t h a n 6 m m behind t h e root a p e x (Fig. 2). The Influence o] the Point o/Application on I A A Transport. Treatm e n t a t t h e r o o t tip or 2 cm from th e tip showed a similar basipetal m o v e m e n t of label (Fig. 3A, C) w i t h a high level of short-distance t r a n s p o r t r a p i d l y decreasing with distance. A c r o p e t a l t r a n s p o r t f r o m 10"

P. J. Davies and E. K. Mitchell:

144 67

5] E E

4~ 5:

!

I

5

,ol

~5 9

}s 5'

i,

. .........

4- !i~i~)~ ~i~i!~i~i~,! :~ 2" ,

0

t,pEx

,

,

,

,

,

,

2

4

6

8

I0

[2

Originat distance from a p e x - r a m

14

I6

BASE

Fig. 2A and B. The growth increments of various areas of the root measured from the apex at the start of measurement. A After 6 h the maximum growth is between 2 and 6 mm from the root apex. B After 16 h the maximum growth is in the zone originally 0-2 mm from the apex

the a p p l i c a t i o n 2 cm from the a p e x was m u c h less t h a n t h e b a s i p e t a l t r a n s p o r t (Fig. 3 B). The r a d i o a c t i v i t y decreased to a low level b e t w e e n t h e p o i n t of a p p l i c a t i o n a n d t h e r o o t apex, a n d t h e n b e c a m e concent r a t e d b y a f a c t o r of 2-3 times a t t h e apex. R a r e l y , a c o n c e n t r a t i o n of label occurred during b a s i p e t a l m o v e m e n t in the region of l a t e r a l r o o t formation. If IAA-~H was a p p l i e d f u r t h e r u p t h e r o o t t h a n 2 era, longer t r e a t m e n t t i m e s were necessary in order to o b t a i n t h e same n u m b e r of counts p e r apical section. This was p r o b a b l y a result of t h e greater distance involved. The Kinetics o] I A A Transport. B o t h a c r o p e t a l a n d b a s i p e t a l movem e n t of label from solutions of I A A - a t t showed a g r a d u a l increase in the a m o u n t of r a d i o a c t i v i t y a t a n y p o i n t w i t h increasing periods from 1 to 6 h (Fig. 3) a n d a n increasing distance of t r a n s p o r t in t h e b a s i p e t a l direction. Calculation of t h e v e l o c i t y of t r a n s p o r t was difficult a t t h e concentrations used because no d i s t i n c t front was observed. A t t h e t i p b a s i p e t a l m o v e m e n t occurred i n i t i a l l y a t a b o u t 7 m m / h slowing to a b o u t 3 m m / h after 6 h. I f roots t r e a t e d a t t h e a p e x w i t h IAA-3I-I were sect i o n e d d o w n w a r d s from a reference p o i n t a b o v e t h e growing zone, different kinetics of b a s i p e t a l t r a n s p o r t were n o t e d (Fig. 4). A t a given distance below t h e reference point, t h e r a d i o a c t i v i t y p e r section rose but, if t h e section was originally in t h e growing zone, i t would t h e n decline as t h e growing zone m o v e d f u r t h e r from t h e reference point.

Auxin Transport in Intact l~oots 160

145

Apex

Base

A

J40 120 I00 8O

4O 20

g (J q.) co

E c,

4o 20 (3

-(3

Distance from a p e x - mm B a

~

t

I

I

I

I

I

I

2

5

4

5

I I 5 4

I 5

140

12o

m

I

I

region of elongation e n ~

I

I

I

6 7 8 9 Distance from

I

I

I

I

I

I

I

I

I0 II 12[ 15 14 15 16 17 18 apex-- mm

o-~ C

IO0

6O 4O

radioactivity below

20 0

I 6

Distance

I I 7 8

I 9

twice background I I I I t r I I I 1 I I0 II 12 15 14 15 16 17 18 19 20

above t r e a t m e n t z o n e - m m

Fig. 3 A-C. Transport patterns of IAA-3H, as radioactivity corrected to dpm/section, after treatment with IAA-3I-I solution at 2 • I0-91~ at: A the root apex; B 2 cm back from the apex measuring acropeta] movement towards the apex; C 2 em back from the apex measuring basipetal movement away from the apex. (~) after 1 h ; (*) a f t e r 3 h; (o) a f t e r 6 h

The Comparative Transport o/8H20 and laC-Sucrose. I n order to determine whether the transport of IAA was specific, the transport of ~HeO and 14C-sucrose applied in aqueous solution at the mid-root application point was also examined (Fig. 5). Movement of sucrose, applied at

146

P . J . Davies and E. K. Mitchell: 120 - Apex

Base

IOO ._~ 80 ~. 60 '~ 40 2o o

18

16

14

12

I0

8

6

4

2

Disl"once(mm) downwards from point originally 2cm from apex

Fig. 4. Basipetal transport pattern of IAA-aH from the apex obtained by sectioning of the root into mm sections downwards from a fixed point above the apex at the start of treatment rather than upwards from the apex as in Fig. 3. (.) 6 h; (~) 8 h

Apex ioo

Bas ? aoo

A

/

80

I

m

co

o

i

60

-'~ D"

40

~

.olO-

.~-o"

/

tO0

'

o, I

,

_~ I

CD

_

0

C/" I

~

~> 600

q

R-

-~

c

I

~

B

I

~

I

~

I

,o

,'2

,'4 ,;

,:3

o

Distance above apex- mm c"

500

EOOO -~ m

~. 40o

E 0_

~

300 I000 20O

,oo

o

V

~

~

'V' i

6

i

o

Oislance above treatment z o n e - mm

Fig. 5A and B. The movement of water-aI-[ (---) and sucrose-14C ( ) (Sucrose 2 • 10-~M; both 3 X the applied dpm of IAA in Fig. 3) from treatment 2 cm up the root. A acropetal movement; B basipetM movement. Treatment time--6 h

Auxin Transport in Intact Roots I00

147

Apex

Base A

8O 6O

d ~

reg.ion of elongation

4O 20 0 800

~

2

4

6 8 IO 12 14 Distance from apex- mm

16

18

7013

~ 60C Q. 50(3 40(3 30C 20C I0(3

Distance above treatment z o n e - m m

Fig. 6. A acropetal; B basipetal transport of IAA from various concentrations of IAAmH applied 2 cm up the root. (,) 2 • 10-1~ M; (.) 2 • 10-9 M; (o) 2 • 10-SM. Treatment t i m e - - 6 h

2 • 10-TM, a p p r o x i m a t e l y 3 t i m e s t h e d p m of I A A a t 2 • 10-gM, r e s e m b l e d t h e p a t t e r n of m o v e m e n t of I A A . A c r o p e t a l w a t e r m o v e m e n t was different f r o m I A A or sucrose in t h a t t h e a m o u n t decreased s t e a d i l y t o w a r d s t h e apex. B a s i p e t a l m o v e m e n t exceeded a c r o p e t a l m o v e m e n t in a m o u n t , w i t h a similar p a t t e r n for I A A , sucrose a n d water. The Influence o/ I A A Concentration on I A A Transport. All of t h e a b o v e e x p e r i m e n t s were p e r f o r m e d w i t h 2 x 10 -9 M I A A . To d e t e r m i n e t h e influence of t h e e x t e r n a l I A A c o n c e n t r a t i o n , t r e a t m e n t s were carried o u t using 2 x 10 -s, 2 • 10 -9 a n d 2 • 10 -l~ M. Similar t r a n s p o r t p a t t e r n s were o b t a i n e d with these concentrations, which are n e a r t h e r o o t - g r o w t h o p t i m u m , t h o u g h t h e a m o u n t of label t r a n s p o r t e d v a r i e d d i r e c t l y w i t h t h e level of r a d i o a c t i v i t y a p p l i e d (Fig. 6).

148

P . J . D~vies and E. K. Mitchell:

18o_Apex

Base

160 140

g

120

o IOC E

8c 6c

40 20 I 25456789 Distance from apex-mm Distanceabovetreatment.zone-ram of IAA-~H (o) contrasted with the transport of radioactive

Fig. 7. Transport impurities (,) isolated from the IAA-3H where they were present to the extent of about 10% of the radioactivity. Left: acropetal movement; right: basipetal movement = IhA

I00 A

o B 5o

o_ IO00 9 C

50C

t

/

§

2

3

4

5

6

2'

8

9

I0 II

12 13

§

ORIGIN Distancefrom origin-cm FRONT :Fig. 8A-C. gadio&etivity from areas of thin layer chromatograms (on Gelman ITLC- SA run in chloroform: 96 % acetic acid, 95 : 5) used to separate ether extracts of different portions of the roots treated 2 cm from the apex with 10-s M IAA. A Zone of treatment to 2 cm above the treating zone; B beyond 2 cm above the treatment up to the cotyledonary stalk; C between the apex and the zone of treatment. More tissue was extracted in B because of the low levels of radioactivity present

Auxin Transport in Intact Roots

149

i,~,

I0"~ 0

B

IAA

--

'~ FRONT

604020-

E c-~ 0

0 200 =

C

I00"

IAA

§ FRONT

1 }FRONT

0

i 2 5 ~- 5 6 "~ 8 9 l() l'l l~ 15 l~-15 16 ORIGIN

Distorlce from

origin-cm

Fig. 9A-C. Radioactivity from areas of thin layer chromatograms (on Gelman ITLC-SA) used to separate components in acidic chloroform extracts of the apical 5 cm of the primary root of dark-grown bean seedlings where the shoot apex had been treated with 10-6 M IAA-att in lanolin. The extracts were run in 3 solvent systems; A methyl acetate:isopropanol:25% ammonium hydroxide, 45:35:20; ]3 n-butanol : ammonium hydroxide: water, 4:1 : 1 ; C isopropanol: ammonium hydroxide : water, 8 : 1 : 1

The Movement o/ Impurities in the Treating Solution. As t h e p u r i t y of the treating solution was about 90% IAA-SH, the movement of the extracted impurities was examined to ensure that the impurities could not account for the observed results when the IAA solution was applied without further purification. The amount of radioactivity (in dpm/section) which moved from the point of application of 2 • i0 9 M IAA was about double that from a solution of impurities at the same concentration (as calculated by dpm/ml) (Fig. 7). The movement of impurities was also generally slower in the aeropetal direction. Thus the error resulting from the movement of applied impurities is unlikely to exceed 10%.

The Influence o/ Water Supply. I n g e n e r a l t h e p r o v i s i o n of a s e c o n d source of w a t e r (0.3 ml), in a d d i t i o n to t h e t r e a t i n g solution, in t h e a r e a

150

P.J. Davies and E. K. Mitchell:

towards which transport was directed reduced by a few percent the absolute amount of label transported but did not alter the transport pattern.

The Nature oI the Transported Label Following I A A - 3 H Treatment. Extraction and chromatography of the transported labeled material showed that the labeled material involved in acropetal movement from the IAA-aH treated area after 24 h was largely I A A (Fig. 8C) while basipetal movement resulted in a partial breakdown of the compound with no measurable IAA beyond 2 cm above the treatment zone (Fig. 8 A, B). Sufficient label for analysis was only obtained from above 2 cm when 60-80 roots were used for extraction. Movement oI I A A lrom Shoot Tip to Root Tip. Chromatography of extracts from the root tip of label transported from the shoot tip showed that some IAA-att was present in unmetabolized form after transport (Fig. 9). Discussion These experiments avoid the damage to tissues caused by the cutting which is required when sections are used for the study of IAA transport, and thus preserve the integrity of the vascular systems while avoiding the activation of IAA-destroying enzymes (Iversen and Aasheim, 1970). I n addition they show I A A movement following treatment with stimulatory rather than inhibitory IAA concentrations (2 • 10-9 M). Under these conditions there is an acropetal (downward) transport of low concentrations (about 10-1~ M) of IAA, from a point above the root tip down of the root tip, and a short distance basipetal (upward) movement of higher amounts of IAA (giving a concentration of about 10-9 M within 3 mm of the zone of treatment). An increase in the applied IAA concentration to 2 • 10-s M resulted in a proportional increase in the amount of label transported but at 2 • 10-1~ M the amount of label detected was too small to draw any conclusions. The amount of label found acropetal to the point of application decreases exponentially, but beyond a certain point there is a constant low level of I A A which persists as far as the root apex, where a concentration of the label occurs in a manner inconsistent with diffusion. The presence of the label predominantly as IAA at a distance from the point of application indicates the separation of I A A from any degradative enzymes. The aeropetal movement of IAA could thus result from either diffusion or active transport into the phloem where it would be carried acropetally from the current carbohydrate source (the seed cotyledons, which were still present in our experimental plants) to a zone of metabolite utilization, namely, the cell division zone in the root apex. Acrepetal transport of sucrose occurs in the same pattern. IAA applied to the stems of intact plants is also transported to the root where it is

Auxin Transport in Intact Roo~s

151

concentrated in the root apex and lateral root primordia (Haissig, 1970; Morris et al., 1969). If endogenous IAA accumulates in the apices in a similar fashion it m a y be a controlling factor in the cell division at these sites. Much IAA is, however, metabolized in the root (Phillips, 1964a, b; Morris et al., 1969) possibly giving a control mechanism for the I A A levels in the plant. The basipctal movement of label from IAA application, which is similar to that of 31120 and sucrose-iaC, presents an exponential pattern as expected for movement by simple diffusion, possibly assisted by cytoplasmic streaming. Transpiration is not a factor in basipetM movement as the shoot was still enclosed in the seed and a water saturated atmosphere was used throughout the experiments. While acropctal transport consists mostly of IAA, degradation of the IAA can be seen during basipetM movement. Over distances of less than two cm above the top of the treatment zone both IAA and another metabolite(s) were found on chromatography while above 2 cm from the treatment zone no IAA and only the metabolite was present. No attempt was made to identify the metabolite. Acropetal transport shows no distinct front, and movement of label is not detectable until after 3 hours of treatment, possibly indicating that time is required for entry of IAA into the transport stream. Continued uptake gives a small overall increase in counts from 3 to 6 hours. The pattern of basipetal transport is dependent on the site of treatment. When treatment is at the apex, increasing amounts and distances of movement are seen with increasing time, indicative of diffusion. This may, however, be partly because of the growth of the root: if label was fixed in the cells immediately above the solution, label higher up the root would become progressively diluted as the cell increased in volume. When sections were measured from an original fixed distance above the apex, rather than by measurement upwards from the apex at the end of the treatment period, the amount of radioactivity per section decreased during the period from 6 to 8 hours, in the growing zone and beyond, as the apex grew away from the fixed point. The growth of the root must therefore exceed the rate at which IAA is transported basipetally above the growing zone so that the radioactivity becomes diluted as the cells elongate. From a treatment zone higher up the root there is no increase in basipetal transport over 5 mm above the treatment zone at times longer than one hour, though there is an increase below 5 mm. This can be tentatively explained by the metabolism of I A A noted during basipetal transport, if it is assumed that the metabolite is less mobile than I A A and so is retained in the area 5 m m or less above the treatment zone. Degradative enzymes might be induced by the transport of endogenous IAA, for it has been shown

152

P.J. Davies and E. K. Mitchell:

t h a t pretreatment of tissues with IAA enhances the metabolism of subsequent applications (Andreae and Van Yssclstein, 1956). These results from intact plants differ slightly from those obtained using short root sections and a higher IAA concentration (10-~M) (Iversen and Aasheim, 1970; Wilkins and Scott, 1968a; Scott and Wilkins, 1968). Though a flux in both directions was lound in root sections of several species, acropetal movement generally exceeded basipetal. By contrast, our experiments showed greater amounts of label, which at less than 2 cm is 50% IAA, moved in a basipctal direction though for shorter distances. Long distance downward transport from the shoot is, however, clearly the means by which the root receives part, if not all, of its IAA supply (Morris et al., 1969, and present work). If IAA distribution is a primary mechanism of control of directional growth in roots, then there is a requirement t h a t the movement of the starch statoliths :in the root cap, which have been implicated as the geosensitive organelles (Hertel et a/.,1968; Iversen et al., 1968; Larsen, 1969; Juniper et al., 1966; Ouitrakul and Hertcl, 1969; Parups, 1970), must directly or indirectly cause the lateral redistribution of IAA which occurs in horizontal roots (Konings, 1967; Parups, 1970; Hertel et al., 1968). Since our results show that the direction of long distance IAA transport in the root is acropetal, the root cap must have either the ability to redistribute and retransport the I A A for a short distance in the basipctal direction (originally proposed by Pilet, 1951), or export another stimulus to influence auxin distribution during its acropetal movement. With an auxin supply from above, however, there is little evidence for a reversal of the movement, as radioactivity accumulates only in the apex with no clear pattern or front of activity returning up the root even for a short distance. Another possibility is t h a t this auxin from the stem is involved only in maintaining the overall growth of the root and t h a t some IAA, probably in very small amounts, is synthesized in the root caps and is exported basipeta]ly. There are indications but no conclusive proof of IAA synthesis in roots (van Overbeck, 1939a, b). Movement of auxin for short distances from the root apex has been clearly shown when an exogenous auxin supply is provided at the apex. Although the distances are short, they are still quite sufficient to reach the growing zone of the root. Auxin synthesized in the root cap might be in a different locale in the cell from t h a t transported downwards and thus tend to behave as the exogenous auxin supplied at the apex. The lateral assymetry of auxin distribution might result from differential production, lateral migration or selective breakdown of this IAA. Lateral distribution of IAA oxidase has been demonstrated by Konings (1967) in pea roots, though it apparently is not related to the distribution of IAA.

Auxin Transport in Intact Roots

153

While we do not, therefore, k n o w the exact m e a n s of control of the geotrophic response i n roots, s t u d y of the m o v e m e n t of IAA, p a r t i c u l a r l y i n i n t a c t plants, is helping to d e m o n s t r a t e the possibilities which exist,, I n such a system it is also i m p o r t a n t to distinguish between t r a n s p o r t of I A A below, in, or above the growing zone of the root. This work was supported in part by grants from Cornell University and NSF grant GB-19639. We wish to thank Dr. Rite Calve for guiding one of us (E.K.M.) into plant research, and Dr. Mary Helen Goldsmith for constructive criticism.

References Andreae, W. A., van Ysselstein, M. W. H. : Studies on 3-indoleacetic acid metabolism. III. The uptake of 3-indoleacetic acid by pea epicotyls and its conversion to 3-indolylacetyl~spartie acid. Plant Physiol. 31, 235-240 (1956). Audus, L. J. : The mechanism of the perception of gravity by plants. Symp. Soc. exp. Biol. 16, 197-226 (1962). Audus, L. J. : Geotrepism and the modified sine rule: an interpretation based e~ the amyloplast statolith theory. Physiol. Plantarum (Cph.) 17, 737-745 (1964). Cane, A. R., Wilkins, M. B.: Auxin transport in roots. VI. Movement through dilferent zones of Zea roots. J. exp. Biol. 21, 212-218 (1970). Diez, J. L., De la Torre, C., Lepez-Saez, J. F.: Auxin deficiency at the onset of root growth in Allium cepa. Planta (Berl.) 97, 364-366 (1971). Fiedler, H. : Entwicklungs~ und reizphysiologische Untersuchungen an Kulturen isolierter Wurzelspitzen. Z. Bet. 30, 385436 (1936/37). Gibbons, G. S. B., Wilkins, M. B.: Growth inhibitor production by root caps in relation to geotropic responses. Nature (Lend.) 226, 558-559 (1970). Gillespie, B., Thimann, K. V. : Transport and distribution of auxin during tropistic response. I. The lateral migration of auxin in geotropism. Plant Physiol. 38, 214-225 (1963). Haissig, B. E. : Influence of IAA on adventitious root primordia of brittle willow. Planta (Berl.) 95, 27-35 (1970). Hertel, R., De la Fuente, R. K., Leopold, A. C. : Geotropism and the lateral transport of auxins in the corn mutant amylomaize. Planta (Berl.) 88, 204-214 (1968). Hertel, R., Leopold, A. C.: Versuche zur Analyse des Auxintransp~rts in der Koleoptile yon Zea mays L. Planta (Berl.) 59, 535-562 (1963). Hillman, S. K., Phillips, I. D. J. : Transport and metabolism of indoL3-yl-(acetie acid-2-1aC) in pea roots. J. exp. Bet. 21, 959-967 (1970). Iversen, T.-H., Aasheim, T. : Decarboxylation and transport of auxin in segments of sunflower and cabbage roots. Planta (Berl.) 93, 354-362 (1970). Iversen, T.-H., Pedersen, K., Larsen, P.: Movement of amyloplasts in the root cap cells of geotrophically sensitive roots. Physiol. Plantarum (Cph.) 21, 81 t-819 (1968). Juniper, B. E., Groves, S., Sachar, ]3. L., Audus, L. J.: The root cap and the perception of gravity. Nature (Lend.) 299, 93-94 (1966). Kirk, S. C., Jacobs, W. P. : Polar movement of indole-3-acetic acid-laC in roots of Lens and Phaseolus. Plant Physiol. 43, 675-682 (1968). Konings, H. : On the mechanism of the transverse distribution of auxin in geotrophicMly exposed pea roots. Acta bet. neerl. 16, 161-176 (1967). Larsen, P. : The optimum angle of geotropic stimulation and its relation to the starch statolith hypothesis. Physiol. Plantarum (Cph.) 22, 469-488 (1969).

154

P . J . Davies and E. K. Mitchell: Auxin Transport in Intact Roots

Leopold, A. C., Guernsey, F. S.: Auxin polarity in the coleus plant. Bot. Gaz. 115, 147-154 (1953). Lepp, N. W., Peel, A. J. : Patterns of translocation and metabolism of 14C labelled IAA in the phloem of willow. Planta (Berl.) 96, 62-73 (1971). Mann, J . P . , Jaworski, E. G.: Minimizing loss of indoleacetic acid during purification of plant extracts. Planta (Berl.) 92, 285-291 (1970). Morris, D. A., Briant, R. E., Thomson, P. G.: The transport and metabolism of l~C-labelled indole acetic acid in intact pea seedlings. Planta (Berl.) 89, 178-192 (1969). Ouitrakul, R., Hertel, R. : Effect of gravity and centrifugal acceleration on auxin transport in corn coleoptiles. Planta (Berl.) 88, 233-243 (1969). Parups, E. V.: Effect of morphactin on gravimorphism and the uptake, translocation and spatial distribution of indol-3-yl-acetic acid in plant tissues in relation to light and gravity. Physiol. Plantarum (Cph.) 23, 1176-1186 (1970). Phillips, I. D. J. : Root-shoot hormone relations. I. The importance of an aerated root system in the regulation of growth hormone levels in the shoot of Helianthua annuua. Ann. Bot. 28, 17-36 (1964a). Phillips, I. D. J. : Root-shoot hormone relations. IT. Changes in endogenous auxin concentration produced by flooding of the root system in Helianthua annuua. Ann. Bot. 28, 37-45 (1964b). Pilet, P. E. : ]~tude de la circulation des auxines dans la racine de Lens. Bull. Soc. Bot. Suisse 61, 410~24 (1951). Pilet, P. E., Kobr, M., Siegenthaler, P. A. : Proposition d'un test ((Lens) pour le dosage auxinique. Rev. gen. Bot. 67, 573-601 (1960). Scott, T. K., Wilkins, M. B. : Auxin transport in roots. II. Polar flux in Zea maya. Planta (Berl.) 83, 323-334 (1968). Scott, T. K., Wilkins, M. B. : Auxin transport in roots. IV. Effect of light on IAA movement and geotrophic responsiveness in Zea roots. Planta (Berl.) 87, 249-258 (1969). Thimann, K. V. : On the nature of inhibitions caused by auxin. Amer. J. Bot. 24, 407-416 (1937). van Overbeek, J. : Evidence for auxin production in isolated roots growing in vitro. Bot. Gaz. 1Ol, 450-456 (1939a). van Overbeek, J.: Is auxin produced in roots ? Proc. nat. Acad. Sci. (Wash.) 25, 245-248 (1939b). Wilkins, M. B., Scott, T. K. : Auxin transport in roots. Nature (Lond.) 219, 13881389 (1968a). Wilkins, M. B., Scott, T. K. : Auxin transport in roots. III. Dependence of polar flux of IAA in Zea maya upon metabolism. Planta (Berl.) 83, 335-346 (1968b). Yeomans, L. M., Audus, L. J. : Auxin transport in roots--Vicia/aba. Nature (Load.) 204, 559-562 (1964). P. J. Davies Section of Genetics, Development and Physiology Division of Biological Sciences Cornell University Ithaca, N.Y. 14850, U.S.A.