Planta

Planta 144, 463-466 1979)

9 by Springer-Verlag 1979

Accumulation of 14C from Exogenous Labelled Auxin in Lateral Root Primordia of Intact Pea Seedlings (Pisum sativum L.) R o s e m a r y A. R o w n t r e e a n d D . A . M o r r i s Department of Biology, Building44, The University, Southampton SO9 5NH, U.K.

Abstract. W h e n [14C]indol-3yl-acetic acid was a p p l i e d to the a p i c a l b u d o f 5-day old d w a r f p e a seedlings which possessed u n b r a n c h e d p r i m a r y roots, a small a m o u n t o f 14C was t r a n s p o r t e d into the r o o t system at a velocity o f 11-14 m m h - 1 . M o s t o f the 14C which e n t e r e d the p r i m a r y r o o t a c c u m u l a t e d in the y o u n g lateral r o o t p r i m o r d i a , i n c l u d i n g the smallest d e t e c t a b l e (20-30 m m f r o m the p r i m a r y r o o t tip). In older (8-d old) seedlings in w h i c h the p r i m a r y r o o t b o r e w e l l - d e v e l o p e d l a t e r a l roots, 14C also a c c u m u l a t e d in the t e r t i a r y r o o t p r i m o r d i a . In contrast, little a4C was detected in the apical region o f the p r i m a r y r o o t or, in o l d e r plants, in the apices o f the l a t e r a l roots.

l a t e r a l r o o t p r i m o r d i a , often l o n g - b e f o r e these have e n l a r g e d sufficiently to emerge t h r o u g h the cortex ( M o r r i s et al., 1969). In contrast, the levels o f 14C d e t e c t e d in the apical regions o f the p r i m a r y r o o t a n d o f w e l l - d e v e l o p e d lateral r o o t s are generally cons i d e r a b l y lower. H e r e we describe e x p e r i m e n t s to investigate in m o r e detail the d i s t r i b u t i o n in the p r i m a r y r o o t o f y o u n g intact pea seedlings o f a4C f r o m [ 1 - I a C ] I A A a n d [2-14C]IAA a p p l i e d to the s h o o t apical bud.

Materials and Methods

Key words: A u x i n t r a n s p o r t - P i s u m - R o o t i n i t i a t i o n -

Plant Material

Root primordia.

Seeds of P. sativum L. cv Meteor were surface sterilized for 15 min in saturated calcium hypochlorite solution and washed overnight in running tap water. They were planted in 10 cm diameter pots of vermiculite and germinated in a growth room maintained at 20~ C_+ 1~ C on a photoperiod of 16 h from fluorescent lamps (intensity 15 klx). Uniform seedlings were selected for transport experiments when 5 days old. At this stage the epicotyl was ca 20 mm long, and the primary root 70-80 mm long and unbranched. In a few experiments 8-day old seedlings were used in which the uppermost laterals had emerged and were ca 40 mm long. The selected seedlings were washed free of vermiculite and were supported individually with their roots dipping into a boiling tube containing Hoagland's mineral nutrient solution.

Introduction W h e n 14C-labelled indol-3yl-acetic acid ([14C]IAA) is a p p l i e d to the apical b u d o f the intact p e a seedling (Pisum sativum L.) a small p r o p o r t i o n o f the ~4C is t r a n s p o r t e d u n c h a n g e d t h r o u g h the stern to the r o o t in a slow, p o l a r t r a n s p o r t system which is p r o b a bly identical to t h a t in which the l o n g - d i s t a n c e transp o r t o f e n d o g e n o u s a u x i n occurs. T h e characteristics o f this t r a n s p o r t have been d e s c r i b e d by G o l d s m i t h (1977), M o r r i s (1977) a n d M o r r i s a n d T h o m a s (1978), a n d in l i t e r a t u r e cited by these authors. T h e e x p o r t o f s h o o t - p r o d u c e d auxin to the r o o t system m a y p l a y a n i m p o r t a n t role in the r e g u l a t i o n o f r o o t g r o w t h a n d d e v e l o p m e n t a n d in the c o n t r o l o f r o o t - s h o o t i n t e r r e l a t i o n s h i p s . One w a y in which this r e g u l a t i o n m i g h t be effected is t h r o u g h an auxinm e d i a t e d c o n t r o l o f lateral r o o t initiation. F o l l o w i n g the a p p l i c a t i o n o f [1-14C]IAA to the apices o f intact d w a r f p e a seedlings a c o n s i d e r a b l e p r o p o r t i o n o f the t4C entering the r o o t system a c c u m u l a t e s in y o u n g Abbreviations: IAA = indol-3yl-acetic acid

Application and Extraction of [I~C]IAA

[1-14C]IAA and [2-14C]IAA (specific activities 52 mCi mM 1 and 56 mCi mM- 1 respectively) were obtained from the Radiochemical Centre, Amersham, and prepared as described by Morris et al. (1969). 3-gl droplets containing 0.05 or 0.1 gCi 1~C according to the experiment were applied to the apical buds with a calibrated micrometer syringe. To prevent accidental contamination of the epicotyl a ring of Vaseline was applied to it just below the apical bud. Treated plants remained in the growth room for the required transport period which varied from 2 to 24 h. At the end of the required transport period the seedlings were carefully blotted dry and their root and epicotyl lengths measured. Each root was then cut into 5 mm segments and each segment immediately transferred individually to labelled counting vials containing 1 ml 85 percent (v/v) aqueous ethanol and 5 ml Bray's scintillation fluid. The segments were extracted for 48 h in darkness at 0~ C, and the amount of 14C in each segment was determined

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R.A. Rowntree and D.A. Morris: Accumulation of Exogenous Auxin in Root Primordia it was possible to estimate both the amount of 14C and the number of developing lateral root primordia in each 5 mm primary root segment,

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Fig. 1 a and b. Distribution of ~4C along the primary root of typical individual plants at various time intervals after the application of [1-14C]IAA (a) or [2J4C]IAA (b) to the apical buds of 5-day old P. sativum seedlings by liquid scintillation counting with corrections for background and quenching. In all experiments a minimum of three replications was used. Autoradiography

In one experiment the distribution of 14C in the root system was investigated by autoradiography 24 h after the application of [14C]IAA to the apical bud of 5- or 8-day old seedlings. After the required transport period the primary root was severed immediately below the cotyledons and gently blotted dry. The root systems were quickly mounted on stiff card, covered with blotting paper, frozen and dried for 48 h at -25 ~ C in an Edwards EF 2 vacuum freeze-drier. The dried roots were exposed to Kodak Industrex-D X-ray film at room temperature for up to 5 weeks. Distribution of Lateral Root Primordia

Lateral root primordia which had not emerged from the primary root were made visible by staining in acetocarmine and clearing in methyl benzoate using minor modifications to the technique described by Hackett and Stewart (1969). This technique allows extremely small primordia to be observed through the cleared tissues of the parent root, and in the present experiments allowed the detection of primordia to within 25-30 mm of the primary root apex, shortly after their formation. Fresh roots were fixed overnight in formalin :acetic acid :ethanol (10:5:85, v/v) and treated as described by Hackett and Stewart (1969). Root segments which had already been counted were removed from the scintillation vials, thorougly rinsed in ethanol and then treated in H20 2 (30%, w/v) for 20 rain prior to staining with acetocarmine. Thereafter differentiation and clearing were carried out in the normal way. Although this technique produced some distortion of the tissues, satisfactory staining and clearing was obtained and individual root primordia could easily be observed and counted under the microscope. Using this technique

T h e changes with time in the d i s t r i b u t i o n o f 14C in the p r i m a r y r o o t s o f 5-day old seedlings following the a p p l i c a t i o n o f either [ 1 J g C ] I A A or [2-14C]IAA to the s h o o t apex are shown for typical i n d i v i d u a l p l a n t s in F i g u r e l a a n d b . 14C was first detected in the r o o t system ca 2.0 h after labelling a n d for the first 6 h well-defined t r a n s p o r t profiles with clear fronts were o b s e r v e d in the older regions o f the root. Estimates o f the speed o f t r a n s p o r t ( f r o m semi-logar i t h m i c profiles) r a n g e d f r o m 11 to 14 m m h - 1, agreeing closely with previous d e t e r m i n a t i o n s o f the velocity o f a u x i n t r a n s p o r t in the stem o f the intact p e a ( M o r r i s et al., 1969). W i t h longer time intervals the speed with which the t r a n s p o r t fronts a d v a n c e d decreased a n d the profiles themselves b e c a m e m o r e variable with regions o f high a n d low r a d i o a c t i v i t y distribu t e d irregularly a l o n g the p r i m a r y r o o t s (Fig. 1 a a n d b). A n e x a m i n a t i o n o f the d i s t r i b u t i o n o f lateral r o o t p r i m o r d i a a l o n g the p r i m a r y r o o t i n d i c a t e d t h a t f r o m a b o u t 6 h after labelling the irregularities o b s e r v e d in the 14C profiles for i n d i v i d u a l r o o t s c o r r e s p o n d e d closely with v a r i a t i o n s in the density o f lateral r o o t p r i m o r d i a (Fig. 2 a - f ) . T h a t lateral r o o t p r i m o r d i a were sites o f a c c u m u l a t i o n o f labelled m a t e r i a l s transp o r t e d f r o m the s h o o t system was c o n f i r m e d by the d i s t r i b u t i o n o f 14C o b s e r v e d in a u t o r a d i o g r a p h s prep a r e d f r o m 5- a n d 8-day o l d seedlings 24 h after the a p p l i c a t i o n o f [ I ~ C ] I A A to the apical b u d o f the s h o o t (Fig. 3 a - d ) . I n 8-day old p l a n t s with w e l l - d e v e l o p e d lateral r o o t s s o m e a c c u m u l a t i o n o f 1~C was also o b s e r v e d in d e v e l o p i n g t e r t i a r y r o o t p r i m o r d i a (Fig. 3 d). I n n o n e o f the seedlings d i d 14C a c c u m u l a t e in the apex o f the p r i m a r y r o o t or in the apices o f seco n d a r y r o o t s which h a d a l r e a d y u n d e r g o n e some e l o n g a t i o n , even after t r a n s p o r t p e r i o d s o f 24 h.

Discussion T h e results p r e s e n t e d a b o v e c o n f i r m earlier o b s e r v a tions t h a t p a r t o f the e x o g e n o u s auxin a p p l i e d to the s h o o t apex of intact p l a n t s o f several species is t r a n s p o r t e d to the r o o t system (for e x a m p l e see M o r r i s et al., 1969; D a v i e s a n d Mitchell, 1972; Bourb o u l o u x a n d B o n n e m a i n , 1974). Since the t r a n s p o r t o f e x o g e n o u s a u x i n f r o m the s h o o t a p e x p r o b a b l y t a k e s place by a m e c h a n i s m a n d in a p a t h w a y characteristic o f e n d o g e n o u s auxin t r a n s p o r t ( M o r r i s a n d K a d i r , 1972; M o r r i s , 1977), it seems likely t h a t the r o o t system m a y n o r m a l l y receive a c o n s i d e r a b l e p r o -

R.A. Rowntree and D.A. Morris: Accumulation of Exogenous Auxin in Root Primordia

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Fig. 2 a-f. Comparison of profiles of 1~C in the primary roots of 5-day old P. sativum seedlings following the application of [I4C]IAA to the shoot apex and the distribution of lateral root primordia along the primary root axis. a-d: [I-14C]IAA 6, 8, 10 and 24 h transport respectively; e and f: [2-14C]IAA 8 and 24 h transport respectively. Arrows indicate position of cotyledonary node

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Fig. 3a-d. Autoradiographs showing distribution of 14C in the primary roots of 5-day old (a and b) and 8-day old (e and d) seedlings of P. sativum following the application of [14C]IAA to the shoot apex, Transport period 24 h. Reduction: x 1/2

466

R.A. Rowntree and D.A. Morris: Accumulation of Exogenous Auxin in Root Primordia

portion of its auxin from shoot tissues. If the patterns of exogenous auxin transport and accumulation described here reflect those Of endogenous auxin, the developing root primordia would appear to constitute a major sink for shoot auxin. The form in which the labelled material accumulated in the lateral root primordia was not determined but previous experiments with P. sativum and with Vicia faba L. have shown that when exogenous auxin is applied as [1- 14C]IAA the transported labelled compound is unchanged IAA (Morris et al., 1969; Bourbouloux and Bonnemain, 1974). However, within 3 to 4 h of the application of [2-1~C]IAA to the shoot apex of Meteor peas ethanol extracts of the root system contain, in addition to [I~C]IAA, considerable quantities of labelled indol-3yl-acetyl aspartic acid and indol-3yl-aldehyde (Morris et al., 1969). These metabolites were also observed by Bourbouloux and Bonnemain (1974) in roots of V.faba following the application of [3H]IAA to the shoot apex, and a number of radioactive metabolites in addition to IAA itself were found by Davies and Mitchell (1972) when [5-3H]IAA was applied to roots of Phaseolus coccineus L. The observed accumulation of 1~C in the lateral root primordia in the present experiments with [2t4C]IAA therefore probably reflected an accumulation of both IAA and its metabolites. It is not clear from these experiments whether the observed accumulation of a4C in the primordia resulted from an involvement of transported auxin in the regulation of lateral root initiation or early development, or whether it was indicative of a general flow of metabolites towards a sink after lateral root initiation had taken place. Several observations support the latter possibility. Firstly, 14C accumulated in considerable quantities in primordia which must have been well-developed long before the application of [t4C]IAA to the shoot apex. Secondly, the probable accumulation of metabolites of IAA in the primordia would tend to argue in favour of a non-specific ' sink' effect. However, the transport of IAA and its metabolites towards a sink in the developing root primordia would not necessarily exclude the possibility that IAA might also play a regulatory role in the initiation or outgrowth of such primordia in the younger regions of the primary root. The results of several investigations employing either intact plants or cultured isolated roots suggest that there may be a specific requirement for auxin in the early stages of lateral root initiation and/or outgrowth in the pea and other species (Street, 1969). In none of the plants studied in the present experiments did arC accumulate to any significant extent in the primary root apices or in the apices of elongated lateral roots (Fig. l a andb; Fig. 3b andd). This ob-

servation is difficult to explain if the observed patterns of accumulation resulted solely from a sink effect. It is possible that auxin transported from the shoot does not pass the elongating zone of the root - recent work has suggested that in addition to receiving auxin from the shoot, the requirement for auxin for root growth may also be met by the local synthesis of auxin in the root tip (Mitchell and Davies, 1975), and that auxin moving basipetally from the root tip may control elongation of the root (Davies et al., 1976; Ohwaki and Tsurumi, 1976). There is an increasing body of evidence that the strict acropetal polarity of auxin transport observed in sub-apical regions of the root close to the tip (Goldsmith, 1977) may be countered in the vicinity of the tip itself by a strong basipetal transport of auxin, probably in the cortical tissues (Nagao and Ohwaki, 1968 ; Mitchell and Davies, 1975; Ohwaki and Tsurumi, 1976; Davies et al., 1976). We thank Mrs. R.P. Bell and Mr. B.A. Lockyer for assistance with the experiments.

References Bourbouloux, A., Bonnemain, J.-L.: Transport, distribution et m&abolisme de l'auxine dans la racine de Vicia faba L. apr6s application de [14C]AIA ou de [3H]AIA sur le bourgeon. Planta 119, 169-182 (1974) Davies, P.J., Mitchell, E.K. : Transport of indoleacetic acid in intact roots of Phaseolus coccineus. Planta 105, 139-154 (1972) Davies, P.J., Doro, J.A., Tarbox, A.W. : The movement and physiological effect of indoleficetic acid following point application to root tips of Zea mays. Physiol. Plant. 36, 333-337 (1976) Goldsmith, M.H.M.: The polar transport of auxin. Ann. Rev. Plant Physiol. 28, 439-478 (1977) Hackett, C., Stewart, H.E. : A method for determining the position and size of lateral root primordia in the axes of roots without sectioning. Ann. Bot. 33, 679-682 (1969) Mitchell, E.K., Davies, P.J. : Evidence for three different systems of movement of indoleacetic acid in intact roots of Phaseolus coccineus. Physiol. Plant. 33, 290-294 (1975) Morris, D.A. : Transport of exogenous auxin in two-branched pea seedlings (Pisurn sativurn L.). Planta 136, 91-96 (1977) Morris, D.A., Briant, R.E., Thompson, P.G.: The transport and metabolism of 14C-labelled indoleacetic acid in intact pea seedlings. Planta 89, 178-197 (1969) Morris, D.A., Kadir, G.O.: Pathways of auxin transport in the intact pea seedling (Pisum sativum L.). Planta 107, 171-182 (1972) Morris, D.A., Thomas, A.G. : A microautoradiographic study of auxin transport in the stem of intact pea seedlings (Pisum satirum L.). J. Exp. Bot. 29, 147-157 (1978) Nagao, M., Ohwaki, Y.: Auxin transport in the elongation zone of Vicia roots. Bot. Mag. Tokyo 81, 44-45 (1968) Ohwaki, Y., Tsurumi, S. : Auxin transport and growth in intact roots of Vicia faba. Plant Cell Physiol. 17, 1329-1342 (1976) Street, H.E. : Factors influencing the initiation and activity of meristems in roots. In: Root growth, pp. 20 41. Whittington, W.J., ed. London: Butterworths 1969 Received 15 August; accepted 26 October 1978