Planta 9 by Springer-Verlag 1978

Planta 142, 211-219 (1978)

Components of Auxin Transport in Stem Segments of Pisum sativum L. 1 Peter J. Davies and Philip H. Rubery Section of Botany, Genetics and Development, Division of Biological Sciences, Cornell University, Ithaca, NY 14853, USA, and Department of Biochemistry, Tennis Court Road, University of Cambridge, Cambridge CB2 1QW, U.K.

Abstract. 1. The uptake of indol-3-yl acetic acid ([1-

1~ C]IAA, 0 - 2 . 0 laM) into light-grown pea stem segments was measured under various conditions to investigate the extent to which mechanisms of auxin transport in crown gall suspension culture cells (Rubery and Sheldrake, Planta 118, 101 121, 1974) are also found in a tissue capable of polar auxin transp o r t . - 2 . I A A uptake increased as the external p H was lowered. I A A uptake was less than that of benzoic acid (BA), naphthylacetic acid (NAA) or 2,4 dichlorophenoxyacetic acid (2,4D) under equivalent conditions. TIBA enhanced net I A A uptake through inhibition of efflux, and to a lesser extent, also increased uptake of N A A and 2,4D while it had no effect on BA uptake. - 3 . Both D N P and, at higher concentrations, BA, reduced I A A uptake probably because of a reduction of cytoplasmic pH. However, low concentrations of both BA and D N P caused a slight enhancement of I A A net uptake, possibly through a reduction of carrier-mediated I A A efflux. In the presence of TIBA, the inhibitory effects of D N P and BA were more severe and there was no enhancement of uptake at low c o n c e n t r a t i o n s . - 4 . Non-radioactive I A A (10 I~M) reduced uptake of labelled I A A but further increases in concentration up to 1.0 m M produced first an inhibition ( 0 - 1 0 min) of labelled I A A uptake, followed by a stimulation at later times. Non-radioactive 2,4 D decreased, but was not observed to stimulate, uptake of labelled IAA. In the presence of T I B A labelled I A A uptake was inhibited by non-radioactive I A A 1 This work was performed in Cambridge during the tenure of a sabbatical leave by P.J.D. Supported by a grant for supplies from the American Philosophical Society to P.J.D. Abbreviations: IAA=indol-3-yl acetic acid; BA=benzoic acid; NAA=l-naphthylacetic acid; 2,4-D=2,4-dichlorophenoxyacetic acid; TIBA=2,3,5-triiodobenzoic acid; DNP=2,4-dinitrophenol; PCMB =p-chloromercuribenzoic acid; PCMBS =p-chloromercuribenzene sulphonic acid

regardless of its c o n c e n t r a t i o n . - 5 . Sulphydryl reagents P C M B and PCMBS promoted or inhibited I A A uptake depending, respectively, on whether they penetrated or were excluded from the cells. The penetrant PCMB also reduced the promotion of labelled I A A uptake by T I B A or by high concentrations of added non-labelled I A A . - 6 . Our findings are interpreted as being consistent with the diffusive entry of unionised I A A into cells together with some carrier-mediated uptake. Auxin efflux from the cells also appears to have a carrier-mediated contribution, at least part of which is inhibited by TIBA, and which has a capacity at least as great as that of the uptake carrier. The data indicate that pea stem segments contain cells whose mechanisms of trans-membrane auxin transport fit the model of polar auxin transport proposed from experiments with crown gall suspension cells, although differences, particularly of carrier specificity, are apparent between the two systems. Key words: Auxin transport

Auxin uptake -- Pisum.

Introduction

Recent investigations of auxin uptake by single cells of Partkenocissus tricuspidata (Virginia creeper) crown gall suspension culture cells (Rubery and Sheldrake, 1973, 1974; Rubery, 1977, 1978) and by cells of the alga Hydrodictyon @icanum (Raven, 1975) have led to a hypothesis to account for the polar auxin transport of auxin (IAA) through stems, coleoptiles and similar plant structures. The features of this hypothesis have been extensively explained and discussed by Goldsmith (1977). Basically, the scheme is as follows : diffusion of unionised I A A molecules (IAA ~ proceeds across the plasma membrane in response to a downhill concentration gradient of

0032-0935/78/0211/$01.80

212

P.J. Davies and P.H. Rubery: Auxin Transport in Stem Segments

I A A ~ P r o v i d e d t h a t the p H o f the c y t o p l a s m is k e p t m o r e alkaline t h a n the external p H , the p a r t i a l ionisation o f the entering I A A ~ to I A A - a n d H § will m a i n tain the I A A ~ g r a d i e n t while the c y t o p l a s m i c concent r a t i o n o f I A A - will rise a b o v e its external c o n c e n t r a tion, since the m e m b r a n e is m u c h less p e r m e a b l e to I A A - t h a n to I A A ~ (Raven, 1975). A u x i n will therefore a c c u m u l a t e within the cells in response to the m e t a b o l i c a l l y m a i n t a i n e d p H g r a d i e n t across the p l a s m a m e m b r a n e . It is h y p o t h e s i s e d that the ratio o f the p e r m e a b i l i t y coefficient o f I A A to t h a t o f I A A ~ is greater at the b a s a l ends o f the cells t h a n at their apical ends. Exit o f I A A will therefore occur preferentially f r o m the b a s a l ends o f the cells by movement down a gradient of electrochemical potential b r o u g h t a b o u t by b o t h the c o n c e n t r a t i o n g r a d i e n t o f I A A - a n d the negative m e m b r a n e p o t e n t i a l . This cellular p o l a r i t y p e r m i t s the c y t o p l a s m i c c o n c e n t r a tion o f I A A to be in e q u i l i b r i u m with a higher external c o n c e n t r a t i o n o f I A A at the b a s a l end o f the cell t h a n at the apical end, a n d can a c c o u n t for the p o l a r ity o f the tissue with respect to auxin t r a n s p o r t ( G o l d s m i t h , 1977). M e t a b o l i c energy is n e e d e d to m a i n t a i n the p H a n d electrical g r a d i e n t across the p l a s m a m e m b r a n e , r a t h e r t h a n being directly a p p l i e d to the " a c t i v e s e c r e t i o n " o f auxin. R u b e r y a n d Sheld r a k e have suggested t h a t an a s y m m e t r i c d i s t r i b u t i o n o f I A A - carriers c o u l d be r e s p o n s i b l e for the p o s t u lated e n h a n c e d p e r m e a b i l i t y o f the cells at their b a s a l ends. U p t a k e carriers m a y also exist for auxin (Rubery a n d Sheldrake, 1974; R u b e r y , 1977, 1978), b u t they are n o t essential to the p r o p o s e d p o l a r t r a n s p o r t m e c h a n i s m o u t l i n e d above. Since the original p r o p o s a l o f this h y p o t h e s i s m u c h w o r k has been d o n e b y R u b e r y (1977, 1978) on the specificity a n d characteristics o f the auxin upt a k e in crown gall suspension culture cells in r e l a t i o n to the t r a n s p o r t model. The p r o b l e m remains, h o w ever, t h a t s u s p e n s i o n c u l t u r e d c r o w n gall cells are n o m i n a l l y s y m m e t r i c a l a n d w o u l d n o t therefore be expected to retain the identical characteristics o f the cells in a tissue actually c a p a b l e o f p o l a r auxin transport. It was therefore d e c i d e d to d e t e r m i n e w h e t h e r those features f o u n d in c r o w n gall suspension culture cells, a n d h y p o t h e s i s e d to be essential for the chemiosm o t i c p o l a r diffusion o f auxin, c o u l d be f o u n d in a tissue that w o u l d be involved in the p o l a r t r a n s p o r t o f auxin, n a m e l y stem segments f r o m l i g h t - g r o w n pea plants (Scott a n d Briggs, 1960; M o r r i s , 1977). In this p a p e r we investigate the u p t a k e o f I A A by light-grown p e a stem sections u n d e r v a r i o u s m o d i fying conditions. The e x p e r i m e n t s d o n o t involve p o lar t r a n s p o r t p e r se, b u t r a t h e r m e a s u r e the u p t a k e characteristics o f p e a stem cells for c o m p a r i s o n with p r e v i o u s results o b t a i n e d with c r o w n gall cells. It m u s t

be e m p h a s i s e d that, because o f the m u l t i c e l l u l a r nature o f the p e a stem segments, there is always the question c o n c e r n i n g the cellular l o c a t i o n o f a n y I A A t a k e n up. This q u e s t i o n c a n n o t at p r e s e n t be answered. H o w e v e r , if the results o b t a i n e d are viewed a g a i n s t the b a c k g r o u n d o f i n f o r m a t i o n f r o m the c r o w n gall cells, we should be able to d e t e r m i n e w h e t h e r the stem cells possess at least some o f the characteristics suggested by the c h e m i o s m o t i c t h e o r y o f p o l a r auxin diffusion.

Materials and Methods Peas (Pisum sativum L. cv 'Alaska') were grown in vermiculite in 10 cm plastic pots in a glasshouse. Under the high light intensities of summer a cylinder of brown paper was placed above the rim of the pot to induce a slight degree of etiolation so that a sufficient length of stem tissue was produced and the plants looked morphologically similar at different seasons. Experiments repeated at different seasons gave similar results. Five mm segments (fresh weight=ca. 10 mg) (or other lengths as noted) were cut from the 3rd expanding internode of 10-day old plants. The segments were pooled in ice cold water until use (about 30 rain). The segments were placed in boiling tubes (15 randomized segments per tube) containing 5 ml of the treating solution containing 0.02 M-citrate/ phosphate or 0.02 M-Na/K phosphate buffer (the composition of the buffers made no difference) of given pH containing 2% sucrose, 10- v M (or as stated) [1-14C]IAAammonium salt (57 mCi/ mmol), or [1-14C]BA (60 mCi/mmol), or [1-14C]2,4-D (54 mCi/ mmol), or [I-I~C]NAA (54mCi/mmol) (Radiochemical Centre, Amersham, U.K.) prepared as in Rubery (1977), and various additives which will be described for each experiment. The tubes were shaken in a water bath in diffuse light at 22~ C. At the designated times a tube was removed and the contents poured through two layers of Whatman No. 1 filter paper in a Buchner funnel held under vacuum so that the drained segments were retained. After 30 s the segments were carefully picked up individually by forceps and placed in a vial containing 5 ml of aqueous scintillation fluid (toluene: Triton X-100 2:1 by volume, with 5 g/1 PPO and 0.1 g/l POPOP, 85% by volume, plus 15% water by volume so that a clear single phase cocktail resulted after the water had been withdrawn from the segments). The segments were left overnight in scintillation fluid, then counted in a Phillips liquid scintillation spectrometer with efficiency determined by automatic externalstandard channels-ratio. All counts were corrected for surface radioactivity and quenching. Tests to determine the efficiency of this method of extraction showed that extraction of the radioactivity from the tissue by the scintillation fluid was essentially complete. In many experiments, replication was precluded because of the number of treatments it was felt desirable to include for comparative purposes. In experiments with smaller numbers of treatments, replication was carried out and the standard error of each treatment seldom exceeded 5% of the total uptake.

Results

Effect of Peeling and Aeration of Pea Stem Segments In m o s t o f the f o l l o w i n g e x p e r i m e n t s the segments were n o t peeled o r aerated. In o r d e r to d e t e r m i n e

P.J. Davies and P.H. Rubery: Auxin Transport in Stem Segments whether this affected auxin uptake, the behaviour of unpeeled unaerated segments was compared with that of peeled or aerated segments. Peeling or aeration were both shown to enhance uptake slightly, but the relative effect of other treatments imposed on these conditions remained constant. Most of the uptake in nonpeeled segments is via the cut ends (see below). Thus uptake into nonpeeled segments probably has a greater component affected by polar transport, which is desirable in these experiments. Peeling enables direct entry into more cells. Over short time periods most segments floated and thus obtained adequate aeration. With longer incubation times segments tended to sink, but were kept circulating to the surface of the solution by shaking.

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Fig. 1. The mean IAA content of different 2 mm zones of a 1 cm

Twenty 1 cm segments were marked at the upper end and shaken in 10 ml 4 • 10 6 M [a4C]IAA solution p H 6.0 in a 100 ml beaker at 2 2 ~ and removed individually at intervals over 3 h. After draining, the segments were cut into 2 m m pieces with a multi razor-blade cutter starting from each end. The central section varied in length due to growth during the incubation. Each dissected section was counted individually. Uptake occurs mainly from the cut ends. Continued uptake is principally the result of I A A accumulating in the end cells and moving from the apical end into the cells further down the tissue (Fig. 1).

Uptake of lAA Compared with that of BA, NAA and 2,4-D Uptake of IAA into pea stem segments from a 0.1 10 gM solution, as has been noted previously (Davies, 1973a, b), is biphasic, with an initial phase of a 1/2 time of about 10 min and completed in 30 rain. An uptake then continues at a constant rate for m a n y hours (see Figs. 2, 10 and 11). The first phase probably represents initial uptake into the cells at the cut surface plus a small component of uptake into the end cell walls. The latter is only a small fraction, as even the early uptake (2 min) is both largely temperature dependent (P.J. Davies, unpublished results) and enhanced by T I B A treatment (see below). The continued uptake probably represents the compounded result of 3 processes: 1) transport to cells further down the tissue, 2) internal compartmentation in areas of the cell such as the vacuole, and 3) metabolic conversions, although over the first hour this is almost negligible (Davies, 1972). Uptake of I A A in 1 h was found to be less than uptake from equimolar solutions of BA, N A A or 2,4-D (Fig. 3). BA is

green pea segment at various times after placing in a solution of [1-14C]IAA (2 pM, pH 6.0). Individual 2 mm segments were cut from i cm segments at 2 min intervals up to 10 rain, 5 min intervals up to 30 min and 10 min intervals thereafter

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Components of auxin transport in stem segments of Pisum sativum L.

1. The uptake of indol-3-yl acetic acid ([1-(14)C]IAA, 0-2.0 μM) into light-grown pea stem segments was measured under various conditions to investiga...
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