Planta (Berl.) 83, 276--281 (1968)

Distribution of 14C-Labelled Sucrose in Seedlings of Pisum sativum L. Treated with Indoleacetic Acid and Kinetin D. A. MORELS and ELUNED E. THOMAS Department of Botany, The University of Southampton, England Received August 24, 1968

Summary. The influence of decapitation and treatment with IAA and/or kinetin on the pattern of distribution of 14C-labelled sucrose applied to the third leaf of 14-day old dwarf pea seedlings was investigated. Decapitation resulted in a diversion of the labelled metabolites to the lateral buds, and greatly increased the radioactivity present in the root system indicating that in these seedlings the roots and apex actively competed for translocates from the third leaf. Application of IAA to the decapitated internode prevented the growth of the lateral buds for the duration of the experiment and restored the pattern of distribution of labelled metabolites found in the intact plant. Application of kinetin alone resulted in a marked accumulation of labelled materials in the lateral buds, but when kinetin was applied with IAA metabolites were once again diverted from the lateral buds to the treated internode. Neither of these treatments had any influence on the proportion of the translocated materials which accumulated in the root system when compared with intact plants. The results are discussed in relation to current concepts of 'hormone-directed transport' of nutrients in plants. Introduction I t is well k n o w n t h a t the application of kinetin (6-furfurylaminopurine) to detached leaves (MoTHES, ENOELB~ECHT and KULAJEWA, 1959; MOTHES, 1960; GUNNING and BARKrmY, 1963), and indoleacetic acid (IAA) to decapitated internodes and pedicels (BOOTH et al., 1962 ; SETH and WAEEIHG, 1964, 1967; DAWES and WAEEI~G, 1965) m a y result in transport of metabolites from elsewhere in the plant towards the treated regions. B o t h kinetin and gibberellie acid act synergistically with I A A in bringing a b o u t the accumulation of labelled compounds in decapitated internodes and pedicels of Phaseolus vulgaris L. and Pisum sativum L., b u t SETH and WAEEl~G (1964, 1967) concluded t h a t kinetin and gibberellie acid applied alone did n o t influence the long distance transport of metabolites. The work on bean and pea cited above was limited to a s t u d y of the effects of the applied growth substances on the accumulation of labelled metabolites in the decapitated organs themselves, and the distribution of radioactivity elsewhere in the plant was n o t investigated. As a complement to work already in progress in this d e p a r t m e n t on factors controlling the distribution of assimilates within plants, and on the

Influence if IAA and Kinetin on Patterns of Translocation


influence of n u t r i t i o n a l factors on l a t e r a l b u d growth, the effect of I A A a n d k i n e t i n on t h e general d i s t r i b u t i o n of ~4C-labelled sucrose a p p l i e d to i n t a c t or d e c a p i t a t e d dwarf p e a p l a n t s (P. sativum cv. Meteor) was investigated. Materials and Methods Seedlings used in the experiment were grown in a seedling compost in 4-inch pots in a greenhouse maintained at approximately 20~ Daylight was supplemented by mercury-vapour lamps giving a total photoperiod of 15 hours. Uniform, 14-day old seedlings, bearing a single expanded foliage leaf (leaf 3) were decapitated above the third node to leave a stump 0.5 cm long to which the growth substances were applied in lanolin paste. The lanolin was applied to the freshly cut stump with a calibrated micro-syringe in 0.01 ml amounts containing 10 fxg of each growth substance to be tested. Plants treated in this way were returned to the greenhouse for 24 hours, after which 1.0 ~Ci of sucrose, uniformly labelled with iaC (specific activity 10.6 mCi per raM), was applied to the adaxial surface of one leaflet of leaf 3 in 0.01 ml of solution containing 1.0% Tween 20 and 100 p.p.m, of boron. The solution was confined, by means of a lanolin ring 0.5 cm in diameter, to a circular spot above the mid-vein of the treated leaflet. After allowing the plants to translocate for 24 hours at room temperature (ca. 21 ~C) in continuous light, the treated leaflets were removed and discarded, and the remainder of four plants in each treatment were dissected into the parts indicated in Table 1. The corresponding parts from the four plants in each treatment were bulked and extracted twice for 15 minutes each time in boiling 80% ethanol, and once in cold 80% ethanol. Extracts and washings were combined and made up to a known volume. Aliquots of the extracts were assayed by liquid scintillation counting at an efficiency for l~C of 84.4 %. All results were checked by standard autoradiographic procedures using treated material from the same batch of plants. Results and l)iseussion T h e r e was considerable v a r i a t i o n b e t w e e n t r e a t m e n t s in t h e t o t a l r a d i o a c t i v i t y r e c o v e r e d in t h e e t h a n o l e x t r a c t s (Table 1), p r o b a b l y resulting f r o m v a r i a t i o n in t h e r a t e a t which t h e a p p h e d d r o p l e t s e v a p o r a t e d to d r y n e s s on t h e leaf surface. A f t e r corrections for quenching a n d b a c k g r o u n d , t h e r a d i o a c t i v i t y e x t r a c t e d f r o m each p l a n t p a r t is expressed in T a b l e 1 as a p e r c e n t a g e of t h e t o t a l a c t i v i t y e x t r a c t e d f r o m t h e whole p l a n t , a n d t h e t o t a l a c t i v i t y p e r p l a n t is i n c l u d e d for c o m p a r a t i v e purposes. I n spite of t h e v a r i a t i o n in t o t a l u p t a k e , t h e t r e a t m e n t s h a d a n u m b e r of clear-cut effects on t h e p a t t e r n of distribut i o n of t h e ethanol-soluble r a d i o a c t i v i t y . These effects are d e s c r i b e d a n d discussed below. D e c a p i t a t i o n caused a m a r k e d increase in t h e a c t i v i t y p r e s e n t in t h e i n t e r n o d e s a n d a x i l l a r y buds, p a r t i c u l a r l y in b u d 2 a n d t h e i n t e r n o d e i m m e d i a t e l y a b o v e it. I t is this b u d which grows m o s t r a p i d l y when p l a n t s of this age are d e c a p i t a t e d , a n d which e v e n t u a l l y brings a b o u t t h e correlative i n h i b i t i o n of b u d s 1 a n d 3 (T~IoMAs, 1968). D e c a p i t a t i o n also g r e a t l y i n c r e a s e d t h e p r o p o r t i o n of t h e t o t a l ethanol-soluble



Table 1. Distribution o] ethanol-soluble 14C 24 hours after applying labelled sucrose to third lea/o/14-day old seedlings o/P. sativum. (Radioactivity is expressed as percentage o/total recovered; buds and internodes are numbered/tom the base) Plant organ

Treatments Intact

Apex 53.1 Internode 1 1.0 Internode 2 1.8 Internode 3 2.6 Epicotyl 2.5 Bud 1 0.2 Bud 2 0 Bud 3 0 Cotyledons 0.2 Root system 34.7 Fed leaf (remainder) 4.0 Radioactivity 49,835 recovered (epm per plant)




. 2.0 6.7 43.5 4.8 0.9 0 0 0.7 37.6 3.8 66,781

5.1 8.9 2.9 3.4 4.2 7.2 2.6 0.3 61.6 3.9 84,014



IAA and Kinetin

7.1 6.8 7.3 5.7 11.9 15.9 3.1 0.5 36.2 5.5 32,379

2.2 10.3 45.3 5.0 0 0.1 0.1 0.6 30.5 5.9 54,640


activity extracted which was present in the root system, indicating that competition for assimilates from the third leaf occurred between the root system and the apex of intact plants at this stage of development. ttUSAIN and LINCK (1966) have demonstrated a similar diversion of labelled phosphate towards the root system of Alaska pea plants shortly after decapitation. Treatment with I A A reduced the activity present in the lateral buds to the very low level recorded in the intact plants, and at the same time resulted in a very pronounced accumulation of radioactive materials in the IAA-trcated internode. The proportion of the total activity accumulated by the root system was restored to the level found in intact plants. Parallel experiments on plants of the same age grown under identical conditions indicated that 10 ~g of I A A applied to the cut stump of internode 4 could replace the apex in maintaining the inhibition of axillary buds for at least 3 days, after which the growth rate of the laterals slowly increased to the rate observed in decapitated control plants. Application of 10 txg of kinetin did not influence the growth of the lateral buds when compared to their growth on decapitated plants treated with lanolin only, but resulted in a marked accumulation of radioactive compounds in the lateral buds, particularly buds 1 and 2. I t did not influence the proportion of the total ethanol-soluble activity which accumulated in the root system when compared with intact

Influence of IAA and Kinetin on Patterns of Translocation


plants. Simultaneous application of 10 ~g each of IAA and kinetin suppressed the growth of lateral buds for 5 to 7 days, and reduced the level of radioactivity in the buds to t h a t found in intact plants and in decapitated plants treated with I A A alone. At the same time this treatment resulted in the accumulation of soluble labelled materials in the treated and adjacent internodes. The proportion of the total soluble activity present in internode 2 and 3 of these plants was slightly greater than t h a t found in plants treated with I A A alone: but less than might have been expected from the results of previous experiments (SETH and WA~v,I~CG, 1964, 1967) and from the results of parallel autoradiographic studies (which in all other respects confirmed our findings). I t is possible that, as a result of the accelerated growth of the treated internodes, a greater proportion of the soluble material translocated to the internodes of these plants was converted to ethanol-insoluble material not detected by the extraction procedure employed. The parallel data on growth of the third internode over the same time interval (see Table 2) indicate a large synergistic effect of IAA and kinetin on the rate of increase in dry weight of this internode. Table 2. Growth o] the third internode o/P. sativum during the 48 hours ]ollowing decapitation and treatment with I A A and~or ]cinetin. Initial length o/internode stump was 5.0 ram; initial dry weight was 1.58 ~ 0.080 rag. Values given are means of 5 intcrnodes


Final dry weight (nag)

Mean increase in dry weight (nag)

Intact Decapitated IAA Kinetin IAA and kinetin S.E. L.S.D. ( P < 0.001)

2.18 1.70 3.14 1.71 3.88 0.205 0.72

0.60 0.12 1.56 0.13 2.30 ---

These results show t h a t whilst kinetin alone appears to have no effect on the mobilization of assimilates towards the decapitated internodes to which it is applied, it can enhance the transport of nutrients towards lateral buds released from dominance by removal of the apex. This suggests t h a t kinetin itself is translocated from the decapitated internode to sites in the lateral buds. Basipetal transport of benzyladenine applied to the cut ends of petioles of Phaseolus vulgaris L. has been demonstrated by BLACK and OSBOrnE (1965), but the extent to which kinetin itself is translocated in the basipetal direction is not clear, and it is generally believed t h a t it is relatively immobile in the



intact plant (cf. THrMANN, 1963; SACHS and TItIMANN, 1964). However, the distances over which kinetin would have to move to reach the lateral buds in the present experiment never exceeded 2.0 cm, and it is possible t h a t in the time between application of the growth substances and extraction of the labelled metabolites (48 hours), sufficient kinetin m a y have reached the buds b y diffusion to bring about the observed effects on nutrient mobilization. The mechanism b y which kinetin acted to increase the accumulation of labelled metabolites in the lateral buds is not known. Recent work by S~TH and W A ~ I ~ G (1967) suggests t h a t all three major classes of plant growth substances (auxins, gibberellins and cytokinins) m a y be involved in the phenomenon of 'hormone-directed transport', and m a y act synergistically to control the metabolic activity of sinks for translocated metabolites. Their results suggest t h a t auxin is essential for directed transport to occur and t h a t part of the kinetin effect m a y be to increase the uptake or translocation of auxin (DAvIEs, S ~ and WAI~rn~G, 1966). However, cytokinins are known to be involved in the control of m a n y cellular processes including cell division (e.g. see GUTTMAN, 1956; DAS, PATAU and SKOOG, 1956), and in subsequent experiments (to be reported in full elsewhere) 10 ~g of kinetin applied to the decapitated intcrnode was found to markedly stimulate mitosis in the apical mcristem of the lateral buds. Mitotic indices 24 hours after treatment were significantly higher in the buds of kinetin-treated plants than in the buds of decapitated controls treated with lanolin alone, and a high frequency of division was maintained for at least 72 hours after treatment. I n the buds of plants receiving a single dose of 1O ~tg of IAA, the occurrence of mitotic figures remained at a consistently low level for at least 48 hours. These results appear to confirm t h a t kinetin was translocated to the lateral buds and it seems possible t h a t the observed effects of kinetin on the polarization of metabolites towards these buds m a y have been associated with their enhanced mitotic activity. The apparent failure of the lateral buds on kinetintreated plants to elongate more than buds on decapitated controls, in spite of the greater frequency of cell division and their greater effect on the polarization of nutrients, m a y have been the result of a limited supply of endogenous auxins. SACHS and TItlMANN (1964, 1967) have shown t h a t inhibited lateral buds on intact pea plants m a y be induced to elongate b y direct application of kinetin, although buds released in this way do not elongate as rapidly as buds on decapitated controls unless I A A is also applied to them, indicating a requirement for both substances in normal bud growth. We are grateful to Professor S. H. CIr for his interest in this work and for helpful comments on the text, and to Mr. 1). R. MAnSfieLD for technical assistance.

Influence of IAA and Kinetin on Patterns of Translocation


References BLACK, M. K., and O. J. OSBORNE: Polarity of transport of benzyladenine, adenine and indote-3-acetie acid in petiole segments of Phaseolus vulgaris. Plant Physiol. 40, 676--680 (1965). BOOTH, A., J. MOORBY, C. R. DAVIES, H. JONES, and P. F. WA~ING: Effects of indolyl-3-acetic acid on the movement of nutrients within plants. Nature (LoAd.) 194, 204--205 (1962). DAs, N. K., K. PATAV, and F. SKooa: Initiation of mitosis and cell division by kinetin and indoleacetie acid in excised tobacco pith tissue. Physiol. Plant. (Kbh.) 9, 640--646 (1956). DAVIES, C. R., A. K. SETH, and P. F. WAREING: Auxin and kinetin interaction in apical dominance. Science 151, 468--469 (1966). - - , and P. F. WAREmG: Auxin directed transport of radiophosphorus in stems. Planta (Berl.) 65, 139--156 (1965). GuNninG, B. E. S., and W. K. BAR~LE~r: Kinin-induced directed transport and senescence in detached oat leaves. Nature (LoAd.) 199, 262--265 (1963). GUTTMA~, R. : Effects of kinetin on cell division with special reference to initiation and duration of mitosis. Chromosoma (Berl.) 8, 341--350 (1956). HvsAIN, S. M., and A. J. LI~OK: Relationship of apical dominance to the nutrient accumulation pattern in Pisum sativum var. Alaska. Physiol. Plant. (Kbh.) 19, 992--1010 (1966). MOT~ES, K.: t)ber das Altern der Blgtter und die M6glichkeit ihrer Wiederverjiingung. Naturwissensehaften 47, 337--350 (1960). - - L. E~GELRRECJ~Tu. O. KVL~JEWA: Uber die Wirkung des Kinetins auf Stickstoffverteilung und Eiweil3synthese in isolierten Blgttern. Flora (Jena) 147, 445--464 (1959). SAo~s, T., and K. V. T i ~ a N s r : Release of lateral buds from apical dominance. Nature (LoAd.) 291, 939--940 (1964). - - - - The role of auxins and cytokinins in the release of buds from dominance. Amer. J. Bot. 54, 136--144 (1967). SET:4, A . K . , C. R. DAVIES, and P. F. WA~EING: Auxin effects on the mobility of kinetin in the plant. Science 151, 587--588 (1966). - - , and P. F. WAREING: Interaction between auxin, gibberellins and kinetin in hormone directed transport. Life Sci. 3, 1483--1486 (1964). - - - - Hormone-directed transport of metabolites and its possible role in plant senescence. J. exp. Bot. 18, 65--77 (1967). TmNANN, K. V. : Plant growth substances; past, present and future. Ann. Rev. Plant Physiol. 14, 1--18 (1963). T~osIAs, E. E. : The influence of nutritional factors on apical dominance in dwarf peas (Pisum sativum L. cv. 5{eteor). B. Sc. Diss., Univ. Southampton (1968). Dr. D. A. MoaRis and Miss E. E. T~OMAS Department of Botany, The University Southampton, S09 5NIt (England)

Distribution of (14)C-labelled sucrose in seedlings of Pisum sativum L. Treated with indoleacetic acid and kinetin.

The influence of decapitation and treatment with IAA and/or kinetin on the pattern of distribution of (14)C-labelled sucrose applied to the third leaf...
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