Planta
Planta (1989)178:242-248
9 Springer-Verlag 1989
Applicability of the chemiosmotic polar diffusion theory to the transport of indol-3yl-acetic acid in the intact pea (Pisum sativum L.) Claire F. Johnson* and D.A. Morris** Department of Biology, Building 44, The University, Southampton S09 5NH, U K
Abstract. The transport of exogenous indol-3ylacetic acid (IAA) from the apical tissues of intact, light-grown pea (Pisum sativum L. cv. Alderman) shoots exhibited properties identical to those associated with polar transport in isolated shoot segments. Transport in the stem of apically applied [1-14C] - or [5-3H]IAA occurred at velocities (approx. 8-15 ram. h - ~) characteristic of polar transport. Following pulse-labelling, IAA drained from distal tissues after passage of a pulse and the rate characteristics of a pulse were not affected by chases of unlabelled IAA. However, transport of [I-14C]IAA was inhibited through a localised region of the stem pretreated with a high concentration of unlabelled I A A or with the synthetic auxins 1-napthaleneacetic acid and 2,4-dichlorophenoxyacetic acid, and label accumulated in more distal tissues. Transport of [1-14C]IAA was also completely prevented through regions of the intact stem treated with N-l-naphthylphthalamic acid (NPA) and 2,3,5-triiodobenzoic acid. Export of IAA from the apical bud into the stem increased with total concentration of IAA applied (labelled+unlabelled) but approached saturation at high concentrations (834 mmol.m-3). Transport velocity increased with concentration up to 83 mmol. m-3 IAA but fell again with further increase in concentration. Stem segments (2 mm) cut from intact plants transporting apically applied [1-~4C]IAA effluxed 93% of their initial radioactivity into buffer (pH 7.0) in 90 min. The half-time for efflux increased from 32.5 to 103.9 min when 3 mmol.m -3 Institute of Horticultural Research, East Malling, Kent ME19 6BJ, U K ** To whom correspondence should be addressed * Present address:
A b b r e v i a t i o n s : IAA = indol-3yl-acetic acid ; 2,4-D = 2,4-dichlor-
ophenoxyacetic acid; N A A = l-naphthaleneacetic acid; N P A = N-l-naphthylphthalamic acid; TIBA = 2,3,5-triiodobenzoic acid
NPA was included in the efflux medium. Long (30 ram) stem sections cut from immediately below an apical bud 3.0 h after the apical application of [1-~4C]IAA effluxed IAA when their basal ends, but not their apical ends, were immersed in buffer (pH 7.0). Addition of 3 mmol.m -3 NPA to the external medium completely prevented this basal efflux. These results support the view that the slow long-distance transport of I A A from the intact shoot apex occurs by polar cell-to-cell transport and that it is mediated by the components of I A A transmembrane transport predicted by the chemiosmotic polar diffusion theory. Key words: Auxin carriers - Auxin transport Chemiosmotic theory (auxin transport) - Pisum (auxin transport) - Transport, polar (auxin)
Introduction
The problem addressed here is whether or not the properties of the slow, root-directed transport of indol-3yl-acetic acid (IAA) from the apex of the intact shoot (Morris and Johnson 1985) are consistent with the "chemiosmotic polar diffusion theory" of polar auxin transport (Rubery and Sheldrake 1974; Raven 1975; see Goldsmith 1977). Polar transport of IAA in isolated tissue segments involves a pH- and membrane-potential-dependent cell-to-cell movement of the compound (reviewed by Goldsmith 1977; Rubery 1980, 1986, 1987). Three component processes cooperate in the transmembrane transport of IAA. These are: (i) diffusion of the relatively lipophilic undissociated molecule (IAAH) down its own electro-chemical gradient (Rubery and Sheldrake 1974; Raven 1975); (ii) saturable uptake of the anion (IAA-) on a specific electro-impelled porter which probably co-
C.F. Johnson and D.A. Morris : Polar diffussion of IAA
transports IAA- with two protons (Hertel 1983, 1986; Lomax et al. 1985; Benning 1986; Sabater and Rubery 1987); and (iii) saturable efflux of IAA via a second specific plasma-membrane porter which is probably also electro-impelled (Sabater and Rubery 1987). The efflux porter is competitively inhibited by auxins and specifically and non-competitively inhibited by 2,3,5-triiodobenzoic acid (TIBA), phytotropins (including N-l-naphthylphthalamic acid, NPA), and morphactins. Differences in the relative permeability to IAAH and IAA- at each end of a cell can account for a net polar flux of IAA through a file of cells and may explain polar transport of IAA through tissue (Rubery and Sheldrake 1974; Raven 1975; Goldsmith 1977; Rubery 1980; Goldsmith and Goldsmith 1981). Although potentially this differential permeability could be achieved in several ways (Milborrow and Rubery 1985), available evidence (reviewed by Rubery 1987) indicates that it results from asymmetry in efflux-carrier distribution. In intact dicotyledonous shoots the slow rootdirected transport of exogenous IAA from young apical leaves exhibits several characteristics typically associated with polar transport in isolated shoot segments (reviewed by Morris and Johnson 1985; Morris 1988). However, there is no direct evidence that this transport is a true polar transport and, apart from its apparent specificity (Morris and Thomas 1974) and sensitivity to specific auxin-efflux inhibitors (Morris et al. 1973), there is little evidence that it is mediated by the carrier systems involved in polar IAA transport through cells. Material and methods Plant material. Seedlings of Pisum sativum L. cv. Alderman were grown as described by Moris and Johnson (1987). Unless otherwise indicated in Results, long-distance transport experiments with intact plants were carried out on uniform 13- to 15-d-old seedlings selected when leaf 5 was near full expansion and leaf 6 was partially unfolded. Chemicals. Radioactive compounds ([I-14C]IAA, 2.26 GBq. mmol 1; [5_3H]IAA, 681 GBq.mmo1-1) were obtained from Amersham International, Amersham, Bucks., UK. Stock solutions in ethanol or water were stored at - 25 ~ C. Radiochemical purity was checked before use by ascending paper chromatography in isopropanol: ammonia: H 2 0 (I 0 : 1 : 1, by vol.). Sources of unlabelled growth regulators were: IAA, TIBA, 2,4-dichlorophenoxyacetic acid (2,4-D) and 1-naphthaleneacetic acid (NAA) - Sigma Chemical Co. Poole, Dorsek, U K ; NPA gifts from Dr. P.H. Rubery, Department of Biochemistry, University of Cambridge, U K and Dr. G.F. Katekar, C.S.I.R.O., Canberra, Australia. Stock solutions of these were stored refridgerated in darkness. General reagents used were all Analar grade from BDH Chemicals, Poole, Dorset, UK. In some experiments, transport inhibitors (TIBA and NPA)
243 or unlabelled auxins (IAA, N A A and 2,4-D) were applied to stems as suspensions in hydrous lanolin (approx. 25% H20). Measured volumes of the suspensions were applied to defined regions of the stem using 2.0-cm 3 disposable plastic syringes. Stems were first lightly abraded with fine carborundum powder, rinsed with distilled water and blotted dry. Control plants received lanolin only.
Transport experiments. The long-distance transport of [1-14C] or [5-3H]IAA from the apical buds of intact plants was investigated following the technique of Morris et al. (1969). In some experiments the plants remained fully intact throughout the transport period. In "pulse" experiments, transport into the stem was allowed to continue for a specified time (usually 2.0 h) and the labelled apical tissues were then excised immediately below node 6. To prevent dehydration the cut end of the stem was sealed with a little hydrous lanolin and transport of label which had entered the stem prior to decapitation was allowed to continue for the required time. At the end of the transport period the foliage leaves and stipules were carefully cut away and the stem was divided into successive 5-ram segments beginning immediately below node 6. The segments were transferred singly to polyethylene counting vials and extracted overnight in darkness at 4 ~ in 5.0-cm 3 scintillation fluid (Ready-Solv EP; Beckman, High Wycombe, Bucks., UK). Radioactivity was determined by liquid scintillation counting (Beckman Model LS7500) and all counts were corrected for counting efficiency (by automatic external standardization) and background. Efflux experiments. Attempts were made to determine if the label that moved downwards through the stem following apical application of [1-14C]IAA to an intact plant was in a polar transport pathway, and one which involved NPA-sensitive elflux carriers. The apical buds of 13-d-old plants were labelled with [1-14C]IAA and transport was allowed to proceed for 6 h in light at 21 ~ C. At the end of this transport period 2.0-mm segments were cut from the stem 35 55 mm below the apical bud (the region in which the bulk of the mobile IAA would have been present - see Fig. I a). Pooled segments from 10 plants (approx. 450 mg fresh weight) were weighed and transferred to 250-cm 3 beakers containing 50 cm 3 efflux medium (Na-phosphate/citrate buffer, pH 7.0, with or without 3 mmolm-3 NPA). Segments were stirred vigorously with a magnetic bar and 250-mm 3 aliquots of medium were withdrawn at frequent intervals over a 90-min efflux period, transferred to 5 cm 3 scintillation fluid and counted as described above. At the end of the experiment the segments were drained, rinsed in fresh buffer and extracted overnight in 5.0 cm 3 ethanol at room temperature in darkness. Aliquots of the extracts were counted and the total soluble 14C present in the tissue at the start of the effiux period was determined. The 14C remaining in the tissue at each sampling time was calculated. Efflux curves were analysed by conventional curve-stripping techniques to resolve efflux compartments. The polarity of efflux from 30-mm stem sections cut from intact plants that were transporting [I-14C]IAA was also investigated. The [I~C]IAA was applied to the apical bud as before and transport was allowed to proceed for 3 h at 21 ~ C + 1~ in light. Sections (30 ram) cut from the stem immediately below the labelled apical bud were supported vertically with either their basal or their apical ends immersed to a depth of 1 mm in 6.0 cm 3 efflux medium (Na-phosphate/citric acid buffer, pH 7.0, with or without 3 m m o l . m 3 NPA) contained in shallow Plexiglas wells (four sections per well). After a further 3 h in darkness four 250-mm 3 aliquots of the effiux medium were transferred to scintillation vials and counted in 5.0 cm 3 scintillation fluid.
244
C.F. Johnson and D.A. Morris: Polar diffussion of IAA
(:1.
30 10 3
~,
~n
g
0.3~'1
0sF
I
I
ttAz.'2_ v
I
I
I
y% ; =n~176176
c.)
0.3 q 0
I
i
50 Distonce
i
I
100
i
150
(mm)
Fig. la, b. Time-course of [1-14C]IAA transport in pea stems. a Distribution of label in the stems of intact pea plants following the application of [1-14C]IAA to the apical bud. Profiles shown are for bulked samples from two plants at each transport time: 2.3 h (o--o), 4.1 h (A --A), 6.3 h (D--c3), 8.3 h(zx-A)and 10.3 (e--e). Distance was measured from the point of application of label in the apical bud, 5 mm above the uppermost stem segment sampled, b Basipetal migration of 14C in the stems of pea plants after "pulse-labelling" the apical bud with [1-]4C]IAA. After the application of [I-14C]IAA, export of label into the stem was allowed to proceed for 2.0 h, after which the apical bud was removed. Transport times (measured from decapitation) were: 0 h (o--o), 2.1 h (A--A), 4.2 h (tz--n), 6.2 h (A--zx), 8.6 h ( e - - e ) and 23.5 h (top). Treatments were replicated three times. Transport profiles shown are for typical individual plants. Distance was measured from point of decapitation Results
Time-course of transport from the shoot apex. The distribution of ~4C in the stem of 14-d-old pea seedlings following the application of [1-14C]IAA to the intact apical bud is shown in Fig. 1. Plants either remained intact throughout (Fig. l a) or were decapitated at node 6 after pulsing for 2.0 h (Fig. 1 b). In intact plants labelled with [I-14C]IAA (1.339 nmol. plant- 1 ; 270 retool, m - 3) transport profiles were characterised by a steep " f r o n t " , the slope of which decreased and became more distinctly curvilinear with increasing duration of
transport. Behind the " p e a k " of the profiles radioactivity fell to a low, almost constant level, indicating that there was a rapid initial release of labelled IAA into the transport system from the apical bud, followed by a prolonged steady export of labelled IAA at a lower rate. Only a small proportion of the total radioactivity applied to the apical bud was exported, ranging from 1.6% at 2.3 h to 2.23% at 10.3 h. Chromatography of ethanol extracts of the stem 6.0 h after the application of [1-14C]IAA indicated that most of the ethanol-soluble 14C was still with IAA (data not shown). The peak of the profiles of labelled IAA migrated through the stem at a steady velocity of approx. 8 mm. h - 1 whilst the leading edge moved at approx. 14 ram. h-1 Following pulse-labelling with [I-14C]IAA (1.078 n m o l . p l a n t - ] ; 216 m m o l . m - 3 ) , 14C which had entered the stem prior to decapitation continued to migrate downwards as a discrete " p u l s e " which tended to broaden and attenuate slightly with increasing duration of transport (Fig. I b). Apart from a small accumulation of 14C in segments bearing nodes and axillary buds, the labelled IAA almost completely drained from the stems following passage of the pulse (see Fig. I b : 23.5 h). Neither the shape of an [1-14C]IAA pulse nor its transport velocity was affected by chases of unlabelled IAA (0, 0.1, 1.0 or 10.0 mg.g -1 in lanolin applied to the cut surface of the stem immediately after decapitation data not shown). Estimated from the time at which IAA was applied to the apical bud, the mean transport velocity of the pulse peaks was approx. 7 mm. h - 1 and this did not change significantly with increasing duration of transport. Velocities of movement of the leading edges of the pulses were more variable (range 9.7-13.5 ram. h - 1) and exhibited no consistent trend with duration of transport. Velocities estimated from the distances moved by the pulse peaks between successive sampling times were also approx. 7.0 mm- h - 1, indicating that in this experiment the rate of uptake of [1-14C]IAA by the apical bud did not limit the rate of transport of IAA in the stem.
Effects of auxins on [1-14C]IAA transport. Transport of [1-14C]IAA in intact plants was strongly inhibited through regions of the stem pretreated for 16 h with a high concentration of unlabelled IAA (10 m g - g - i lanolin), both the velocity of migration of the profile front and the amount of label transported decreasing substantially (Fig. 2 a). Below the point of application of unlabelled IAA, radioactivity in the stem tended to decrease exponentially with distance. Unlabelled IAA caused pro-
C.F. Johnson and D.A. Morris : Polar diffussion of IAA
245
10 ~
U}
(z 2
g r |
E
o
0 I
~
b.
:o
(1
cr m
6
GL 3
0 0
50
100
0
20 Distance
Distance (ram) Fig. 2a, b. The inhibition of [1-14C]IAA transport in the stem of intact pea plants following pretreatment of the stem with unlabelled auxins, a Profiles of radioactivity in the stem 6.0 h after the apical application of [1-14C]IAA to plants ringed 16 h previously with lanolin ( o - - o ) or with lanolin containing 10 m g - g - ~ unlabelled IAA ( n - - n ) . b Profiles of radioactivity in the stem 8.0 h after apical applications of [1-14C]IAA to plants ringed 2.5 h previously with 1 mg.g -1 N A A (zx A), 1 mg. g- 1 2,4-D ( e - - e ) or plain lanolin ( o - - o ) . Results are means of three plants per treatment. Horizontal bars indicate positions of lanolin rings
nounced bending of the stem at its point of application and stimulated elongation and radial expansion of the stem above this point. This is consistent with the saturation of IAA transport at the point of application of unlabelled IAA and auxin accumulation in more distal tissues. Transport was also markedly inhibited by local applications to the stem of N A A and 2,4-D (1 mg, g-1 in lanolin applied 2.5 h before labelling, and [I-14C]IAA accumulated immediately above the point of application of these synthetic auxins (Fig. 2b).
Effects of concentration of applied IAA on transport. To investigate the effects of applied-IAA concentration on the characteristics of export of labelled IAA from the apical bud and its transport in the stem, [5-3H]IAA (sp. act. 681 GBq. mmo1-1) was combined with unlabelled IAA to give total concentrations ranging from 0.45 mmol. m -3 to 833 m m o l . m -3 and a constant concentra-
40 from
apex
60 80 (mm)
'7
E60
c.
0
400
IAA
800
c~pplied (mmol~ m-3)
Fig. 3 a-e. Effect of applied-IAA concentration on the transport of apically applied [3H]IAA in the intact pea plant. Plants were labelled with 5 mm 3 of solution containing a constant concentration of [3H]IAA (0.45 m m o l . m -3) and unlabelled IAA to give final total concentrations of 0.45 (o o), 1.29 ( e - - e ) , 8.88 (zx--zx), 83.79 ( ~ A ) and 833.79 ( o - - . ) mmol. m -3 IAA. Transport period was 6.0 h. a Distribution of label in the stem at the end of the transport period; b distribution in the stem of total applied IAA (labelled plus unlabelled) at the end of the transport period (results for 0.45 mmol-m 3 omitted for clarity); and e the relationship between total IAA recovered from each stem at the end of the transport period and the concentration of IAA applied. All results are means of five plants per treatment
tion of [3H]IAA of 0.45 m m o l . m -3. These solutions were applied to the apical buds of intact plants (5 mm 3- plant - 1) and the distribution of 3H in the stem was determined after 6.0 h transport at 22 ~ C.
246
C.F. Johnson and D.A. Morris : Polar diffussion of IAA
30
E
cn
,~o
9
10
r
o.
D
m
3
6
E.
I
I
I
1
0
5
10
15
cr
1
111 .m
r "--
0.3 ;"
0
I 50 Distance
o
5
. NPA
e. ~
O
r
100 (ram)
Fig. 4. Inhibition of IAA transport in intact pea plants by TIBA and NPA. TIBA (zx, A) and NPA (n, m) were applied to the stem in lanolin (I0 mg.g 1) either 16 h before (solid symbols) or immediately before (open symbols) the application of [1~4C]IAA to the apical buds. Control plants received lanolin only (o--o). Transport period was 6.0 h. The results shown are means of three plants per treatment. Horizontalbars indicate the positions of the lanolin rings. Those for the 16-h pretreatment are displaced downwards as a result of the stem extension which occurred betweenapplication of the inhibitors and termination of the experiment The total amount of IAA (3H-labelled plus unlabelled) exported into the stem increased over the whole range of concentrations applied to the apical bud (Fig. 3 b), but appeared to approach saturation at the highest concentration applied (833.8 m m o l . m - 3 ) with an estimated half-saturation at 22.2 mmol. m - 3 (Fig. 3 c). The proportion of the applied IAA that entered the stem was low (range 12.9% at 0 . 4 5 m m o l . m -3 to 1.3% at 833.8 m m o l . m -3) and decreased as a linear function of ln[IAA] (corr. c o e f f . = - 0 . 9 9 8 5 ; r 2= 0.9971; P = 0.00007). Up to 8 3 . 8 m m o l . m -3 the velocity of movement of the peaks of the transport profiles in the stem increased significantly with concentration of applied IAA (Fig. 3a; velocity 4 . 4 + 0 . 4 m m . h -1 at 0 . 4 5 m m o l . m 3 to 6.7_+ 0.5 m m . h - 1 at 83.8 m m o l - m - 3 ) ; further increase in the concentration of IAA to 833.8 m m o l . m -3 reduced the velocity of the peak once more. The velocity of the profile front was not significantly affected by the concentration of IAA applied. Inhibition of transport by N P A and TIBA. Movement of [14C]IAA in the intact plant was strongly inhibited by localised applications to the stem of the inhibitors of polar auxin transport, N P A and TIBA (Fig. 4). When N P A or TIBA was applied
E =, (J
O~ 4
I
I O
I
I 20
I Time
I 40
I
I 60
I
I 80
I
(mln)
Fig. 5. Effect of NPA (3 mmol-m -3) on the efflux of 14C from 2-mm segments cut from the stems of intact pea plants transporting apically applied [I-14C]IAA. Conventional curve-stripping techniques were used to resolve fast (inset) and slow efflux compartments. In the presence of NPA (zx), the half-time of effiux from the slow compartment was increased from 32.5 min to 103.9 min. Effiux from the fast (initial) compartment was unaffected by NPA (half-time approx. 6 min in both treatments)
to the stem of intact plants at the same time that [1-14C]IAA was applied to the apical bud, radioactivity migrated through the stem and accumulated in tissues immediately above the point of application of the inhibitor. Prolonged (16 h) pretreatment of the stem with N P A or TIBA inhibited the subsequent export of labelled IAA from the apical tissues, possibly by causing the accumulation in the stem of sufficiently high concentrations of endogenous auxin as to interfere with the subsequent basipetal movement of [14C]IAA (Fig. 4; compare Morris et al. 1973). This interpretation is supported by the observed inhibition of IAA transport following pretreatment of the stem with high concentrations of unlabelled IAA itself (Fig. 2a; compare Goldsmith 1982). Furthermore, the 16-h pretreatments with N P A and TIBA caused bending and radial expansion of the stem above the point of their application comparable to that observed following prolonged pretreatment with high concentrations of unlabelled IAA.
C.F. Johnson and D.A. Morris: Polar diffussion of IAA Table 1. Polar efflux of 14C-labelled IAA from 30-mm sections removed from immediately below the apical bud of intact, 13-dold seedlings of Pisurn sativurn 3.0 h after the application of [1-1~C]IAA to the bud. Sections were placed with either their apical or their basal ends immersed to a depth of 1.0 m m in 6 cm a Na-phosphate/citrate buffer (pH 7.0) and were allowed to transport for a further 3.0 h. Four sections per treatment; results are Bq 14C effluxed per section • SE End of segment in efflux medium
Control + N P A (3 m m o l . m 3)
Basal
Apical
4.36 • 1.07 0.15 +0.02
0.20 + 0.03 0.24-+0.04
Efflux of IAA from the transport system. Efflux of [1-14C]IAA into Na-phosphate/citrate buffer (pH 7.0) from 2-mm segments cut from just behind the front of the transport profile in plants which had been transporting apically applied IAA for 6 h exhibited two distinct components: (i) a fast component, half-time approx. 6 min, which was unaffected by NPA; and (ii) a slower component, the half-time of which increased from 32.5min to 103.9 min when 3 m m o l . m -3 NPA was included in the efflux medium (Fig. 5). In a second experiment the polarity of efflux from long (30 mm) segments cut from just below the apical bud of intact plants which had been transporting [I-a4C]IAA for 3 h was investigated. In the absence of NPA, segments with their basal ends immersed in efflux medium lost 22.5 times more a4C to the medium than those with their apical ends immersed (Table 1). This basipetal efflux was strongly inhibited by the inclusion of NPA in the medium. Discussion
Previous uptake studies (Davies and Rubery 1978; Johnson and Morris 1987) have indicated that the transmembrane transport of IAA in cells of the pea stem involves all the components of transport predicted by the chemiosmotic polar diffusion hypothesis (Goldsmith 1977; Rubery 1986, 1987). The results reported here confirm that these components of transmembrane transport also mediate in the slow, root-directed transport of IAA from the apical bud of the intact plant. The export of exogenous IAA from the apical bud of the intact pea was found to be saturable, consistent with carrier participation, although suprisingly high concentrations of IAA (in excess of 800 mmol. m - 3) could be applied to the apical bud (as a 5-mm 3 droplet) before saturation of ex-
247
port into the transport system was approached (Fig. 3 b, c). The fact that the total amount of IAA transported was very low compared with that applied (see Results) and that the total amount of IAA in the transport stream increased over the whole range of concentrations studied (Fig. 3), indicates that in the intact plant the IAA-transport system may normally function well below its potential maximum capacity. Thus the amount of IAA transported may be limited by the rate of auxin loading into the polar transport stream rather than by the capacity of the stream itself. Indol3yl-acetic acid drains from tissues after passage of a pulse (Fig. 1 a), and pulse shape and velocity is not affected by chases of unlabelled IAA. Therefore, once IAA has entered the transport pathway in the stem of an intact plant, its transport does not require continued auxin loading into the transport stream and must be driven by the transporting cells themselves. The fronts of transport profiles of [1-14C]IAA in the stem of intact plants and the peaks of 14C in pulse-labelled plants both migrated downwards at velocities ( 7 - 1 5 m m . h -1) characteristic of polar transport in isolated segments (Goldsmith 1969; Kaldewey 1984). Transport velocity was sensitive to the concentration of IAA in the transport stream, At low concentrations velocity increased with auxin concentration; high total concentrations reduced transport velocity, or completely inhibited transport (see Results; and Figs. 2, 3). Similar effects of auxin concentration on the velocity of transport of pulses of [3H]IAA in isolated corn coleoptile sections have been reported by Goldsmith (1982), who suggested that at low concentrations IAA may stimulate IAA efflux. As in polar transport in excised shoot or coleoptile sections (e.g. Goldsmith 1982) transport of IAA in intact plants was strongly inhibited through regions of the stem pretreated with either specific non-competitive inhibitors of IAA transmembrane efflux (NPA and TIBA; Fig. 4), or with high concentrations of unlabelled auxins (IAA, NAA and 2,4-D; Fig. 2). In both cases, labelled [AA accumulated in and above the treated region of the stem. This is consistent with the inhibition of a mediated efflux component of long-distance IAA transport in the stem. With both treatments there was a near exponential decline of radioactive IAA with distance below the auxin- or inhibitortreated region of the stem, indicating that a slow non-mediated migration of label through the tissues could occur (compare Goldsmith 1982). It is impossible to determine directly whether the long-distance transport of endogenous IAA synthesised at the shoot apex of the intact plant
248
is a true polar transport since transport can only occur in one direction, namely towards the root. The efflux experiments reported above, however, provide compelling indirect evidence that the transport of [I-14C]IAA applied to the apical bud of the intact is both a polar transport and is mediated by carrier systems sensitive to NPA (Fig. 5; Table 1). The slow, NPA-sensitive, component of effiux had a half-time (approx. 30 min) of the same order as that (approx. 60 min) observed for the efflux of [3H]IAA from corn coleoptile segments by Edwards and Goldsmith (1980). The fast component of efflux (half-time approx. 6 rain) probably represents diffusion of [1-14C]IAA from the wall space and consequently auxin must enter this space during cell-to-cell transport through the stem (see also Cande and Ray 1976). Considered together, the results presented here provide strong evidence that the slow long-distance transport of IAA from the apical bud of the intact pea plant occurs by a mechanism which involves diffusive and carrier-mediated uptake components and a polar, mediated, NPA-sensitive efflux of IAA into the apoplast. The mechanism appears to be identical to that which drives the polar transport of IAA in isolated tissue segments. This work was supported by Grant No. GR/D/08760 from the UK Science and Engineering Research Council. We are grateful to Dr. M.H.M. Goldsmith (Yale University, New Haven, Conn., USA) for constructive comments on a draft of this paper and Drs. G.F. Katekar and P.H. Rubery for gifts of NPA. We thank Mrs. R.P. Bell for able technical assistance.
References Benning, C. (1986) Evidence supporting a model of voltagedependent uptake of auxin into Cucurbita vesicles. Planta 169, 228537 Cande, W.Z., Ray, P.M. (1976) Nature of cell-to-cell transfer of auxin in polar transport. Planta 129, 43 52 Davies, P.J., Rubery, P.H. (1978) Components of auxin transport in stem segments of Pisum sativum L. Planta 142, 211219 Edwards, K.L., Goldsmith, M.H.M. (1980) pH-dependent accumulation of indoleacetic acid by corn coleoptile sections. Planta 147, 457~,66 Goldsmith, M.H.M. (1969) Transport of plant growth regulators. In: Physiology of plant growth and development, pp. 127-162, Wilkins, M.B., ed. McGraw-Hill, London Goldsmith, M.H.M. (1977) The polar transport of auxin. Annu. Rev. Plant Physiol. 28, 439-478 Goldsmith, M.H.M. (1982) A saturable site responsible for polar transport of indole-3-acetic acid in sections of maize coleoptiles. Planta 155, 68-75 Goldsmith, M.H.M., Goldsmith, T.H. (1981) Quantitative predictions for the chemiosmotic uptake of auxin. Planta 153, 25-33 Hertel, R. (1983) The mechanism of auxin transport as a model for auxin action. Z. Pflanzenphysiol. 112, 53 67 Hertel, R. (1986) Two comments on auxin transport: the up-
C.F. Johnson and D.A. Morris : Polar diffussion of IAA take/efflux mechanism and the problem of adaption. In: Plant growth substances 1985, pp. 214-217, Bopp, M., ed. Springer, Berlin Heidelberg New York Tokyo Johnson, C.F., Morris, D.A. (1987) Regulation of auxin transport in pea (Pisum sativum L.) by phenylacetic acid: effects on the components of transmembrane transport of indol3yl-acetic acid. Planta 172, 400-407 Kaldewey, H. (1984) Transport and other modes of movement of hormones (mainly auxins). In: Encyclopedia of plant physiology N.S., vol. 10, pp. 80-148, Scott, T.K., ed. Springer, Berlin Heidelberg New York Tokyo Lomax, T.L., Mehlhorn, R.J., Briggs, W.R. (1985) Active auxin uptake by zucchini membrane vesicles: quantitation using ESR volume and pH determinations. Proc. Natl. Acad. Sci. USA 82, 6541-6545 Milborrow, B.V., Rubery, P.H. (1985) The specificity of the carrier-mediated uptake of ABA by root segments of Phaseolus coccineus L. J. Exp. Bot. 36, 807-822 Morris, D.A. (1988) Cartier-mediated transmembrane auxin transport: possibilities for regulation and some implications for the control of growth and development. In: Physiology and biochemistry of auxins in plants, pp. 195-203, Kutacek, M., Bandurski, R.S., Krekule, J., eds. Academia, Prague Morris, D.A., Briant, R.E., Thomson, P.G. (1969) The transport and metabolism of ~4C-labelled indoleacetic acid in intact pea seedlings. Planta 89, 178-197 Morris, D.A., Johnson, C.F. (1985) Characteristics and mechanisms of long-distance auxin transport in intact plants. Acta Univ. Agric. Brno Fac. Agron. 33, 377-383 Morris, D.A., Johnson, C.F. (1987) Regulation of auxin transport in pea (Pisum sativum L.) by phenylacetic acid: inhibition of polar auxin transport in intact stems and stem segments. Planta 172, 408-416 Morris, D.A., Kadir, G.O., Barry, A.J. (1973) Auxin transport in intact pea seedlings (Pisum sativum L.): the inhibition of transport by 2,3,5-triiodobenzoic acid. Planta 110, 173 182 Morris, D.A., Thomas, A.G. (1974) The specificity of auxin transport in intact pea seedlings. Planta 118, 225-234 Raven, J.A. (1975) Transport of indoleacetic acid in plant cells in relation to pH and electrical potential gradients, and its significance for polar IAA transport. New Phytol. 74, 163172 Rubery, P.H. (1980) The mechanism of transmembrane auxin transport and its relation to the chemiosmotic hypothesis of the polar transport of auxin. In: Plant growth substances 1979, pp. 50-60, Skoog, F., ed. Springer, Berlin Heidelberg New York Tokyo Rubery, P.H. (1986) The evolution of polar transport models, and some possibilities for the regulation of auxin carriers. In: Plant growth substances 1985, pp. 197 202, Bopp., M., ed. Springer, Berlin Heidelberg New York Tokyo Rubery, P.H. (1987) Manipulation of hormone transport in physiological and developmental studies. In: Hormone action in plant development - a critical appraisal, pp. 161-174, Hoad, G.V., Lenton, J.R., Jackson, M.B., Atkin, R.K., eds. Butterworths, London Rubery, P.H., Sheldrake, A.R. (1974) Carrier-mediated auxin transport. Planta 118, 101-121 Sabater, M., Rubery, P.H. (1987) Auxin carriers in Cucurbita vesicles. II. Evidence that carrier-mediated routes of both indole-3-acetic acid influx and efflux are electroimpelled. Planta 171, 507-513
Received 28 September 1988; accepted 1 February 1989
Planta (1989)178:242-248
9 Springer-Verlag 1989
Applicability of the chemiosmotic polar diffusion theory to the transport of indol-3yl-acetic acid in the intact pea (Pisum sativum L.) Claire F. Johnson* and D.A. Morris** Department of Biology, Building 44, The University, Southampton S09 5NH, U K
Abstract. The transport of exogenous indol-3ylacetic acid (IAA) from the apical tissues of intact, light-grown pea (Pisum sativum L. cv. Alderman) shoots exhibited properties identical to those associated with polar transport in isolated shoot segments. Transport in the stem of apically applied [1-14C] - or [5-3H]IAA occurred at velocities (approx. 8-15 ram. h - ~) characteristic of polar transport. Following pulse-labelling, IAA drained from distal tissues after passage of a pulse and the rate characteristics of a pulse were not affected by chases of unlabelled IAA. However, transport of [I-14C]IAA was inhibited through a localised region of the stem pretreated with a high concentration of unlabelled I A A or with the synthetic auxins 1-napthaleneacetic acid and 2,4-dichlorophenoxyacetic acid, and label accumulated in more distal tissues. Transport of [1-14C]IAA was also completely prevented through regions of the intact stem treated with N-l-naphthylphthalamic acid (NPA) and 2,3,5-triiodobenzoic acid. Export of IAA from the apical bud into the stem increased with total concentration of IAA applied (labelled+unlabelled) but approached saturation at high concentrations (834 mmol.m-3). Transport velocity increased with concentration up to 83 mmol. m-3 IAA but fell again with further increase in concentration. Stem segments (2 mm) cut from intact plants transporting apically applied [1-~4C]IAA effluxed 93% of their initial radioactivity into buffer (pH 7.0) in 90 min. The half-time for efflux increased from 32.5 to 103.9 min when 3 mmol.m -3 Institute of Horticultural Research, East Malling, Kent ME19 6BJ, U K ** To whom correspondence should be addressed * Present address:
A b b r e v i a t i o n s : IAA = indol-3yl-acetic acid ; 2,4-D = 2,4-dichlor-
ophenoxyacetic acid; N A A = l-naphthaleneacetic acid; N P A = N-l-naphthylphthalamic acid; TIBA = 2,3,5-triiodobenzoic acid
NPA was included in the efflux medium. Long (30 ram) stem sections cut from immediately below an apical bud 3.0 h after the apical application of [1-~4C]IAA effluxed IAA when their basal ends, but not their apical ends, were immersed in buffer (pH 7.0). Addition of 3 mmol.m -3 NPA to the external medium completely prevented this basal efflux. These results support the view that the slow long-distance transport of I A A from the intact shoot apex occurs by polar cell-to-cell transport and that it is mediated by the components of I A A transmembrane transport predicted by the chemiosmotic polar diffusion theory. Key words: Auxin carriers - Auxin transport Chemiosmotic theory (auxin transport) - Pisum (auxin transport) - Transport, polar (auxin)
Introduction
The problem addressed here is whether or not the properties of the slow, root-directed transport of indol-3yl-acetic acid (IAA) from the apex of the intact shoot (Morris and Johnson 1985) are consistent with the "chemiosmotic polar diffusion theory" of polar auxin transport (Rubery and Sheldrake 1974; Raven 1975; see Goldsmith 1977). Polar transport of IAA in isolated tissue segments involves a pH- and membrane-potential-dependent cell-to-cell movement of the compound (reviewed by Goldsmith 1977; Rubery 1980, 1986, 1987). Three component processes cooperate in the transmembrane transport of IAA. These are: (i) diffusion of the relatively lipophilic undissociated molecule (IAAH) down its own electro-chemical gradient (Rubery and Sheldrake 1974; Raven 1975); (ii) saturable uptake of the anion (IAA-) on a specific electro-impelled porter which probably co-
C.F. Johnson and D.A. Morris : Polar diffussion of IAA
transports IAA- with two protons (Hertel 1983, 1986; Lomax et al. 1985; Benning 1986; Sabater and Rubery 1987); and (iii) saturable efflux of IAA via a second specific plasma-membrane porter which is probably also electro-impelled (Sabater and Rubery 1987). The efflux porter is competitively inhibited by auxins and specifically and non-competitively inhibited by 2,3,5-triiodobenzoic acid (TIBA), phytotropins (including N-l-naphthylphthalamic acid, NPA), and morphactins. Differences in the relative permeability to IAAH and IAA- at each end of a cell can account for a net polar flux of IAA through a file of cells and may explain polar transport of IAA through tissue (Rubery and Sheldrake 1974; Raven 1975; Goldsmith 1977; Rubery 1980; Goldsmith and Goldsmith 1981). Although potentially this differential permeability could be achieved in several ways (Milborrow and Rubery 1985), available evidence (reviewed by Rubery 1987) indicates that it results from asymmetry in efflux-carrier distribution. In intact dicotyledonous shoots the slow rootdirected transport of exogenous IAA from young apical leaves exhibits several characteristics typically associated with polar transport in isolated shoot segments (reviewed by Morris and Johnson 1985; Morris 1988). However, there is no direct evidence that this transport is a true polar transport and, apart from its apparent specificity (Morris and Thomas 1974) and sensitivity to specific auxin-efflux inhibitors (Morris et al. 1973), there is little evidence that it is mediated by the carrier systems involved in polar IAA transport through cells. Material and methods Plant material. Seedlings of Pisum sativum L. cv. Alderman were grown as described by Moris and Johnson (1987). Unless otherwise indicated in Results, long-distance transport experiments with intact plants were carried out on uniform 13- to 15-d-old seedlings selected when leaf 5 was near full expansion and leaf 6 was partially unfolded. Chemicals. Radioactive compounds ([I-14C]IAA, 2.26 GBq. mmol 1; [5_3H]IAA, 681 GBq.mmo1-1) were obtained from Amersham International, Amersham, Bucks., UK. Stock solutions in ethanol or water were stored at - 25 ~ C. Radiochemical purity was checked before use by ascending paper chromatography in isopropanol: ammonia: H 2 0 (I 0 : 1 : 1, by vol.). Sources of unlabelled growth regulators were: IAA, TIBA, 2,4-dichlorophenoxyacetic acid (2,4-D) and 1-naphthaleneacetic acid (NAA) - Sigma Chemical Co. Poole, Dorsek, U K ; NPA gifts from Dr. P.H. Rubery, Department of Biochemistry, University of Cambridge, U K and Dr. G.F. Katekar, C.S.I.R.O., Canberra, Australia. Stock solutions of these were stored refridgerated in darkness. General reagents used were all Analar grade from BDH Chemicals, Poole, Dorset, UK. In some experiments, transport inhibitors (TIBA and NPA)
243 or unlabelled auxins (IAA, N A A and 2,4-D) were applied to stems as suspensions in hydrous lanolin (approx. 25% H20). Measured volumes of the suspensions were applied to defined regions of the stem using 2.0-cm 3 disposable plastic syringes. Stems were first lightly abraded with fine carborundum powder, rinsed with distilled water and blotted dry. Control plants received lanolin only.
Transport experiments. The long-distance transport of [1-14C] or [5-3H]IAA from the apical buds of intact plants was investigated following the technique of Morris et al. (1969). In some experiments the plants remained fully intact throughout the transport period. In "pulse" experiments, transport into the stem was allowed to continue for a specified time (usually 2.0 h) and the labelled apical tissues were then excised immediately below node 6. To prevent dehydration the cut end of the stem was sealed with a little hydrous lanolin and transport of label which had entered the stem prior to decapitation was allowed to continue for the required time. At the end of the transport period the foliage leaves and stipules were carefully cut away and the stem was divided into successive 5-ram segments beginning immediately below node 6. The segments were transferred singly to polyethylene counting vials and extracted overnight in darkness at 4 ~ in 5.0-cm 3 scintillation fluid (Ready-Solv EP; Beckman, High Wycombe, Bucks., UK). Radioactivity was determined by liquid scintillation counting (Beckman Model LS7500) and all counts were corrected for counting efficiency (by automatic external standardization) and background. Efflux experiments. Attempts were made to determine if the label that moved downwards through the stem following apical application of [1-14C]IAA to an intact plant was in a polar transport pathway, and one which involved NPA-sensitive elflux carriers. The apical buds of 13-d-old plants were labelled with [1-14C]IAA and transport was allowed to proceed for 6 h in light at 21 ~ C. At the end of this transport period 2.0-mm segments were cut from the stem 35 55 mm below the apical bud (the region in which the bulk of the mobile IAA would have been present - see Fig. I a). Pooled segments from 10 plants (approx. 450 mg fresh weight) were weighed and transferred to 250-cm 3 beakers containing 50 cm 3 efflux medium (Na-phosphate/citrate buffer, pH 7.0, with or without 3 mmolm-3 NPA). Segments were stirred vigorously with a magnetic bar and 250-mm 3 aliquots of medium were withdrawn at frequent intervals over a 90-min efflux period, transferred to 5 cm 3 scintillation fluid and counted as described above. At the end of the experiment the segments were drained, rinsed in fresh buffer and extracted overnight in 5.0 cm 3 ethanol at room temperature in darkness. Aliquots of the extracts were counted and the total soluble 14C present in the tissue at the start of the effiux period was determined. The 14C remaining in the tissue at each sampling time was calculated. Efflux curves were analysed by conventional curve-stripping techniques to resolve efflux compartments. The polarity of efflux from 30-mm stem sections cut from intact plants that were transporting [I-14C]IAA was also investigated. The [I~C]IAA was applied to the apical bud as before and transport was allowed to proceed for 3 h at 21 ~ C + 1~ in light. Sections (30 ram) cut from the stem immediately below the labelled apical bud were supported vertically with either their basal or their apical ends immersed to a depth of 1 mm in 6.0 cm 3 efflux medium (Na-phosphate/citric acid buffer, pH 7.0, with or without 3 m m o l . m 3 NPA) contained in shallow Plexiglas wells (four sections per well). After a further 3 h in darkness four 250-mm 3 aliquots of the effiux medium were transferred to scintillation vials and counted in 5.0 cm 3 scintillation fluid.
244
C.F. Johnson and D.A. Morris: Polar diffussion of IAA
(:1.
30 10 3
~,
~n
g
0.3~'1
0sF
I
I
ttAz.'2_ v
I
I
I
y% ; =n~176176
c.)
0.3 q 0
I
i
50 Distonce
i
I
100
i
150
(mm)
Fig. la, b. Time-course of [1-14C]IAA transport in pea stems. a Distribution of label in the stems of intact pea plants following the application of [1-14C]IAA to the apical bud. Profiles shown are for bulked samples from two plants at each transport time: 2.3 h (o--o), 4.1 h (A --A), 6.3 h (D--c3), 8.3 h(zx-A)and 10.3 (e--e). Distance was measured from the point of application of label in the apical bud, 5 mm above the uppermost stem segment sampled, b Basipetal migration of 14C in the stems of pea plants after "pulse-labelling" the apical bud with [1-]4C]IAA. After the application of [I-14C]IAA, export of label into the stem was allowed to proceed for 2.0 h, after which the apical bud was removed. Transport times (measured from decapitation) were: 0 h (o--o), 2.1 h (A--A), 4.2 h (tz--n), 6.2 h (A--zx), 8.6 h ( e - - e ) and 23.5 h (top). Treatments were replicated three times. Transport profiles shown are for typical individual plants. Distance was measured from point of decapitation Results
Time-course of transport from the shoot apex. The distribution of ~4C in the stem of 14-d-old pea seedlings following the application of [1-14C]IAA to the intact apical bud is shown in Fig. 1. Plants either remained intact throughout (Fig. l a) or were decapitated at node 6 after pulsing for 2.0 h (Fig. 1 b). In intact plants labelled with [I-14C]IAA (1.339 nmol. plant- 1 ; 270 retool, m - 3) transport profiles were characterised by a steep " f r o n t " , the slope of which decreased and became more distinctly curvilinear with increasing duration of
transport. Behind the " p e a k " of the profiles radioactivity fell to a low, almost constant level, indicating that there was a rapid initial release of labelled IAA into the transport system from the apical bud, followed by a prolonged steady export of labelled IAA at a lower rate. Only a small proportion of the total radioactivity applied to the apical bud was exported, ranging from 1.6% at 2.3 h to 2.23% at 10.3 h. Chromatography of ethanol extracts of the stem 6.0 h after the application of [1-14C]IAA indicated that most of the ethanol-soluble 14C was still with IAA (data not shown). The peak of the profiles of labelled IAA migrated through the stem at a steady velocity of approx. 8 mm. h - 1 whilst the leading edge moved at approx. 14 ram. h-1 Following pulse-labelling with [I-14C]IAA (1.078 n m o l . p l a n t - ] ; 216 m m o l . m - 3 ) , 14C which had entered the stem prior to decapitation continued to migrate downwards as a discrete " p u l s e " which tended to broaden and attenuate slightly with increasing duration of transport (Fig. I b). Apart from a small accumulation of 14C in segments bearing nodes and axillary buds, the labelled IAA almost completely drained from the stems following passage of the pulse (see Fig. I b : 23.5 h). Neither the shape of an [1-14C]IAA pulse nor its transport velocity was affected by chases of unlabelled IAA (0, 0.1, 1.0 or 10.0 mg.g -1 in lanolin applied to the cut surface of the stem immediately after decapitation data not shown). Estimated from the time at which IAA was applied to the apical bud, the mean transport velocity of the pulse peaks was approx. 7 mm. h - 1 and this did not change significantly with increasing duration of transport. Velocities of movement of the leading edges of the pulses were more variable (range 9.7-13.5 ram. h - 1) and exhibited no consistent trend with duration of transport. Velocities estimated from the distances moved by the pulse peaks between successive sampling times were also approx. 7.0 mm- h - 1, indicating that in this experiment the rate of uptake of [1-14C]IAA by the apical bud did not limit the rate of transport of IAA in the stem.
Effects of auxins on [1-14C]IAA transport. Transport of [1-14C]IAA in intact plants was strongly inhibited through regions of the stem pretreated for 16 h with a high concentration of unlabelled IAA (10 m g - g - i lanolin), both the velocity of migration of the profile front and the amount of label transported decreasing substantially (Fig. 2 a). Below the point of application of unlabelled IAA, radioactivity in the stem tended to decrease exponentially with distance. Unlabelled IAA caused pro-
C.F. Johnson and D.A. Morris : Polar diffussion of IAA
245
10 ~
U}
(z 2
g r |
E
o
0 I
~
b.
:o
(1
cr m
6
GL 3
0 0
50
100
0
20 Distance
Distance (ram) Fig. 2a, b. The inhibition of [1-14C]IAA transport in the stem of intact pea plants following pretreatment of the stem with unlabelled auxins, a Profiles of radioactivity in the stem 6.0 h after the apical application of [1-14C]IAA to plants ringed 16 h previously with lanolin ( o - - o ) or with lanolin containing 10 m g - g - ~ unlabelled IAA ( n - - n ) . b Profiles of radioactivity in the stem 8.0 h after apical applications of [1-14C]IAA to plants ringed 2.5 h previously with 1 mg.g -1 N A A (zx A), 1 mg. g- 1 2,4-D ( e - - e ) or plain lanolin ( o - - o ) . Results are means of three plants per treatment. Horizontal bars indicate positions of lanolin rings
nounced bending of the stem at its point of application and stimulated elongation and radial expansion of the stem above this point. This is consistent with the saturation of IAA transport at the point of application of unlabelled IAA and auxin accumulation in more distal tissues. Transport was also markedly inhibited by local applications to the stem of N A A and 2,4-D (1 mg, g-1 in lanolin applied 2.5 h before labelling, and [I-14C]IAA accumulated immediately above the point of application of these synthetic auxins (Fig. 2b).
Effects of concentration of applied IAA on transport. To investigate the effects of applied-IAA concentration on the characteristics of export of labelled IAA from the apical bud and its transport in the stem, [5-3H]IAA (sp. act. 681 GBq. mmo1-1) was combined with unlabelled IAA to give total concentrations ranging from 0.45 mmol. m -3 to 833 m m o l . m -3 and a constant concentra-
40 from
apex
60 80 (mm)
'7
E60
c.
0
400
IAA
800
c~pplied (mmol~ m-3)
Fig. 3 a-e. Effect of applied-IAA concentration on the transport of apically applied [3H]IAA in the intact pea plant. Plants were labelled with 5 mm 3 of solution containing a constant concentration of [3H]IAA (0.45 m m o l . m -3) and unlabelled IAA to give final total concentrations of 0.45 (o o), 1.29 ( e - - e ) , 8.88 (zx--zx), 83.79 ( ~ A ) and 833.79 ( o - - . ) mmol. m -3 IAA. Transport period was 6.0 h. a Distribution of label in the stem at the end of the transport period; b distribution in the stem of total applied IAA (labelled plus unlabelled) at the end of the transport period (results for 0.45 mmol-m 3 omitted for clarity); and e the relationship between total IAA recovered from each stem at the end of the transport period and the concentration of IAA applied. All results are means of five plants per treatment
tion of [3H]IAA of 0.45 m m o l . m -3. These solutions were applied to the apical buds of intact plants (5 mm 3- plant - 1) and the distribution of 3H in the stem was determined after 6.0 h transport at 22 ~ C.
246
C.F. Johnson and D.A. Morris : Polar diffussion of IAA
30
E
cn
,~o
9
10
r
o.
D
m
3
6
E.
I
I
I
1
0
5
10
15
cr
1
111 .m
r "--
0.3 ;"
0
I 50 Distance
o
5
. NPA
e. ~
O
r
100 (ram)
Fig. 4. Inhibition of IAA transport in intact pea plants by TIBA and NPA. TIBA (zx, A) and NPA (n, m) were applied to the stem in lanolin (I0 mg.g 1) either 16 h before (solid symbols) or immediately before (open symbols) the application of [1~4C]IAA to the apical buds. Control plants received lanolin only (o--o). Transport period was 6.0 h. The results shown are means of three plants per treatment. Horizontalbars indicate the positions of the lanolin rings. Those for the 16-h pretreatment are displaced downwards as a result of the stem extension which occurred betweenapplication of the inhibitors and termination of the experiment The total amount of IAA (3H-labelled plus unlabelled) exported into the stem increased over the whole range of concentrations applied to the apical bud (Fig. 3 b), but appeared to approach saturation at the highest concentration applied (833.8 m m o l . m - 3 ) with an estimated half-saturation at 22.2 mmol. m - 3 (Fig. 3 c). The proportion of the applied IAA that entered the stem was low (range 12.9% at 0 . 4 5 m m o l . m -3 to 1.3% at 833.8 m m o l . m -3) and decreased as a linear function of ln[IAA] (corr. c o e f f . = - 0 . 9 9 8 5 ; r 2= 0.9971; P = 0.00007). Up to 8 3 . 8 m m o l . m -3 the velocity of movement of the peaks of the transport profiles in the stem increased significantly with concentration of applied IAA (Fig. 3a; velocity 4 . 4 + 0 . 4 m m . h -1 at 0 . 4 5 m m o l . m 3 to 6.7_+ 0.5 m m . h - 1 at 83.8 m m o l - m - 3 ) ; further increase in the concentration of IAA to 833.8 m m o l . m -3 reduced the velocity of the peak once more. The velocity of the profile front was not significantly affected by the concentration of IAA applied. Inhibition of transport by N P A and TIBA. Movement of [14C]IAA in the intact plant was strongly inhibited by localised applications to the stem of the inhibitors of polar auxin transport, N P A and TIBA (Fig. 4). When N P A or TIBA was applied
E =, (J
O~ 4
I
I O
I
I 20
I Time
I 40
I
I 60
I
I 80
I
(mln)
Fig. 5. Effect of NPA (3 mmol-m -3) on the efflux of 14C from 2-mm segments cut from the stems of intact pea plants transporting apically applied [I-14C]IAA. Conventional curve-stripping techniques were used to resolve fast (inset) and slow efflux compartments. In the presence of NPA (zx), the half-time of effiux from the slow compartment was increased from 32.5 min to 103.9 min. Effiux from the fast (initial) compartment was unaffected by NPA (half-time approx. 6 min in both treatments)
to the stem of intact plants at the same time that [1-14C]IAA was applied to the apical bud, radioactivity migrated through the stem and accumulated in tissues immediately above the point of application of the inhibitor. Prolonged (16 h) pretreatment of the stem with N P A or TIBA inhibited the subsequent export of labelled IAA from the apical tissues, possibly by causing the accumulation in the stem of sufficiently high concentrations of endogenous auxin as to interfere with the subsequent basipetal movement of [14C]IAA (Fig. 4; compare Morris et al. 1973). This interpretation is supported by the observed inhibition of IAA transport following pretreatment of the stem with high concentrations of unlabelled IAA itself (Fig. 2a; compare Goldsmith 1982). Furthermore, the 16-h pretreatments with N P A and TIBA caused bending and radial expansion of the stem above the point of their application comparable to that observed following prolonged pretreatment with high concentrations of unlabelled IAA.
C.F. Johnson and D.A. Morris: Polar diffussion of IAA Table 1. Polar efflux of 14C-labelled IAA from 30-mm sections removed from immediately below the apical bud of intact, 13-dold seedlings of Pisurn sativurn 3.0 h after the application of [1-1~C]IAA to the bud. Sections were placed with either their apical or their basal ends immersed to a depth of 1.0 m m in 6 cm a Na-phosphate/citrate buffer (pH 7.0) and were allowed to transport for a further 3.0 h. Four sections per treatment; results are Bq 14C effluxed per section • SE End of segment in efflux medium
Control + N P A (3 m m o l . m 3)
Basal
Apical
4.36 • 1.07 0.15 +0.02
0.20 + 0.03 0.24-+0.04
Efflux of IAA from the transport system. Efflux of [1-14C]IAA into Na-phosphate/citrate buffer (pH 7.0) from 2-mm segments cut from just behind the front of the transport profile in plants which had been transporting apically applied IAA for 6 h exhibited two distinct components: (i) a fast component, half-time approx. 6 min, which was unaffected by NPA; and (ii) a slower component, the half-time of which increased from 32.5min to 103.9 min when 3 m m o l . m -3 NPA was included in the efflux medium (Fig. 5). In a second experiment the polarity of efflux from long (30 mm) segments cut from just below the apical bud of intact plants which had been transporting [I-a4C]IAA for 3 h was investigated. In the absence of NPA, segments with their basal ends immersed in efflux medium lost 22.5 times more a4C to the medium than those with their apical ends immersed (Table 1). This basipetal efflux was strongly inhibited by the inclusion of NPA in the medium. Discussion
Previous uptake studies (Davies and Rubery 1978; Johnson and Morris 1987) have indicated that the transmembrane transport of IAA in cells of the pea stem involves all the components of transport predicted by the chemiosmotic polar diffusion hypothesis (Goldsmith 1977; Rubery 1986, 1987). The results reported here confirm that these components of transmembrane transport also mediate in the slow, root-directed transport of IAA from the apical bud of the intact plant. The export of exogenous IAA from the apical bud of the intact pea was found to be saturable, consistent with carrier participation, although suprisingly high concentrations of IAA (in excess of 800 mmol. m - 3) could be applied to the apical bud (as a 5-mm 3 droplet) before saturation of ex-
247
port into the transport system was approached (Fig. 3 b, c). The fact that the total amount of IAA transported was very low compared with that applied (see Results) and that the total amount of IAA in the transport stream increased over the whole range of concentrations studied (Fig. 3), indicates that in the intact plant the IAA-transport system may normally function well below its potential maximum capacity. Thus the amount of IAA transported may be limited by the rate of auxin loading into the polar transport stream rather than by the capacity of the stream itself. Indol3yl-acetic acid drains from tissues after passage of a pulse (Fig. 1 a), and pulse shape and velocity is not affected by chases of unlabelled IAA. Therefore, once IAA has entered the transport pathway in the stem of an intact plant, its transport does not require continued auxin loading into the transport stream and must be driven by the transporting cells themselves. The fronts of transport profiles of [1-14C]IAA in the stem of intact plants and the peaks of 14C in pulse-labelled plants both migrated downwards at velocities ( 7 - 1 5 m m . h -1) characteristic of polar transport in isolated segments (Goldsmith 1969; Kaldewey 1984). Transport velocity was sensitive to the concentration of IAA in the transport stream, At low concentrations velocity increased with auxin concentration; high total concentrations reduced transport velocity, or completely inhibited transport (see Results; and Figs. 2, 3). Similar effects of auxin concentration on the velocity of transport of pulses of [3H]IAA in isolated corn coleoptile sections have been reported by Goldsmith (1982), who suggested that at low concentrations IAA may stimulate IAA efflux. As in polar transport in excised shoot or coleoptile sections (e.g. Goldsmith 1982) transport of IAA in intact plants was strongly inhibited through regions of the stem pretreated with either specific non-competitive inhibitors of IAA transmembrane efflux (NPA and TIBA; Fig. 4), or with high concentrations of unlabelled auxins (IAA, NAA and 2,4-D; Fig. 2). In both cases, labelled [AA accumulated in and above the treated region of the stem. This is consistent with the inhibition of a mediated efflux component of long-distance IAA transport in the stem. With both treatments there was a near exponential decline of radioactive IAA with distance below the auxin- or inhibitortreated region of the stem, indicating that a slow non-mediated migration of label through the tissues could occur (compare Goldsmith 1982). It is impossible to determine directly whether the long-distance transport of endogenous IAA synthesised at the shoot apex of the intact plant
248
is a true polar transport since transport can only occur in one direction, namely towards the root. The efflux experiments reported above, however, provide compelling indirect evidence that the transport of [I-14C]IAA applied to the apical bud of the intact is both a polar transport and is mediated by carrier systems sensitive to NPA (Fig. 5; Table 1). The slow, NPA-sensitive, component of effiux had a half-time (approx. 30 min) of the same order as that (approx. 60 min) observed for the efflux of [3H]IAA from corn coleoptile segments by Edwards and Goldsmith (1980). The fast component of efflux (half-time approx. 6 rain) probably represents diffusion of [1-14C]IAA from the wall space and consequently auxin must enter this space during cell-to-cell transport through the stem (see also Cande and Ray 1976). Considered together, the results presented here provide strong evidence that the slow long-distance transport of IAA from the apical bud of the intact pea plant occurs by a mechanism which involves diffusive and carrier-mediated uptake components and a polar, mediated, NPA-sensitive efflux of IAA into the apoplast. The mechanism appears to be identical to that which drives the polar transport of IAA in isolated tissue segments. This work was supported by Grant No. GR/D/08760 from the UK Science and Engineering Research Council. We are grateful to Dr. M.H.M. Goldsmith (Yale University, New Haven, Conn., USA) for constructive comments on a draft of this paper and Drs. G.F. Katekar and P.H. Rubery for gifts of NPA. We thank Mrs. R.P. Bell for able technical assistance.
References Benning, C. (1986) Evidence supporting a model of voltagedependent uptake of auxin into Cucurbita vesicles. Planta 169, 228537 Cande, W.Z., Ray, P.M. (1976) Nature of cell-to-cell transfer of auxin in polar transport. Planta 129, 43 52 Davies, P.J., Rubery, P.H. (1978) Components of auxin transport in stem segments of Pisum sativum L. Planta 142, 211219 Edwards, K.L., Goldsmith, M.H.M. (1980) pH-dependent accumulation of indoleacetic acid by corn coleoptile sections. Planta 147, 457~,66 Goldsmith, M.H.M. (1969) Transport of plant growth regulators. In: Physiology of plant growth and development, pp. 127-162, Wilkins, M.B., ed. McGraw-Hill, London Goldsmith, M.H.M. (1977) The polar transport of auxin. Annu. Rev. Plant Physiol. 28, 439-478 Goldsmith, M.H.M. (1982) A saturable site responsible for polar transport of indole-3-acetic acid in sections of maize coleoptiles. Planta 155, 68-75 Goldsmith, M.H.M., Goldsmith, T.H. (1981) Quantitative predictions for the chemiosmotic uptake of auxin. Planta 153, 25-33 Hertel, R. (1983) The mechanism of auxin transport as a model for auxin action. Z. Pflanzenphysiol. 112, 53 67 Hertel, R. (1986) Two comments on auxin transport: the up-
C.F. Johnson and D.A. Morris : Polar diffussion of IAA take/efflux mechanism and the problem of adaption. In: Plant growth substances 1985, pp. 214-217, Bopp, M., ed. Springer, Berlin Heidelberg New York Tokyo Johnson, C.F., Morris, D.A. (1987) Regulation of auxin transport in pea (Pisum sativum L.) by phenylacetic acid: effects on the components of transmembrane transport of indol3yl-acetic acid. Planta 172, 400-407 Kaldewey, H. (1984) Transport and other modes of movement of hormones (mainly auxins). In: Encyclopedia of plant physiology N.S., vol. 10, pp. 80-148, Scott, T.K., ed. Springer, Berlin Heidelberg New York Tokyo Lomax, T.L., Mehlhorn, R.J., Briggs, W.R. (1985) Active auxin uptake by zucchini membrane vesicles: quantitation using ESR volume and pH determinations. Proc. Natl. Acad. Sci. USA 82, 6541-6545 Milborrow, B.V., Rubery, P.H. (1985) The specificity of the carrier-mediated uptake of ABA by root segments of Phaseolus coccineus L. J. Exp. Bot. 36, 807-822 Morris, D.A. (1988) Cartier-mediated transmembrane auxin transport: possibilities for regulation and some implications for the control of growth and development. In: Physiology and biochemistry of auxins in plants, pp. 195-203, Kutacek, M., Bandurski, R.S., Krekule, J., eds. Academia, Prague Morris, D.A., Briant, R.E., Thomson, P.G. (1969) The transport and metabolism of ~4C-labelled indoleacetic acid in intact pea seedlings. Planta 89, 178-197 Morris, D.A., Johnson, C.F. (1985) Characteristics and mechanisms of long-distance auxin transport in intact plants. Acta Univ. Agric. Brno Fac. Agron. 33, 377-383 Morris, D.A., Johnson, C.F. (1987) Regulation of auxin transport in pea (Pisum sativum L.) by phenylacetic acid: inhibition of polar auxin transport in intact stems and stem segments. Planta 172, 408-416 Morris, D.A., Kadir, G.O., Barry, A.J. (1973) Auxin transport in intact pea seedlings (Pisum sativum L.): the inhibition of transport by 2,3,5-triiodobenzoic acid. Planta 110, 173 182 Morris, D.A., Thomas, A.G. (1974) The specificity of auxin transport in intact pea seedlings. Planta 118, 225-234 Raven, J.A. (1975) Transport of indoleacetic acid in plant cells in relation to pH and electrical potential gradients, and its significance for polar IAA transport. New Phytol. 74, 163172 Rubery, P.H. (1980) The mechanism of transmembrane auxin transport and its relation to the chemiosmotic hypothesis of the polar transport of auxin. In: Plant growth substances 1979, pp. 50-60, Skoog, F., ed. Springer, Berlin Heidelberg New York Tokyo Rubery, P.H. (1986) The evolution of polar transport models, and some possibilities for the regulation of auxin carriers. In: Plant growth substances 1985, pp. 197 202, Bopp., M., ed. Springer, Berlin Heidelberg New York Tokyo Rubery, P.H. (1987) Manipulation of hormone transport in physiological and developmental studies. In: Hormone action in plant development - a critical appraisal, pp. 161-174, Hoad, G.V., Lenton, J.R., Jackson, M.B., Atkin, R.K., eds. Butterworths, London Rubery, P.H., Sheldrake, A.R. (1974) Carrier-mediated auxin transport. Planta 118, 101-121 Sabater, M., Rubery, P.H. (1987) Auxin carriers in Cucurbita vesicles. II. Evidence that carrier-mediated routes of both indole-3-acetic acid influx and efflux are electroimpelled. Planta 171, 507-513
Received 28 September 1988; accepted 1 February 1989
