Planta (1990)181:117-124
Pl~__t~ 9 Springer-Verlag1990
The role of auxin efflux carriers in the reversible loss of polar auxin transport in the pea (Pisum sativum L.) stem D a v i d A . M o r r i s * and C l a i r e F. J o h n s o n * *
Department of Biology,Building44, The University,Southampton, SO9 5NH, UK
Abstract. Correlatively inhibited pea shoots ( P i s u m s a t i r u m L.) did not transport apically applied 14C-labelled
Key words: Auxin carrier - Auxin transport - Carrier (membrane electrophoresis) - Cell polarity - P i s u m (aux-
indol-3yl-acetic acid ([14C]IAA), and polar IAA transport did not occur in internodal segments cut from these shoots. Polar transport in shoots and segments recovered within 24 h of removing the dominant shoot apex. Decapitation of growing shoots also resulted in the loss of polar transport in segments from internodes subtending the apex. This loss was prevented by apical applications of unlabelled IAA, or by low temperatures (approx. 2~ C) after decapitation. Rates of net uptake of [14C]IAA by 2-mm segments cut from subordinate or decapitated shoots were the same as those in segments cut from dominant or growing shoots. In both cases net uptake was stimulated to the same extent by competing unlabelled IAA and by N-l-naphthylphthalamic acid. Uptake of the pH probe [14C]-5,5-dimethyloxazolidine-2,4-dione from unbuffered solutions was the same in segments from both types of shoot. Patterns of [14C]IAA metabolism in shoots in which polar transport had ceased were the same as those in shoots capable of polar transport. The reversible loss of polar IAA transport in these systems, therefore, was not the result of loss or inactivation of specific IAA effiux carriers, loss of ability of cells to maintain transmembrane pH gradients, or the result of a change in IAA metabolism. Furthermore, in tissues incapable of polar transport, no evidence was found for the occurrence of inhibitors of IAA uptake or efflux. Evidence is cited to support the possibility that the reversible loss of polar auxin transport is the result of a gradual randomization of effluxcarrier distribution in the plasma membrane following withdrawal of an apical auxin supply and that the recovery of polar transport involves reestablishment of effluxcarrier asymmetry under the influence of vectorial gradients in auxin concentration.
in transport) - Transport, polar (auxin)
* To whom correspondence should be addressed ** Present address: Institute of Horticultural Research, East Mailing, Maidstone,Kent MEI9 6BJ, UK Abbreviations: DMO=5,5-dimethyloxazolidine-2,4-dione;IAA= indol-3yl-acetic acid; NPA=N-l-naphthylphthalamic acid; TIBA= 2,3,5-triiodobenzoicacid
Introduction
Transport of indol-3yl-acetic acid (IAA) and related growth-active auxins across the plasma membrane of plant cells is mediated by specific uptake and effiux carriers distinguishable by their differing sensitivity to phytotropins such as N-l-naphthylphthalamic acid (NPA; Rubery 1986, 1987a, b). The characteristic polar transport of IAA results from asymmetry in the distribution in the plasma membrane of one of these carriers, probably the NPA-sensitive effiux carrier (Jacobs and Gilbert 1983; Jacobs and Short 1986; Rubery 1987b; but see Morris 1988). The long-distance transport of IAA from the shoot apex of intact plants is a polar transport mediated by the same uptake and effiux carrier systems (Johnson and Morris 1989). Polar auxin transport through plant cells is essential for the initiation and-or maintenance of spatially organised patterns of cell division, cell enlargement and cell differentiation (Sachs 1981 a, b, 1986; Gersani and Sachs 1984; Aloni 1987; and references cited by these authors). The mechanisms regulating the turnover of IAA carrier proteins, their targeting to and insertion into the plasma membrane, their localization in this membrane with respect to the direction of cellular axes, and their catalytic activity may therefore exert a profound influence on the organized development of tissues and organs around a recognizable shoot-root axis. Little is known about the mechanisms involved in these processes. Circumstantial evidence indicates that the ability of cells to carry out polar auxin transport is regulated by auxin itself and requires the continued presence of auxin in the transporting cells (reviewed by Goldsmith 1977). In isolated stem or coleoptile segments the capacity for polar (basipetal) transport declines with time after cutting but may be maintained or restored by applications of exogenous IAA (Goldsmith 1977; Rubery 1987b). We have identi-
118
fled two systems in pea (Pisum sativum L.), one natural and one experimental, in which polar IAA transport ceases reversibly if the supply of IAA from the shoot apex fails. These are, respectively, correlatively inhibited shoots on two-branched plants (Morris 1977) and decapitated shoots (described below). We show here that the reversible loss of polar transport in these systems following withdrawal of an apical auxin source is not caused by loss of specific auxin carriers from the plasma membrane, but probably results from randomization of carrier distribution.
Material and methods Plant material. Seedlings of P. sativum cv. Alderman (Booker Seeds, Sleaford, Lincs., UK) were grown in vermiculite in a growth cabinet (21~176 constant; photoperiod 16h from "warm white" fluorescent lamps; radiation approx. 400 ~tmol photons. m - 2 " s - 1), either singly in 90-mm plastic pots or in seed trays (210 mm. 320 mm; approx. 100 plants per tray). They were watered with Hoagland's mineral nutrient solution as necessary. "Twobranched" plants were obtained by removing the epicotyl immediately above the cotyledonary node 6 d after sowing to induce growth of the two axillary buds at this node (Sachs 1966; Morris 1977). In the majority of plants so treated, this resulted in plants bearing one actively growing " d o m i n a n t " shoot and one correlatively inhibited "subordinate" shoot which usually ceased further growth approx. 14 d after epicotyl removal. Renewed growth of the subordinate shoot can be induced by decapitation of the dominant shoot (Morris 1977). Chemicals. Stock solutions of 3-[5(n)-3H]IAA (specific activity 681GBq'mmo1-1) and [1-14C]IAA (specific activity 2.042.26 GBq.mmol 1), both from Amersham International, Amersham, Bucks., UK, were stored in ethanol (uptake experiments) or in distilled water containing 0.01% Tween 20 (polyoxyethylene sorbitan monolaurate; transport experiments) in darkness at - 2 5 ~ C. These stock solutions were diluted as required for individual experiments (see Results). 5,5-Dimethyl[2-14C]oxazolidine-2,4dione ([2-14C]DMO; specific activity 2.00 G B q . m m o l 1) was obtained from Amersham International and stored in aqueous solution at - 2 5 ~ C. Unlabelled growth regulators used were: 2,3,5triiodobenzoic acid (TIBA) and IAA (Sigma Chemical Co., Poole, Dorset, UK); and NPA (gift from Dr. G.F. Katekar, C.S.I.R.O., Canberra, Australia). Stock solutions in ethanol were stored refrigerated in darkness. In some experiments these compounds were applied to plants after incorporation of appropriate weights of the solid compound into hydrous lanolin (approx. 25% H20).
D.A. Morris and C.F. Johnson: Polar auxin transport in the pea stem determined by liquid scintillation spectrometry (Beckman Model LS5000). Authentic [1-14C]IAA was chromatogrammed as a marker for comparison with test samples.
Results
Two branched plants. In intact two-branched plants, [114C]IAA applied to the apical bud of dominant shoots was transported in the stem at an estimated velocity of 13.6_+0.9 mm.h -1, but little transport occurred in correlatively inhibited subordinate shoots (Fig. 1 A). In the subordinate shoots 14C decreased exponentially with distance from the labelled apex (correlation coefficient for the computed curve [y = exp{a + b}x] = - 0.9940_+ 0.0519; r2=0.9880), indicating that diffusion made a major contribution to the limited movement observed. Removal of the dominant shoot apex 96 h before the application of [ 1 - 1 4 C ] I A A to the subordinate shoot resulted in a full recovery of basipetal transport in the latter (Fig. 1A; mean velocity 12.7_+0.1 mm.h-1). In a second experiment transport in the subordinate shoot
A 10
8 6 '- 4
E
o-0 Cr en
~10
B
"-- 8 o
~5 6 O
2 Procedures. Techniques used to study long-distance transport of [I-14C]IAA from the apical buds of intact plants were identical to those described previously (Morris et al. 1969; Morris and Johnson 1987). Each plant received approx. 1.8 kBq 1~C (approx. 1.0 nmol IAA in 3.0 mm-3). Polar transport of [1-14C]IAA in 30mm-long internodal segments and uptake of [1-14C]IAA by short (2 mm) stem segments were investigated using the techniques described by Johnson and Morris (1987). In some experiments, metabolism of the applied [ 1-I~C]IAA was investigated by paper chromatography of ethanol extracts of tissue. Extracts were either first concentrated by rotary evaporation under reduced pressure at 30 ~ C, or spotted directly onto Whatman (Maidstone, Kent, UK) No. 1 chromatography paper strips, and chromatograms were developed in the ascending direction in isopropanol:ammonia:H20 (10:1:1, by vol.) at room temperature. Developed chromatograms were air-dried and cut into 10 or 15 equal sequential segments. Radioactive compounds were eluted in scintillation fluid (ReadySolv EP; Beckman, High Wycombe, Bucks., UK) and 14C was
0
1 20
40
60
80
Distance (ram)
Fig. 1 A, B. Transport of [1-14C]IAA from the apical buds of dominant and subordinate shoots of two-branched pea plants and the effect on transport in the subordinate shoot of removing the apex of the dominant shoot. A Approximately 1.8 kBq [1-x4C]IAA (328 mmol. m - 3) was applied to the apical buds in 3 mm s of solution, transport was allowed to proceed for 6.0 h and 14C was determined in sequential 5-mm segments. 9 dominant shoot; zx subordinate shoot; o, subordinate shoot 96 h after removal of dominant shoot apex. Means of five replications per treatment. B Methods as in A except that 1.5 kBq (339 m m o l . m -3) was applied to each plant. Transport period was 4.0 h and results are means of three replications, 9 dominant shoots; A, o, [] subordinate shoot 0, 24 and 48 h, respectively, after decapitation of dominant shoot
D.A. Morris and C.F. Johnson: Polar auxin transport in the pea stem
119
c
3.0
E
~3.o I/~
tl
%
~2.0 m
g ..g
o 1.0 O
o
II
ISg
0
I
I
0
10 Distance
20 (mm)
30
Fig. 2. Polar transport of IAA in 30-mm-internodal segments cut from dominant and subordinate shoots of two-branched pea plants. Segments were pulse-labelled with [1-14C]IAA (7.4 kBq; 3.4 mmol-m -3) for 30 min at pH 5.0 (Na-phosphate/citric acid) in 1.5% sucrose solution and chased for 2.0 h in the presence of 1 m m o l . m - 3 unlabelled IAA. Where used, NPA was included in pulse and chase at 3.0 m m o l ' m -3. Means of two (zx, A) or three (o, 9 segments per treatment, o Dominant basipetal; zx dominant acropetal; 9 subordinate basipetal; 9 subordinate basipetal + NPA
substantially recovered within 24 h of removal of the dominant shoot apex (Fig. 1 B). A strictly basipetal polar transport of pulses of [114C]IAA was observed in 30 m m long segments cut from dominant shoots, but only a weak basipetal m o v e m e n t of I A A occurred in segments from subordinate shoots (Fig. 2). In the latter the [14C]IAA taken up during pulsing decayed exponentially with distance from the apical end. However, a small residual a m o u n t of NPA-sensitive m o v e m e n t of label occurred (Fig. 2). Polar transport through segments from subordinate shoots recovered quickly after removal of the dominant shoot apex and a basipetally moving pulse of t4C was apparent in segments cut 16 h after decapitation of the dominant shoot (Fig. 3). Basipetal transport had recovered fully by 24 h after decapitation. To test whether the failure of correlatively inhibited shoots to mediate polar transport resulted from a loss of effiux carriers for IAA, characteristics of uptake of [1-14C]IAA (0.2 m m o l . m-3) from buffered solutions by 2-mm-long internodal segments from dominant and subordinate shoots were compared. In the absence of additives, I A A net uptake was generally lower (by approx. 10-20%) in segments from subordinate shoots than in those from dominant shoots (Tables 1, 2), although this difference disappeared when segments cut from older regions of each stem type were c o m p a r e d (Table 2; Experiment 1). Net uptake by segments from both shoot types was significantly stimulated by high concentrations ( 1 0 0 m m o l . m -3) of unlabelled I A A (Table 1) and by
o
20 Distance (ram) 10
30
Fig. 3. Time-course of recovery of basipetal transport in 30-mmsegments cut from subordinate shoots of two-branched pea plants following removal of the dominant shoot apex. Experimental conditions as in Fig. 2. Means of three segments per treatment. 9 6.5 h, o 16 h, zx 24 h, [] 72 h after excision of dominant shoot apex
Table 1. Effect of unlabelled IAA (100 mmol.m -3) on the net uptake of [1-14C]IAA (0.2 mmol.m 3) by 2-mm pea stem segments cut from dominant and subordinate shoots of two-branched plants. Net uptake at pH 4.5 was measured over 30 min at 25~ C. Results are expressed as Bq. (g FW) 1. (30 min)- 1+ SE (means of five observations per treatment) Shoot type a
Control
+ IAA b
+ IAA as % of control
Dominant Subordinate Subordinate as % of dominant
878.3+21.7 1056.7-t-33.3 120.3 731.7-I-16.7 961.7_+16.7 131.4 83.3 91.0
Net uptake of [I'*C]IAA by segments from subordinate shoots was significantly lower than that by segments from dominant shoots (P
Pl~__t~ 9 Springer-Verlag1990
The role of auxin efflux carriers in the reversible loss of polar auxin transport in the pea (Pisum sativum L.) stem D a v i d A . M o r r i s * and C l a i r e F. J o h n s o n * *
Department of Biology,Building44, The University,Southampton, SO9 5NH, UK
Abstract. Correlatively inhibited pea shoots ( P i s u m s a t i r u m L.) did not transport apically applied 14C-labelled
Key words: Auxin carrier - Auxin transport - Carrier (membrane electrophoresis) - Cell polarity - P i s u m (aux-
indol-3yl-acetic acid ([14C]IAA), and polar IAA transport did not occur in internodal segments cut from these shoots. Polar transport in shoots and segments recovered within 24 h of removing the dominant shoot apex. Decapitation of growing shoots also resulted in the loss of polar transport in segments from internodes subtending the apex. This loss was prevented by apical applications of unlabelled IAA, or by low temperatures (approx. 2~ C) after decapitation. Rates of net uptake of [14C]IAA by 2-mm segments cut from subordinate or decapitated shoots were the same as those in segments cut from dominant or growing shoots. In both cases net uptake was stimulated to the same extent by competing unlabelled IAA and by N-l-naphthylphthalamic acid. Uptake of the pH probe [14C]-5,5-dimethyloxazolidine-2,4-dione from unbuffered solutions was the same in segments from both types of shoot. Patterns of [14C]IAA metabolism in shoots in which polar transport had ceased were the same as those in shoots capable of polar transport. The reversible loss of polar IAA transport in these systems, therefore, was not the result of loss or inactivation of specific IAA effiux carriers, loss of ability of cells to maintain transmembrane pH gradients, or the result of a change in IAA metabolism. Furthermore, in tissues incapable of polar transport, no evidence was found for the occurrence of inhibitors of IAA uptake or efflux. Evidence is cited to support the possibility that the reversible loss of polar auxin transport is the result of a gradual randomization of effluxcarrier distribution in the plasma membrane following withdrawal of an apical auxin supply and that the recovery of polar transport involves reestablishment of effluxcarrier asymmetry under the influence of vectorial gradients in auxin concentration.
in transport) - Transport, polar (auxin)
* To whom correspondence should be addressed ** Present address: Institute of Horticultural Research, East Mailing, Maidstone,Kent MEI9 6BJ, UK Abbreviations: DMO=5,5-dimethyloxazolidine-2,4-dione;IAA= indol-3yl-acetic acid; NPA=N-l-naphthylphthalamic acid; TIBA= 2,3,5-triiodobenzoicacid
Introduction
Transport of indol-3yl-acetic acid (IAA) and related growth-active auxins across the plasma membrane of plant cells is mediated by specific uptake and effiux carriers distinguishable by their differing sensitivity to phytotropins such as N-l-naphthylphthalamic acid (NPA; Rubery 1986, 1987a, b). The characteristic polar transport of IAA results from asymmetry in the distribution in the plasma membrane of one of these carriers, probably the NPA-sensitive effiux carrier (Jacobs and Gilbert 1983; Jacobs and Short 1986; Rubery 1987b; but see Morris 1988). The long-distance transport of IAA from the shoot apex of intact plants is a polar transport mediated by the same uptake and effiux carrier systems (Johnson and Morris 1989). Polar auxin transport through plant cells is essential for the initiation and-or maintenance of spatially organised patterns of cell division, cell enlargement and cell differentiation (Sachs 1981 a, b, 1986; Gersani and Sachs 1984; Aloni 1987; and references cited by these authors). The mechanisms regulating the turnover of IAA carrier proteins, their targeting to and insertion into the plasma membrane, their localization in this membrane with respect to the direction of cellular axes, and their catalytic activity may therefore exert a profound influence on the organized development of tissues and organs around a recognizable shoot-root axis. Little is known about the mechanisms involved in these processes. Circumstantial evidence indicates that the ability of cells to carry out polar auxin transport is regulated by auxin itself and requires the continued presence of auxin in the transporting cells (reviewed by Goldsmith 1977). In isolated stem or coleoptile segments the capacity for polar (basipetal) transport declines with time after cutting but may be maintained or restored by applications of exogenous IAA (Goldsmith 1977; Rubery 1987b). We have identi-
118
fled two systems in pea (Pisum sativum L.), one natural and one experimental, in which polar IAA transport ceases reversibly if the supply of IAA from the shoot apex fails. These are, respectively, correlatively inhibited shoots on two-branched plants (Morris 1977) and decapitated shoots (described below). We show here that the reversible loss of polar transport in these systems following withdrawal of an apical auxin source is not caused by loss of specific auxin carriers from the plasma membrane, but probably results from randomization of carrier distribution.
Material and methods Plant material. Seedlings of P. sativum cv. Alderman (Booker Seeds, Sleaford, Lincs., UK) were grown in vermiculite in a growth cabinet (21~176 constant; photoperiod 16h from "warm white" fluorescent lamps; radiation approx. 400 ~tmol photons. m - 2 " s - 1), either singly in 90-mm plastic pots or in seed trays (210 mm. 320 mm; approx. 100 plants per tray). They were watered with Hoagland's mineral nutrient solution as necessary. "Twobranched" plants were obtained by removing the epicotyl immediately above the cotyledonary node 6 d after sowing to induce growth of the two axillary buds at this node (Sachs 1966; Morris 1977). In the majority of plants so treated, this resulted in plants bearing one actively growing " d o m i n a n t " shoot and one correlatively inhibited "subordinate" shoot which usually ceased further growth approx. 14 d after epicotyl removal. Renewed growth of the subordinate shoot can be induced by decapitation of the dominant shoot (Morris 1977). Chemicals. Stock solutions of 3-[5(n)-3H]IAA (specific activity 681GBq'mmo1-1) and [1-14C]IAA (specific activity 2.042.26 GBq.mmol 1), both from Amersham International, Amersham, Bucks., UK, were stored in ethanol (uptake experiments) or in distilled water containing 0.01% Tween 20 (polyoxyethylene sorbitan monolaurate; transport experiments) in darkness at - 2 5 ~ C. These stock solutions were diluted as required for individual experiments (see Results). 5,5-Dimethyl[2-14C]oxazolidine-2,4dione ([2-14C]DMO; specific activity 2.00 G B q . m m o l 1) was obtained from Amersham International and stored in aqueous solution at - 2 5 ~ C. Unlabelled growth regulators used were: 2,3,5triiodobenzoic acid (TIBA) and IAA (Sigma Chemical Co., Poole, Dorset, UK); and NPA (gift from Dr. G.F. Katekar, C.S.I.R.O., Canberra, Australia). Stock solutions in ethanol were stored refrigerated in darkness. In some experiments these compounds were applied to plants after incorporation of appropriate weights of the solid compound into hydrous lanolin (approx. 25% H20).
D.A. Morris and C.F. Johnson: Polar auxin transport in the pea stem determined by liquid scintillation spectrometry (Beckman Model LS5000). Authentic [1-14C]IAA was chromatogrammed as a marker for comparison with test samples.
Results
Two branched plants. In intact two-branched plants, [114C]IAA applied to the apical bud of dominant shoots was transported in the stem at an estimated velocity of 13.6_+0.9 mm.h -1, but little transport occurred in correlatively inhibited subordinate shoots (Fig. 1 A). In the subordinate shoots 14C decreased exponentially with distance from the labelled apex (correlation coefficient for the computed curve [y = exp{a + b}x] = - 0.9940_+ 0.0519; r2=0.9880), indicating that diffusion made a major contribution to the limited movement observed. Removal of the dominant shoot apex 96 h before the application of [ 1 - 1 4 C ] I A A to the subordinate shoot resulted in a full recovery of basipetal transport in the latter (Fig. 1A; mean velocity 12.7_+0.1 mm.h-1). In a second experiment transport in the subordinate shoot
A 10
8 6 '- 4
E
o-0 Cr en
~10
B
"-- 8 o
~5 6 O
2 Procedures. Techniques used to study long-distance transport of [I-14C]IAA from the apical buds of intact plants were identical to those described previously (Morris et al. 1969; Morris and Johnson 1987). Each plant received approx. 1.8 kBq 1~C (approx. 1.0 nmol IAA in 3.0 mm-3). Polar transport of [1-14C]IAA in 30mm-long internodal segments and uptake of [1-14C]IAA by short (2 mm) stem segments were investigated using the techniques described by Johnson and Morris (1987). In some experiments, metabolism of the applied [ 1-I~C]IAA was investigated by paper chromatography of ethanol extracts of tissue. Extracts were either first concentrated by rotary evaporation under reduced pressure at 30 ~ C, or spotted directly onto Whatman (Maidstone, Kent, UK) No. 1 chromatography paper strips, and chromatograms were developed in the ascending direction in isopropanol:ammonia:H20 (10:1:1, by vol.) at room temperature. Developed chromatograms were air-dried and cut into 10 or 15 equal sequential segments. Radioactive compounds were eluted in scintillation fluid (ReadySolv EP; Beckman, High Wycombe, Bucks., UK) and 14C was
0
1 20
40
60
80
Distance (ram)
Fig. 1 A, B. Transport of [1-14C]IAA from the apical buds of dominant and subordinate shoots of two-branched pea plants and the effect on transport in the subordinate shoot of removing the apex of the dominant shoot. A Approximately 1.8 kBq [1-x4C]IAA (328 mmol. m - 3) was applied to the apical buds in 3 mm s of solution, transport was allowed to proceed for 6.0 h and 14C was determined in sequential 5-mm segments. 9 dominant shoot; zx subordinate shoot; o, subordinate shoot 96 h after removal of dominant shoot apex. Means of five replications per treatment. B Methods as in A except that 1.5 kBq (339 m m o l . m -3) was applied to each plant. Transport period was 4.0 h and results are means of three replications, 9 dominant shoots; A, o, [] subordinate shoot 0, 24 and 48 h, respectively, after decapitation of dominant shoot
D.A. Morris and C.F. Johnson: Polar auxin transport in the pea stem
119
c
3.0
E
~3.o I/~
tl
%
~2.0 m
g ..g
o 1.0 O
o
II
ISg
0
I
I
0
10 Distance
20 (mm)
30
Fig. 2. Polar transport of IAA in 30-mm-internodal segments cut from dominant and subordinate shoots of two-branched pea plants. Segments were pulse-labelled with [1-14C]IAA (7.4 kBq; 3.4 mmol-m -3) for 30 min at pH 5.0 (Na-phosphate/citric acid) in 1.5% sucrose solution and chased for 2.0 h in the presence of 1 m m o l . m - 3 unlabelled IAA. Where used, NPA was included in pulse and chase at 3.0 m m o l ' m -3. Means of two (zx, A) or three (o, 9 segments per treatment, o Dominant basipetal; zx dominant acropetal; 9 subordinate basipetal; 9 subordinate basipetal + NPA
substantially recovered within 24 h of removal of the dominant shoot apex (Fig. 1 B). A strictly basipetal polar transport of pulses of [114C]IAA was observed in 30 m m long segments cut from dominant shoots, but only a weak basipetal m o v e m e n t of I A A occurred in segments from subordinate shoots (Fig. 2). In the latter the [14C]IAA taken up during pulsing decayed exponentially with distance from the apical end. However, a small residual a m o u n t of NPA-sensitive m o v e m e n t of label occurred (Fig. 2). Polar transport through segments from subordinate shoots recovered quickly after removal of the dominant shoot apex and a basipetally moving pulse of t4C was apparent in segments cut 16 h after decapitation of the dominant shoot (Fig. 3). Basipetal transport had recovered fully by 24 h after decapitation. To test whether the failure of correlatively inhibited shoots to mediate polar transport resulted from a loss of effiux carriers for IAA, characteristics of uptake of [1-14C]IAA (0.2 m m o l . m-3) from buffered solutions by 2-mm-long internodal segments from dominant and subordinate shoots were compared. In the absence of additives, I A A net uptake was generally lower (by approx. 10-20%) in segments from subordinate shoots than in those from dominant shoots (Tables 1, 2), although this difference disappeared when segments cut from older regions of each stem type were c o m p a r e d (Table 2; Experiment 1). Net uptake by segments from both shoot types was significantly stimulated by high concentrations ( 1 0 0 m m o l . m -3) of unlabelled I A A (Table 1) and by
o
20 Distance (ram) 10
30
Fig. 3. Time-course of recovery of basipetal transport in 30-mmsegments cut from subordinate shoots of two-branched pea plants following removal of the dominant shoot apex. Experimental conditions as in Fig. 2. Means of three segments per treatment. 9 6.5 h, o 16 h, zx 24 h, [] 72 h after excision of dominant shoot apex
Table 1. Effect of unlabelled IAA (100 mmol.m -3) on the net uptake of [1-14C]IAA (0.2 mmol.m 3) by 2-mm pea stem segments cut from dominant and subordinate shoots of two-branched plants. Net uptake at pH 4.5 was measured over 30 min at 25~ C. Results are expressed as Bq. (g FW) 1. (30 min)- 1+ SE (means of five observations per treatment) Shoot type a
Control
+ IAA b
+ IAA as % of control
Dominant Subordinate Subordinate as % of dominant
878.3+21.7 1056.7-t-33.3 120.3 731.7-I-16.7 961.7_+16.7 131.4 83.3 91.0
Net uptake of [I'*C]IAA by segments from subordinate shoots was significantly lower than that by segments from dominant shoots (P
