Planta (Berl.) 124, 177--189 (1975) 9 by Springer-Verlag 1975
The Movement of 2,4-Dichlorophenoxy Acetic Acid in Root Segments of Pisum sativum L. H e n r y Wilkins* and Malcolm B.Wilkins Department of Botany, The University, Glasgow, G12 8 Q Q, U.K. Received 3 March; accepted 14 March 1975 Summary. The movement of 2,4-Dichlorophenoxy acetic acid (2,4-D) through subapieal segments of the primary roots of Pisum seedlings has been investigated using [1-14C]2,4-D. Donation of [1-14C]2,4-Dto the apical or basal ends of Pisum root segments at 25 ~ C in darkness revealed a preferential movement of the compound towards the root apex i.e. an aeropetal polarisation. Thus the movement of [1-14C]2,4-D into receiver blocks applied to the apical ends of the segments is greater than that into receiver blocks applied to the basal ends of the segments. The low level of basipetal transport appears to be associated with a restriction of the movement of [1-14C]2,4-Dto the half of the segment nearest the donor block. Acropetal transport of 2,4-]) is faster than basipetal transport in root segments maintained at 15~ and 35~ C but is slower than basipetal transport if the segments are maintained at 25~ C. Maximum velocities are 0.71 and 0.83 mm h-1 for acropetal and basipetal transport respectively. Evidence from experiments carried out (a) in an anaerobic environment in the presence or absence of sodium fluoride and (b) over a range of temperatures from 1-35 ~ C, indicates that the movement of [1-1~C]2,4-Dis dependent on the metabolic activity of the Pisum root segments. Release of 14C0~ during transport of [1-14C]2,4-Dis small and supports chromatographic evidence that negligible degradation of the 2,4-D molecules takes place during transport through the root segments.
Introduction Detailed investigations have shown that the movement of exogenously applied 3-indolyl-acetic acid (IAA) is acropetally polarised in intact roots and root segments of several plant species (Pilet, 1964; Bonnett and Torrey, 1965; Kirk and Jaeobs, 1968; Wilkins and Scott, 1968 a, 1968 b; Hillman and Phillips, 1970; Iversen and Aasheim, 1970; Iversen etal., 1971; Konings and Gayadin, 1971; Davies and Mitchell, 1972). The movement of IAA, labelled with 14C in the earboxyl group of the acetic acid side chain (i.e. [1-14C]IAA), in segments of the primary roots of P i s u m seedlings, however, is less clearly defined due to the failure of 1~C to move into receiver blocks following donation of the auxin to either end of the segments (Wilkins and Scott, 1968). This apparent absence of transport might be related to degradation of IAA by the root tissues since the transport of IAA, labelled with 14C in the methylene group of the side chain (i.e. [2-14C]IAA), is distinctly acropetally polarised in P i s u m root segments (Hillman and Phillips, 1970). A rapid degradation of [2-~4C]IAA in P i s u m root tissue suggests that the * Present address: Agricultural Research Council Plant Growth Substance and Systemic Fungicide Unit, Wye College (University of London), Wye, Ashford, Kent TN25 5AH.
178
H.Wilkins and M. B.Wilkins
m o v e m e n t of lag reflects t h e m o v e m e n t of r a d i o a c t i v e m e t a b o l i t e s r a t h e r t h a n t h e m o v e m e n t of I A A and, f u r t h e r m o r e , these m e t a b o l i t e s m i g h t influence t h e n o r m a l characteristics of I A A t r a n s p o r t . I t was i m p o r t a n t , therefore, t o s t u d y t h e a u x i n t r a n s p o r t s y s t e m of P i s u m r o o t s e g m e n t s using t h e m o r e stable s y n t h e t i c a u x i n 2,4-dichlorophenoxyacetic acid (2,4-D). Materials and Methods
Plant Material. Seeds of Pisum sativum L. cv. Alaska were soaked for 8 h in flowing tap water prior to germination on moist Whatman No. 1 filter paper in covered plastic boxes at 254-1 ~ C in darkness. After 72 h, seedlings were selected in green light (510-530 ran) for straight radicles 20-30 mm in length and 10 ram-long segments were excised 1.0 mm behind the root tip. 2,4-Diohlorophenoxy Acetic Acid. Transport of 2,4-D in Pisum root segments was monitored using 2,4-D labelled with i4C in the earboxyl group of the acetic acid side chain of the molecule. The compound, referred to as [1-i4C]2,4-D, was supplied by the Radioehemical Centre, Amersham, Bucks., U.K., and had a specific activity of 153 txCi/mg. Chromatographic analysis revealed a chemical and radiochemical purity which exceeded 99%. The sodium salt of [1-1~C]2,4-D was prepared and supplied to the root segments at a concentration of 1.0 ~tM incorporated in 1.5% (w/v) agar donor blocks (Ionagar No. 2) which were 8 mm in diameter and 2 mm thick. Agar receiver blocks were prepared in the same way as donor blocks but did not contain [1-14C]2,4-D. Detection o] Radioactivity. i~C in root tissue, donor and receiver blocks was extracted in 1.5 ml 95% ethanol for 30h at 2~ in darkness in scintillation vials. After drying under vacuum, 10 ml scintillation fluid (4 g 2,5-diphenyloxazole/1000 ml toluene) was added to each vial and the iaC assessed by liquid scintillation counting (Nuclear Chicago "Unilux 3" or Packard Triearb liquid scintillation spectrometer with automatic activity analyser). Data are corrected for machine efficiency, quenching and background radiation. Transport Experiments. Movement of i4C towards the root tip (acropetal) or away from the root tip (basipetal) was determined by supplying donor blocks of 1.0 ttM [1-i4C]2,4-D to the basal or apical ends respectively of sets of 4 Pisum root segments, which were held vertically in perspex holders. Receiver blocks were applied to the opposite ends of the segments. 12 independent sets of root segments were set up in order that the level of ~4C in successive 2 mm sections of the segments and in the receiver blocks could be assessed every 5 h from 0-30 h using a different set of segments at each 5-h step. Green light was used during experimental manipulations but the transport periods were in total darkness at 25 4- 1~ C in humidified growth chambers. Dependence o/ Transport on Metabolism. The techniques of Wilkins and Whyte (1968) were used to establish whether the movement of [1-iaC]2,4-D through Pisum root segments was affected by (a) an anaerobic environment, (b) the metabolic inhibitor sodium fluoride (NaF) or (c) a combination of treatments (a) and (b). Anaerobic conditions were achieved by enclosing the normal transport experiments in vacuum desiccators, evacuating to 250 mm mercury followed by release in humidified, oxygen-free nitrogen (N2). T h e evacuation procedure was repeated six times to ensure complete exclusion of oxygen. Air controls were subjected to the same procedure but were released in air. NaF was supplied to the root segments, in donor and/or receiver blocks, at a concentration of 2.0 raM. Further investigations were carried out in which the transport of [1-iaC]2,4-D through Pisum root segments was monitored at 1~ 5 ~ 15 ~ 25 ~ and 35 ~ C. Seedlings were grown at 25~ 1~ in darkness for 72 h and equilibrated for 30 min prior to experimentation in a dark room maintained at the required temperature. Identification o/ Radioactive Compounds. Root segments, donor blocks or receiver blocks were extracted at 2 4- 1~ C in darkness for 24 h in 95 % ethanol and for a further 24 h in ethanol/acetone (6:4 v/v). Appropriate extracts were combined, reduced to dryness under vacuum, redissolved in 1.0 ml absolute ethanol and applied to the origin of pre-eluted paper chromatograms (Whatman No. 1 ; descending) together with marker spots of authentic 2,4-])
Movement of 2,4 D in Root Segments
179
and [1-14C]2,4-D. Experiments were carried out on three separate occasions so that extracts from the root tissue and agar blocks could be analysed using three different solvent systems for chromatography: (A) isopropanol: 0.88 ammonium hydroxide: water (10:1:1 v/v), (B) n-butanol: acetic acid: water (5:1:2.2 v/v) and (C) n-butanol: acetone: water (5:2:3 v/v). When developed, the chromatograms were dried, cut into 20 equal zones from the origin to the solvent front and the radioactivity in each zone determined by liquid scintillation counting. Detection o] ~adioactive Carbon Dioxide (1~C02). Experiments were carried out in sealed chambers in order that 14C02 released from [1-1aC]2,4-D by 80 t~isum root segments could be trapped by bubbling the air from the chambers through 7.5 ml CO~ trapping-agent (ethanolamine: ethylene glycol monomethyl ether: toluene (1 : 8:10 v/v). Results
Polarity o/2fl-D Movement The movement of 2,4-D has been monitored by supplying donor blocks of 1.0 ~M [1-14C]2,4-D to the apical or basal ends of sets of 4 Pisum root segments and receiver blocks to the opposite ends of the segments. Radioactivity in the segments and receiver blocks was assessed at 5-hourly intervals during a period of 30 h at 25 ~ C in darkness. laC was not detected in receiver blocks applied to either end of the Pisum root segments until 10-15 h after the application of [1-14C]2,4-D donor blocks (Fig. 1). A subsequent increase in the laC-content of the receiver blocks applied to the apical ends of the segments was accompanied by a smaller increase in the basal receiver blocks. In fact, the basal blocks never contained more than 10 dpm whilst the apical receiver blocks contained 128 dpm after 30 h, which shows a distinct acropetal polarity of laC movement. The total accumulation of 14C in root segments and receiver blocks, i.e. the total uptake, was not significantly different during donation of [1-14C]2,4-D to either the apical or basal end of the segments for 25 h (Fig. 2). After a 30-h transport period, the level of laC increased to 1440 dpm following basal donation whilst only 800 dpm could be extracted following donation to the apical end of the segments. At this stage the loss of 14C from donor blocks, (initial 14C-content 5661.1• dpm) was 1 3 3 3 . 4 • dpm after basal donation and 924.6 • 43.7 dpm after donation to the apical end of the segments. On cutting the root segments into 2 mm sections progressively from the point of donation (A) to the receiver block end (F), the acropetal polarity of 14G movement was detectable in the pattern of distribution of radioactivity along the segments. Apical donation of [1-1aC]2,4-D resulted in less movement of 14C along the segments than that found after basal donation (Fig. 3). For example, after basal donation the level of radioactivity in sections D, E and F was 3.5, 4.0 and 11.0 times higher, respectively, than in the corresponding sections following apical donation. The level of ~4C in section A, on the other hand, was higher following apical rather than basal donation, whilst the level in section B was not greatly influenced by the site of donation. Expression of the data as percentage of the total uptake of radioactivity shows that the distribution of 14C along the segments from the donor block to the receiver block end was: (A) 25 %, (B) 15 %, (C) 11%, (D) 8%, (E) 7% and (F) 4% after 30 h basal donation. The corresponding distribution after 3 0 h apical donation was: (A) 55%, (B) 22%, (C) 10%, (D) 6%, (E) 3% and (F) 1%. Thus it is clear that an acropetal polarity of lag
180
H.Wilkins and M. B.Wilkins !
I
I
I
I 10
l
~ 20
'
I I~
"
120
>= I-
80
I-
~
4o
? 0
TIME
I
I 30
(h)
Fig. 1. Time.course for aeropetal (, ,) and basipetal (c o) movement of radioactivity from sets of 4 Pisum root segments into receiver blocks (white blocks) at 25 ~ C in darkness when the donor (dark blocks) concentration of [1-1~C]2,4-D was 1.0 ~ I . Data are the mean of 6 independent experiments and twice standard errors are represented by vertical lines
1200 E
~"
800
FU 0
a a:
400
0
!
I 4.0
I TIME
I 20
I
I 30
(h)
Fig. 2. The total accumulation of radioactivity in root segments and receiver blocks (white blocks) i.e. total uptake, as a function of time during donation of 1.0 [zM [1-14C]2,4-D (dark blocks) to the apical (o o)or basal ends (o ,) of P i s u m root segments at 25 ~ C in darkness. Data arc the mean of 6 independent experiments in which each replicate consisted of 4 root segments in contact with communal donor and receiver blocks. Vertical lines represent twice the standard error
Movement of 2,4 D in Root Segments
181
600
A I-
400
I--
o o
200
C
f~
0 O
10
20
30 1"
!
O M
E
I 10
!
I 20
I
I 30
{ h )
Fig. 3. The distribution of radioactivity in successive 2 mm pieces of Pisum root segments, from the point of donation (A) to the receiver block end (F), as a function of time during apical or basal donation of 1.0 ~tM [1-14C]2,4-D to sets of 4 segments at 25 ~ C in darkness. Data are the mean of experiments carried out on 6 different occasions and twice standard errors are represented by vertical lines
movement can be detected whether the data axe expressed as absolute levels of radioactivity or as percentages and both methods of presentation reveal a restriction of movement in the 4 m m of tissue nearest to the apical donor block (i.e. sections A and B).
Dependence ol 2,4-D Movement on Metabolism The radioactivity in receiver blocks has been determined following the application of donor blocks of 1.0 91VI [1-14C]2,4-D to Pisum root segments for 30 h in air or an atmosphere of N~ at 25 ~ C in darkness. Additional treatments involved the incorporation of N a F in donor blocks and/or receiver blocks and the data are presented in Fig. 4. Exposure of the root segments to Iq2 (treatment 2) resulted in a 37.5% reduction in the level oi 14C in the apical receiver blocks compared with the air control (treatment 1). The N~/NaF in donor block treatment (4) gave a 67% reduction and the N~/NaF in donor and receiver block t r e a t m e n t (8) a 77 % reduction in the laC-content of the apical receiver blocks compared with t h a t of the respective air controls (treatments 3, 7). Radioactivity in apical receiver blocks to which N a F had been incorporated was vh~ually identical following air and N~ treatments (5, 6 respectively). I n the aerobic treatments, acropetal transport of 14C into the apical receiver blocks increased, compared with the air control (treatment 1), b y 141.5% when N a F was incorporated in the donor blocks (treatment 3) and b y 82.9% when
182
It. Wilkins and M. B. Wilkins 120
[
---~AIR
o.
BE 80 I-
i.~ 0 o
12
34
I 8
56
ACROPET
L
12
34
BAS
56 I PETA
78 L
Fig. 4. Acropetal and basipetal movement of radioactivity into receiver blocks during treatment in air (unshaded histogram) or N~ (shaded histogram) for 30 h at 25 ~ C in darkness as follows: (1-2) Air or N~ only, (3-4) NaF in donor block, (5-6) NaF in receiver block, (7-8) NaF in donor and receiver block. Data are the mean of experiments carried out on 2 different occasions using 2 replicate samples/treatment. Each sample consisted of 4 Pisum root segments supplied with communal receiver and donor block of 1.0 ~M [1-14C]2,4-D. LSD (P0.001) acropetal = 16.44, basipetal = 10.86
incorporation was in donor and receiver blocks (treatment 7). Incorporation of N a F in the receiver blocks only (treatment 5), however, produced a 58.5% decrease in the level of 14C in the receiver blocks. Basipetal transport of 14C into the basal receiver blocks was enhanced, compared with the air control (treatment 1), by the incorporation of N a F in the donor and receiver block (treatment 7), and was significantly reduced only by the N2/NaF in receiver block t r e a t m e n t (6). I t appears, therefore, t h a t both acropetal and basipetal transport of 44C from [1-~4C]2,4-D is dependent, to a certain extent, on the metabolic activity of the Pisum root segments. However, clear interpretation of the results is difficult because of the stimulation of the transport systems caused b y NaF.
Dependence o/ 2,4-D Transport on Temperature The movement of 14C was determined after supplying 1.0 9M [1-1aC]2,4-D to
Pisum root segments maintained at 1 ~ 5 ~ 15 ~ 25 ~ or 35 ~ C in darkness. At 15 ~ 25 ~ and 35~ the 14C-content of apical and basal receiver blocks increased steadily with time after an initial temperature-dependent lag-phase of 10-25 h (Fig. 5A, 5B). A strong acropetal polarisation of movement was evident at each of the three temperatures, with the apical receiver blocks containing 14, 19 and 26 times more radioactivity respectively than the basal receiver blocks after a 30.h transport period. Detectable levels of 14C could not be found in receiver blocks applied to either end of the segments when experiments were carried out at 1 ~ or 5 ~ C.
Movement of 2 , 4 D i n R o o t Segments
300
183
35 ~
~:~B
12
2~o
200
100
E
4
.o
--
0
1200
- 3000 U 0
The Movement of 2,4-Dichlorophenoxy Acetic Acid in Root Segments of Pisum sativum L. H e n r y Wilkins* and Malcolm B.Wilkins Department of Botany, The University, Glasgow, G12 8 Q Q, U.K. Received 3 March; accepted 14 March 1975 Summary. The movement of 2,4-Dichlorophenoxy acetic acid (2,4-D) through subapieal segments of the primary roots of Pisum seedlings has been investigated using [1-14C]2,4-D. Donation of [1-14C]2,4-Dto the apical or basal ends of Pisum root segments at 25 ~ C in darkness revealed a preferential movement of the compound towards the root apex i.e. an aeropetal polarisation. Thus the movement of [1-14C]2,4-D into receiver blocks applied to the apical ends of the segments is greater than that into receiver blocks applied to the basal ends of the segments. The low level of basipetal transport appears to be associated with a restriction of the movement of [1-14C]2,4-Dto the half of the segment nearest the donor block. Acropetal transport of 2,4-]) is faster than basipetal transport in root segments maintained at 15~ and 35~ C but is slower than basipetal transport if the segments are maintained at 25~ C. Maximum velocities are 0.71 and 0.83 mm h-1 for acropetal and basipetal transport respectively. Evidence from experiments carried out (a) in an anaerobic environment in the presence or absence of sodium fluoride and (b) over a range of temperatures from 1-35 ~ C, indicates that the movement of [1-1~C]2,4-Dis dependent on the metabolic activity of the Pisum root segments. Release of 14C0~ during transport of [1-14C]2,4-Dis small and supports chromatographic evidence that negligible degradation of the 2,4-D molecules takes place during transport through the root segments.
Introduction Detailed investigations have shown that the movement of exogenously applied 3-indolyl-acetic acid (IAA) is acropetally polarised in intact roots and root segments of several plant species (Pilet, 1964; Bonnett and Torrey, 1965; Kirk and Jaeobs, 1968; Wilkins and Scott, 1968 a, 1968 b; Hillman and Phillips, 1970; Iversen and Aasheim, 1970; Iversen etal., 1971; Konings and Gayadin, 1971; Davies and Mitchell, 1972). The movement of IAA, labelled with 14C in the earboxyl group of the acetic acid side chain (i.e. [1-14C]IAA), in segments of the primary roots of P i s u m seedlings, however, is less clearly defined due to the failure of 1~C to move into receiver blocks following donation of the auxin to either end of the segments (Wilkins and Scott, 1968). This apparent absence of transport might be related to degradation of IAA by the root tissues since the transport of IAA, labelled with 14C in the methylene group of the side chain (i.e. [2-14C]IAA), is distinctly acropetally polarised in P i s u m root segments (Hillman and Phillips, 1970). A rapid degradation of [2-~4C]IAA in P i s u m root tissue suggests that the * Present address: Agricultural Research Council Plant Growth Substance and Systemic Fungicide Unit, Wye College (University of London), Wye, Ashford, Kent TN25 5AH.
178
H.Wilkins and M. B.Wilkins
m o v e m e n t of lag reflects t h e m o v e m e n t of r a d i o a c t i v e m e t a b o l i t e s r a t h e r t h a n t h e m o v e m e n t of I A A and, f u r t h e r m o r e , these m e t a b o l i t e s m i g h t influence t h e n o r m a l characteristics of I A A t r a n s p o r t . I t was i m p o r t a n t , therefore, t o s t u d y t h e a u x i n t r a n s p o r t s y s t e m of P i s u m r o o t s e g m e n t s using t h e m o r e stable s y n t h e t i c a u x i n 2,4-dichlorophenoxyacetic acid (2,4-D). Materials and Methods
Plant Material. Seeds of Pisum sativum L. cv. Alaska were soaked for 8 h in flowing tap water prior to germination on moist Whatman No. 1 filter paper in covered plastic boxes at 254-1 ~ C in darkness. After 72 h, seedlings were selected in green light (510-530 ran) for straight radicles 20-30 mm in length and 10 ram-long segments were excised 1.0 mm behind the root tip. 2,4-Diohlorophenoxy Acetic Acid. Transport of 2,4-D in Pisum root segments was monitored using 2,4-D labelled with i4C in the earboxyl group of the acetic acid side chain of the molecule. The compound, referred to as [1-i4C]2,4-D, was supplied by the Radioehemical Centre, Amersham, Bucks., U.K., and had a specific activity of 153 txCi/mg. Chromatographic analysis revealed a chemical and radiochemical purity which exceeded 99%. The sodium salt of [1-1~C]2,4-D was prepared and supplied to the root segments at a concentration of 1.0 ~tM incorporated in 1.5% (w/v) agar donor blocks (Ionagar No. 2) which were 8 mm in diameter and 2 mm thick. Agar receiver blocks were prepared in the same way as donor blocks but did not contain [1-14C]2,4-D. Detection o] Radioactivity. i~C in root tissue, donor and receiver blocks was extracted in 1.5 ml 95% ethanol for 30h at 2~ in darkness in scintillation vials. After drying under vacuum, 10 ml scintillation fluid (4 g 2,5-diphenyloxazole/1000 ml toluene) was added to each vial and the iaC assessed by liquid scintillation counting (Nuclear Chicago "Unilux 3" or Packard Triearb liquid scintillation spectrometer with automatic activity analyser). Data are corrected for machine efficiency, quenching and background radiation. Transport Experiments. Movement of i4C towards the root tip (acropetal) or away from the root tip (basipetal) was determined by supplying donor blocks of 1.0 ttM [1-i4C]2,4-D to the basal or apical ends respectively of sets of 4 Pisum root segments, which were held vertically in perspex holders. Receiver blocks were applied to the opposite ends of the segments. 12 independent sets of root segments were set up in order that the level of ~4C in successive 2 mm sections of the segments and in the receiver blocks could be assessed every 5 h from 0-30 h using a different set of segments at each 5-h step. Green light was used during experimental manipulations but the transport periods were in total darkness at 25 4- 1~ C in humidified growth chambers. Dependence o/ Transport on Metabolism. The techniques of Wilkins and Whyte (1968) were used to establish whether the movement of [1-iaC]2,4-D through Pisum root segments was affected by (a) an anaerobic environment, (b) the metabolic inhibitor sodium fluoride (NaF) or (c) a combination of treatments (a) and (b). Anaerobic conditions were achieved by enclosing the normal transport experiments in vacuum desiccators, evacuating to 250 mm mercury followed by release in humidified, oxygen-free nitrogen (N2). T h e evacuation procedure was repeated six times to ensure complete exclusion of oxygen. Air controls were subjected to the same procedure but were released in air. NaF was supplied to the root segments, in donor and/or receiver blocks, at a concentration of 2.0 raM. Further investigations were carried out in which the transport of [1-iaC]2,4-D through Pisum root segments was monitored at 1~ 5 ~ 15 ~ 25 ~ and 35 ~ C. Seedlings were grown at 25~ 1~ in darkness for 72 h and equilibrated for 30 min prior to experimentation in a dark room maintained at the required temperature. Identification o/ Radioactive Compounds. Root segments, donor blocks or receiver blocks were extracted at 2 4- 1~ C in darkness for 24 h in 95 % ethanol and for a further 24 h in ethanol/acetone (6:4 v/v). Appropriate extracts were combined, reduced to dryness under vacuum, redissolved in 1.0 ml absolute ethanol and applied to the origin of pre-eluted paper chromatograms (Whatman No. 1 ; descending) together with marker spots of authentic 2,4-])
Movement of 2,4 D in Root Segments
179
and [1-14C]2,4-D. Experiments were carried out on three separate occasions so that extracts from the root tissue and agar blocks could be analysed using three different solvent systems for chromatography: (A) isopropanol: 0.88 ammonium hydroxide: water (10:1:1 v/v), (B) n-butanol: acetic acid: water (5:1:2.2 v/v) and (C) n-butanol: acetone: water (5:2:3 v/v). When developed, the chromatograms were dried, cut into 20 equal zones from the origin to the solvent front and the radioactivity in each zone determined by liquid scintillation counting. Detection o] ~adioactive Carbon Dioxide (1~C02). Experiments were carried out in sealed chambers in order that 14C02 released from [1-1aC]2,4-D by 80 t~isum root segments could be trapped by bubbling the air from the chambers through 7.5 ml CO~ trapping-agent (ethanolamine: ethylene glycol monomethyl ether: toluene (1 : 8:10 v/v). Results
Polarity o/2fl-D Movement The movement of 2,4-D has been monitored by supplying donor blocks of 1.0 ~M [1-14C]2,4-D to the apical or basal ends of sets of 4 Pisum root segments and receiver blocks to the opposite ends of the segments. Radioactivity in the segments and receiver blocks was assessed at 5-hourly intervals during a period of 30 h at 25 ~ C in darkness. laC was not detected in receiver blocks applied to either end of the Pisum root segments until 10-15 h after the application of [1-14C]2,4-D donor blocks (Fig. 1). A subsequent increase in the laC-content of the receiver blocks applied to the apical ends of the segments was accompanied by a smaller increase in the basal receiver blocks. In fact, the basal blocks never contained more than 10 dpm whilst the apical receiver blocks contained 128 dpm after 30 h, which shows a distinct acropetal polarity of laC movement. The total accumulation of 14C in root segments and receiver blocks, i.e. the total uptake, was not significantly different during donation of [1-14C]2,4-D to either the apical or basal end of the segments for 25 h (Fig. 2). After a 30-h transport period, the level of laC increased to 1440 dpm following basal donation whilst only 800 dpm could be extracted following donation to the apical end of the segments. At this stage the loss of 14C from donor blocks, (initial 14C-content 5661.1• dpm) was 1 3 3 3 . 4 • dpm after basal donation and 924.6 • 43.7 dpm after donation to the apical end of the segments. On cutting the root segments into 2 mm sections progressively from the point of donation (A) to the receiver block end (F), the acropetal polarity of 14G movement was detectable in the pattern of distribution of radioactivity along the segments. Apical donation of [1-1aC]2,4-D resulted in less movement of 14C along the segments than that found after basal donation (Fig. 3). For example, after basal donation the level of radioactivity in sections D, E and F was 3.5, 4.0 and 11.0 times higher, respectively, than in the corresponding sections following apical donation. The level of ~4C in section A, on the other hand, was higher following apical rather than basal donation, whilst the level in section B was not greatly influenced by the site of donation. Expression of the data as percentage of the total uptake of radioactivity shows that the distribution of 14C along the segments from the donor block to the receiver block end was: (A) 25 %, (B) 15 %, (C) 11%, (D) 8%, (E) 7% and (F) 4% after 30 h basal donation. The corresponding distribution after 3 0 h apical donation was: (A) 55%, (B) 22%, (C) 10%, (D) 6%, (E) 3% and (F) 1%. Thus it is clear that an acropetal polarity of lag
180
H.Wilkins and M. B.Wilkins !
I
I
I
I 10
l
~ 20
'
I I~
"
120
>= I-
80
I-
~
4o
? 0
TIME
I
I 30
(h)
Fig. 1. Time.course for aeropetal (, ,) and basipetal (c o) movement of radioactivity from sets of 4 Pisum root segments into receiver blocks (white blocks) at 25 ~ C in darkness when the donor (dark blocks) concentration of [1-1~C]2,4-D was 1.0 ~ I . Data are the mean of 6 independent experiments and twice standard errors are represented by vertical lines
1200 E
~"
800
FU 0
a a:
400
0
!
I 4.0
I TIME
I 20
I
I 30
(h)
Fig. 2. The total accumulation of radioactivity in root segments and receiver blocks (white blocks) i.e. total uptake, as a function of time during donation of 1.0 [zM [1-14C]2,4-D (dark blocks) to the apical (o o)or basal ends (o ,) of P i s u m root segments at 25 ~ C in darkness. Data arc the mean of 6 independent experiments in which each replicate consisted of 4 root segments in contact with communal donor and receiver blocks. Vertical lines represent twice the standard error
Movement of 2,4 D in Root Segments
181
600
A I-
400
I--
o o
200
C
f~
0 O
10
20
30 1"
!
O M
E
I 10
!
I 20
I
I 30
{ h )
Fig. 3. The distribution of radioactivity in successive 2 mm pieces of Pisum root segments, from the point of donation (A) to the receiver block end (F), as a function of time during apical or basal donation of 1.0 ~tM [1-14C]2,4-D to sets of 4 segments at 25 ~ C in darkness. Data are the mean of experiments carried out on 6 different occasions and twice standard errors are represented by vertical lines
movement can be detected whether the data axe expressed as absolute levels of radioactivity or as percentages and both methods of presentation reveal a restriction of movement in the 4 m m of tissue nearest to the apical donor block (i.e. sections A and B).
Dependence ol 2,4-D Movement on Metabolism The radioactivity in receiver blocks has been determined following the application of donor blocks of 1.0 91VI [1-14C]2,4-D to Pisum root segments for 30 h in air or an atmosphere of N~ at 25 ~ C in darkness. Additional treatments involved the incorporation of N a F in donor blocks and/or receiver blocks and the data are presented in Fig. 4. Exposure of the root segments to Iq2 (treatment 2) resulted in a 37.5% reduction in the level oi 14C in the apical receiver blocks compared with the air control (treatment 1). The N~/NaF in donor block treatment (4) gave a 67% reduction and the N~/NaF in donor and receiver block t r e a t m e n t (8) a 77 % reduction in the laC-content of the apical receiver blocks compared with t h a t of the respective air controls (treatments 3, 7). Radioactivity in apical receiver blocks to which N a F had been incorporated was vh~ually identical following air and N~ treatments (5, 6 respectively). I n the aerobic treatments, acropetal transport of 14C into the apical receiver blocks increased, compared with the air control (treatment 1), b y 141.5% when N a F was incorporated in the donor blocks (treatment 3) and b y 82.9% when
182
It. Wilkins and M. B. Wilkins 120
[
---~AIR
o.
BE 80 I-
i.~ 0 o
12
34
I 8
56
ACROPET
L
12
34
BAS
56 I PETA
78 L
Fig. 4. Acropetal and basipetal movement of radioactivity into receiver blocks during treatment in air (unshaded histogram) or N~ (shaded histogram) for 30 h at 25 ~ C in darkness as follows: (1-2) Air or N~ only, (3-4) NaF in donor block, (5-6) NaF in receiver block, (7-8) NaF in donor and receiver block. Data are the mean of experiments carried out on 2 different occasions using 2 replicate samples/treatment. Each sample consisted of 4 Pisum root segments supplied with communal receiver and donor block of 1.0 ~M [1-14C]2,4-D. LSD (P0.001) acropetal = 16.44, basipetal = 10.86
incorporation was in donor and receiver blocks (treatment 7). Incorporation of N a F in the receiver blocks only (treatment 5), however, produced a 58.5% decrease in the level of 14C in the receiver blocks. Basipetal transport of 14C into the basal receiver blocks was enhanced, compared with the air control (treatment 1), by the incorporation of N a F in the donor and receiver block (treatment 7), and was significantly reduced only by the N2/NaF in receiver block t r e a t m e n t (6). I t appears, therefore, t h a t both acropetal and basipetal transport of 44C from [1-~4C]2,4-D is dependent, to a certain extent, on the metabolic activity of the Pisum root segments. However, clear interpretation of the results is difficult because of the stimulation of the transport systems caused b y NaF.
Dependence o/ 2,4-D Transport on Temperature The movement of 14C was determined after supplying 1.0 9M [1-1aC]2,4-D to
Pisum root segments maintained at 1 ~ 5 ~ 15 ~ 25 ~ or 35 ~ C in darkness. At 15 ~ 25 ~ and 35~ the 14C-content of apical and basal receiver blocks increased steadily with time after an initial temperature-dependent lag-phase of 10-25 h (Fig. 5A, 5B). A strong acropetal polarisation of movement was evident at each of the three temperatures, with the apical receiver blocks containing 14, 19 and 26 times more radioactivity respectively than the basal receiver blocks after a 30.h transport period. Detectable levels of 14C could not be found in receiver blocks applied to either end of the segments when experiments were carried out at 1 ~ or 5 ~ C.
Movement of 2 , 4 D i n R o o t Segments
300
183
35 ~
~:~B
12
2~o
200
100
E
4
.o
--
0
1200
- 3000 U 0
